Contractor's Quarterly Progress_ Status and Management Report

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          Contractor's Quarterly Progress, Status and Management Report
                        9th Quarter 2009 Reporting Period

Contractor’s Name and Address:
                           Wil Milhous, Ph.D.
                           College of Public Health
                           University of South Florida
                           13201 Bruce B. Downs Blvd., MDC 56
                           Tampa, Florida 33612-3805

Contract Number:               W911SR-07-C-0084

Report Date:                   1 February 2010

Period Covered by Report:      1 October 2009 – 1 January 2010

Title of Report:               Florida Biodefense Research Consortium, Task Area 1:
                               Countermeasures to Biological and Chemical Threats,
                               Laboratory Component

CDRL Item Number:              A003

Security Classification:       Unclassified Sensitive

Issuing Government Activity:
                               AMC Acquisition Center – Edgewood
                               AMSSB-ACC/Bldg. E04455
                               5183 Blackhawk Road
                               Aberdeen Proving Ground, MD 21010-5424
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Item b. Description of Progress Made Against Milestones During Reporting Period.

SUMMARY
Section 1: Research Activities Related to Identification and Typing of Classic
           Biological Threat Agents, Food pathogens and Emerging Infectious
           Disease Agents
            Development of a Spore-less Protocol for RiboPrint and PFGE of Bacillus
              anthracis
            Attempts to enhance performance of DNA extraction from soil using
              Three Manual Soil Kits
            Enhancing PLET medium for use in soil cultures
            Testing efficacy of two novel copper solutions against USA300 MRSA
              isolates
            Testing soils from USGS for presence of B. anthracis
            Staphylococcal Cassette Chromosome mec (SCCmec) Typing Using a
              Multiplex Bead-based Suspension Array
            Molecular Characterization of USA300 and USA400 Methicillin-
              Sensitive Staphylococcus aureus
            Characterization of Methicillin-Resistant Staphylococcus aureus
              Epidemic Clone USA100 Isolates Identified Among Strains Collected for
              Community-associated Staphylococcus Studies
            Pulsed Field Gel Electrophoresis of S. Typhi with Spe-1
            Isolation of E. coli O157:H7 from artificially contaminated lettuce
            Isolation of Shigella sonnei by immunomagnetic separation
            FERN Proficiency Test- Listeria monocytogenes testing
            Broad Range PCR for Detection of BT Agents
            Pyrosequencing of Bacillus anthracis
            Bead validation for Detection of Pathogens on the Luminex System



Section 2:       Support Activities
                Culture Collection Development

   Subcontract assistance was provided by FDOH personnel, for work performed
   under Sections 1 and 2.
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Section 1: Research Activities Related to Identification and Typing of Classic
           Biological Threat Agents, Food pathogens and Emerging Infectious
           Disease Agents

      Development of a Spore-less Protocol for RiboPrint and PFGE of B. anthracis
       (Debbie King and Vicki Luna)

Our goals were to (i) develop a protocol that would eliminate or greatly reduce
sporulation within Bacillus anthracis vegetative cells, and (ii) harvest an adequate
number of cells and sufficient DNA suitable for molecular methods including Riboprint®
analysis and PFGE. Seven strains of B. anthracis (Ames, French B2, Heluky, Kruger,
Pasteur, Sterne, and Vollum) were grown at 37, 42 and 45°C under normal air, enhanced
CO2, microaerophilic, and anaerobic conditions on solid media and subcultured in two
broths with and without supplements. The bacterial cells were centrifuged and washed.
Slides made from the cell pellets were stained with Malachite Green and observed for the
presence of spores. Cell preparations were subjected to 80°C for 30 minutes and
processed for, and analyzed by either Riboprinter® or PFGE. Multiple pellets of each
strain were processed, stained, placed onto solid culture media, incubated for seven days
and observed for growth. The cell preparations yielded clear and reproducible results
with both molecular methods. None of the cell preparations yielded growth on the
culture media. This method eliminated viable spores in cell preparations of B. anthracis
yet still allowed the growth of vegetative cells to provide sufficient DNA suitable for
analysis by Riboprinter® and PFGE. This method will provide safe cell preparations,
prevent instrument contamination, and may be useful for other aerobic and anaerobic
spore-formers.

A paper was submitted and accepted in October of 2009.

King, Luna, Cannons, and Amuso. 2009. Journal of Applied Microbiology.


      Attempts to enhance performance of DNA extraction from soil using Three
       Manual Soil Kits (Jenny Gulledge and Vicki Luna)

The previous work with the DNA kits and automated instruments compared the different
protocols with and without a pretreatment step. Due to the extremely high content of
DNA of other organisms present in all soil, all the kits were faced with a high
background of various DNA. This is a problem for all soil kits. Thus, the detection
limits of the different methods were very disappointing. Therefore, we have started work
in trying to increase the number of B. anthracis cells in a sample before proceeding with
the extraction. This enrichment step involves growing the spores in a broth (PLET) that is
inhibitory to many soil organisms yet allows B. anthracis to grow. Previous work in our
lab has shown that the addition of low amounts of Trimethoprim/sulfamethoxazole
inhibited or delayed the growth of most Bacillus species. In order to determine the
sensitivity limits of the automated systems, soils from Florida, Texas and store bought
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garden soil were spiked with varying amounts of spores of CBD 63 (B. anthracis
Pasteur). The amount of soil spiked was 0.5 grams. Reviewing the work, we identified
a few areas that needed to be repeated to give a clearer picture.


Methods:
Soils and Sampling Sites. Three different soils were used to examine the different DNA
extraction methods. One soil sample was taken at a site located on the grounds of the
University of South Florida at Tampa, Florida) (GPS location = N28º 03.475min; W82º
25.203min). A top layer of soil covering 12 cm x 15 cm in area and 2.5 cm in depth was
removed from the test site to avoid contaminants and results of UV action on the exposed
soil. A second layer (10 cm x 12.5 cm) of soil of equal depth was removed and placed
into a plastic Nalgene bottle (catalogue number 02-893C) (Fisher Scientific, Inc.,
Pittsburg, PA). This layer was expected to have the highest concentration of DNA
compared to deeper layers (Mergel, et al.). A second soil sample was obtained in Texas
at GPS location (N32 7.404min; W102 27.776min). The third soil sample was
obtained from a commercially prepared garden soil purchased from a local Tampa area
home improvement store. The soils were vigorously shaken to mix them thoroughly. A
portion (20-25 mL) of each soil was analyzed for its physical characteristics. Samples
were described utilizing the Unified Soil Classification System. Sample testing included
a visual inspection on a contrasting surface, and a hydrometer test to help in determining
the approximate volumes of each grain size. Grain Size Analysis using sieves was not an
option at the time of testing, so percentages given are close approximations using field
expedient hydrometer methods
        Ten soils from different sites in Texas were also obtained at different GPS
locations that all fell within the geographical area endemic for B. anthracis and resultant
anthrax in livestock and wild herbivores (Table 1).
Soil cultures. Aliquots of each of the three initial soils (1 to 5 g) were cultured as
previously described (Dragon and Elkin, 2001; Dunbar, 1999; Hollender, 2003). B.
anthracis was never isolated in any culture attempts. Aliquots of soil (0.1 to 1g) were
tested with or without the pre-treatment steps below. Dot blots made from crude soil
extracts were also consistently negative in DNA-DNA hybridization assays for target
genes (lef, pag, and capC) of B. anthracis plasmids pX01 and pX02 (Luna et al 2006).
Therefore, the soils were considered negative for B. anthracis and safe for use in this
study. The soil aliquots that later had B. anthracis spores added to them and were to be
used on automated instruments were autoclaved before testing to preclude contamination
of the instruments.
Bacterial Spores. B. anthracis Pasteur (CDC BC 3233) spores of known concentration
(10 8 CFU / mL) in saline were obtained from the Florida Department of Health. A
known amount of spores was added to an aliquot of soil before either directly processing
with one of the manual kits and automatic methods or before pretreatment and / or
enrichment steps. All tests were performed in triplicate. Spores were counted using a
Rosenfuch’s hemacytometer or heat-shocked (60°C for 15 minutes), serially diluted ten-
fold, and plated onto tryptic soy agar supplemented with 5% sheep red blood cells (BA)
(Remel, Lenexa, KS) to confirm the visually counted and /or calculated amount of spores
added to the different tests. The plates were incubated at 30°C. At 24 hours and each
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subsequent day, colonies were counted and the colony forming units (CFU) per mL were
calculated. Plates were held up to 7 days. The average number ± standard deviation of
spores added to the soil aliquots were: 1.33 ± 0.32 x107; 1.58 ± 0.95 x106; 2.5 ± 1.84
x105; 1.84 ± 1.51 x104; 3.8 ± 0.33 x 103; 2.4 ± 0.77 x 102; 6.7 ± 2.1 x 101; and 5.0 ± 3.1 x
100.
Soil Pretreatment. In order to remove humic acids and free DNA from the test samples,
aliquots of soil (0.1 to 0.5 g) with spores were suspended in 5 mL (10 mL for garden
soil) of a Pretreatment Solution (0.1% sodium pyrophosphate tetrabasic [Na4P2O7], 1
mM EDTA, 10 mM Tris-Cl) and vortexed hard 2-3 minutes at least three times to
thoroughly break up the soil spore mixture (Rosch et al 2002) The mixture was allowed
to settle for 10 minutes at room temperature and centrifuged for 10 minutes at 4500 × g.
The supernatant was discarded and pellet processed with one of the five protocols.
Testing Kits with Sterilized Soil treated with UV. To confirm that the B. anthracis
target gene was not already in the soil samples and that the PCR was detecting the added
spores, twelve aliquots (2 – 5 g) of the three soils were placed into 1.5 mL screw-topped
autoclavable tubes (056698) (Fisher Scientific, Suwanee, GA) moistened with 5-10µL
sterile water, sonicated for 30 minutes in a Branson 1500 sonicating water bath (Branson
Ultrasonics Corp., Danbury, CT.), and autoclaved as previously described (Luna, et al
2002). The amount of soil used was specified by the different DNA methods. The tubes
were opened, placed into a Bio-Safety Cabinet and exposed to ultraviolet (UV) light (254
nm) overnight to destroy any DNA within the sample. The next day a small portion of
each soil (up to one-half of soil aliquot) was cultured for any bacterial growth. The
remainder of the sonicated and UV-exposed soil was placed into 1.5 mL screw-topped
autoclavable tubes which were divided into two different groups. One group was
processed as is while the other group had 106 or 107 B. anthracis Pasteur spores added
before processing. The amount of soil in each tube that was used for DNA extraction
ranged from 0.1 to 1 g following the manufacturer’s requirements. DNA was extracted
from all of the aliquots.
Soil Enrichment in Modified PLET Broth. Aliquots of soil (0.25 to 1 g) with spores
were suspended into 25 mL of a selective medium polymyxin B-lysozyme-EDTA-
thallium acetate broth (PLET) supplemented with sulfamethoxazole (38 g/mL) and
trimethoprim (2 g/mL) (Luna et al 2009). The soil mixture was vortexed hard for 10 –
20 seconds three times, heated to 65 or 80C for 30 minutes and incubated overnight in a
30C shaking water bath. The bacterial growth was vortexed hard twice for 10 – 20
seconds, filtered through one layer of gauze (Fisher Scientific, Pittsburg, PA) to remove
large soil particles and centrifuged for 1 hour at 5500 x g. The supernatant was discarded
and the pellet washed with dH2O and then centrifuged again for 10 minutes at 4500 x g.
The bacterial pellet was then immediately used in the DNA extraction steps with one of
the five tested methods.
DNA Extractions. Three commercially available soil kits for manual DNA extractions
were examined: MO BIO UltraClean™ Soil DNA Isolation Kit (12800-50) (MO BIO
Laboratories, Inc., Solana Beach, CA), Epicentre SoilMaster™ DNA Extraction Kit
(SM02050) (Epicentre Biotechnologies, Madison, WI) and Q-BIOgene Fast DNA®
SPIN Kit for Soil (6560-200) (Q-BIOgene, Irvine, CA). The two automatic systems
evaluated were: MagNaPure® (Roche Diagnostics Corp., Indianapolis, IN) and
Qiagen® BioRobot M48 Workstation (Qiagen,Valencia, CA.
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         Whole cell DNA was extracted using the three manual kits and two automatic
instruments following the various manufacturers’ instructions. The two automated kits
had extra instructions for processing the soil. The Roche MagNA Pure LC DNA Kit III
was used for extracting DNA from soil with the MagNA Pure LC automated system. The
MagNAPure®LC directions required autoclaving of the soil aliquot and a pre-lysis step
in the lysis buffer found in their MagNaPure®LC DNA Isolation Kit III before a 10
minute centrifugation at 13,000 × g. The entire amount of supernatant was then used
with the rest of the kit solutions as directed by the manufacturer. The Qiagen®
BioRobot M48 Workstation directions required the soil samples to be pre-treated with
components from two Quiagen® kits (QIAamp®DNA Stool Mini Kit and MagAttract
DNA Mini 48 Kit). QIAamp® DNA Stool Mini Kit Water (600 ul) was added to each
soil aliquot (up to 0.5g), incubated for 10 minutes at 95°C and centrifuged 2 minutes at
4000 x g. The supernatant was transferred to a 2 ml tube and 190 ul of buffer G2 from
the MagAttract DNA Mini M48 Kit was added. One InhibitEX tablet from the
QIAamp® DNA Stool Mini Kit was then added and incubated for 1 minute before being
mixed and centrifuged 2 minutes at 10,000 x g. The supernatant was then loaded into
sample tubes in the BioRobot M48 Workstation instrument.
         Following the manufacturer’s recommendations, portions of 0.1 to 1 g of
untreated soil with and without spores were directly processed by each method and tested
in parallel with the pre-treated soil pellets. DNA was stored at -20 until needed.
PCR. Primers for capC were previously designed using LaserGene
(DNAStar,Madison, WI) and B. anthracis pX02 sequence obtained from GenBank
(accession number NC_ 002146 (Luna et al 2006). This produced a 1535 bp amplicon.
Primers for lef and pag genes were designed using the pX01 sequence obtained form
GenBank(accession number NC_001496)
         Using MegAlign® (DNAStar®), an alignment of the 16S sequences of different
Bacillus species, including B. atrophaeus (X60607), B. anthracis (AE017334 and
AE017225), B. cereus (X55060 and X55063), B mycoides (X55061), B. subtilis
(EF422864) and B. thuringiensis (X55062) was used to derive primers that would work
with various Bacillus species. The resulting 16S assay 403 bp amplicon was used as a
control to demonstrate that the PCR chemistry was not inhibited.
         PCR reactions of 10 L [1.5 mM MgCl2; 1X buffer; 2 M dNTP; 1.3 M
primers; 0.025U Taq DNA polymerase (Takara, Madison, WI)] were carried out using
0.5 to 2 ng of extracted DNA as template in a T1 Thermocycler (Biometra, Horsham,
PA). For the 16S, pag and lef assays, the following parameters were followed: initial
heating for 2 minutes at 94C, followed by 40 cycles of 20 seconds at 94C, 20 seconds
at 58C, and 3 minutes at 72C, with a final extension at 72C for 5 minutes. For the
capC assay, the annealing temperature was raised to 68C while the denaturing and
annealing times were lengthened to 1 minute and the final extension time to 7 minutes.
PCR reactions were performed in triplicate. Positive and negative controls were used for
all assays (B. anthracis Pasteur CBD 63 (+); B. anthracis CBD 131(+); B. anthracis
Sterne BB001 (+), B. cereus CBD 58 (-) and / or dH2O (-). In addition, positive control
DNA was mixed with an equal amount of the extracted DNA obtained by each of the
methods and used as template to show that the PCR reactions worked in that particular
soil. The PCR products and the “DNA” from the UV-treated and sterilized soils were
electrophoresed on a 1 % agarose gel (0.5X TBE with 0.05 g/mL ethidium bromide) for
                                                                                       7


50 minutes at 80v constant voltage. Staining with additional ethidium bromide and
destaining were not necessary. The DNA bands were visualized with ultraviolet light and
photographs were made using the GelDoc (Bio-Rad, Hercules, CA).
DNA –DNA Hybridization. DNA dot blots containing 10 – 100 g purified whole cell
DNA and Southern blots from gels of the electrophoresed PCR amplicons were made
using Immobilon-nYplus nylon membrane (Millipore, Bedford, MA) or Roche nylon
membrane (Roche Diagnostics, Indianapolis, IN) and prepared following standard
protocols (Sambrook). The DNA was bound to the membrane by UV irradiation using
the Spectrolinker XL1000 (Spectronics Corporation, Westbury, NY). Oligonucleotide
probes specific for internal portions of the targeted genes were designed with
LaserGene (DNAStar) and labeled with digoxigenin using the DIG Oligonucleotide
Tailing Kit (Roche) as per the manufacturer’s instructions. The different probes detected
the gene targets in as low as 2 ng of whole cell DNA from the positive control. Positive
and negative DNA were used with each hybridization.
RESULTS
Soil characteristics. The Texas soil sample TX-A3 was described as alkaline, dry, very
loose, tan medium-grained silica sand containing silt, organics and minor sub-rounded
gravel. The composition of the soil was roughly 8% coarse-grained sand, 30% medium-
grained sand, 55% fine-grained sand, 6% silt and clay and 1% organic compounds.
Using the Unified Soil Classification System (USCS) method, the soil TX-A3 was
described as Poorly-Graded SAND with ≥ 50% of coarse fraction larger than 4.75 mm.
The ten additional Texas soils resembled Texas TX-A3 having the same characteristics
as described above. The Florida soil sample FL-51 was acidic, dry, very loose, tan, fine-
grained calcareous sand, with silt. It also contained minor coarse-grained sand and minor
fine sub-rounded gravel. FL-51 was composed of roughly 87% fine-grained sand, 1%
silt and clay, and 12% organics and by the USCS method was classified as Poorly-
Graded SAND with ≥ 50% of coarse fraction less than 4.75 mm. The commercial garden
soil sample Com-A was acidic, black, damp, and very loose peat with fine to medium-
grained sand. The soil composition was roughly 4% fine to medium-grained sand, about
1% silt and clay, and 95% organics. The soil Com-A was described as PEAT when
following the USCS method.
Initial Evaluation of Extraction Kits. The sterilized and UV-treated soil samples did
not produce any bacterial growth after seven days incubation. The soils were processed
with one of the three manual kits (Epicentre SoilMasterTM DNA Extraction Kit, Q-
BIOgene Fast DNA® SPIN Kit for Soil or MoBio UltraCleanTM Soil DNA Isolation Kit).
Spectroscopy of the potential DNA product extracts from the UV-treated soils produced
readings equal to a blank sample and DNA was not visually detectable on an agarose gel.
When DNA from the positive control (CBD 63) was added to these preparations, PCR
assays yielded positive reactions. In addition, the UV-sterilized soil that had spores
added and then processed also produced DNA that yielded positive PCR amplicons for
both 16S and capC.
         The different methods extracted a wide range of DNA concentrations from the
test soil samples that were spiked with B. anthracis spores (Table 2). The
MagNaPure®LC instrument gave the highest concentrations of DNA and was the most
inconsistent in the amount of DNA extracted from a single soil type. The extracted DNA
was consistently negative for capC and positive for 16S when multiple PCR assays were
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performed from the same extracted DNA sample. With the other methods, no pattern
correlating the DNA concentrations and PCR results could be discerned. Surprisingly,
the garden soil did not always yield more total DNA than the other two soils. For
example, the Epicentre SoilMaster™ DNA Extraction Kit produced 300 µl of DNA from
untreated Florida, untreated Texas and untreated garden soils, an average of 18 ± 1.9
ng/ul (range = 14-21 ng/µl), 7.8 ± 4.5 ng/µl (range = 3-15 ng/µl), and 10.5 ± 6.1 ng/µl
(range = 4.3 – 22 ng/µl), respectively (Table 2).
         The 16S PCR assay was not specific for Bacillus anthracis and gave positive
results in reactions with template DNA from other Bacillus and Staphylococcus spp.
Although B. anthracis was never cultured from any of the three soils, numerous other
Bacillus spp, Staphylococcus and other Gram-positive bacteria were present in the soil
cultures (results not shown). All of the methods and soils produced DNA that yielded
positive results for the 16S PCR assay.
         Although capC has been noted in other species of Bacillus isolates, repeated PCR
assays performed on the soils without any added spores were consistently negative.21, 24
As noted earlier, the unspiked soils had previously tested negative for the lef and pag
genes found on the pX01 plasmid by both PCR and DNA-DNA hybridization studies.
After the addition of spores to the soil, capC gene was detected in the Florida and Texas
soil samples (both pretreated and untreated) using the DNA extracted by both the
Epicentre SoilMaster™ DNA Extraction Kit and the automated Qiagen® BioRobot M48
Workstation method (Table 2). However, the gene target was never detected in the
garden soil samples that were processed by either protocol. The MoBio UltraClean™
Soil DNA Isolation Kit extracted DNA in which the capC gene was detected for the pre-
treated Florida soil (106 spores), untreated and pre-treated Texas soil samples (105 and
104 spores, respectively). The DNA extracted with the Q-BIOgene Fast DNA® SPIN Kit
for Soil produced positive PCR results for capC only with high concentrations (107) of
spores in the Texas and garden soil. However, the DNA produced by the same kit did not
yield positive PCR results for capC in any of the Florida soil samples. In contrast, none
of the DNA extracted by the MagNaPure®LC from the three soil types produced positive
PCR results for the capC gene even though the 16S assays were positive. Only the
Qiagen® BioRobot M48 Workstation extracted DNA that gave positive results for the
target gene when 103 spores were present in the soil sample. Because the kits stipulated
different optimal amounts of soil to use in their procedures, the limit of detection (LOD)
was calculated per gram of soil for each method for comparison. The detection limit for
the Epicentre SoilMasterTM DNA Extraction Kit increased by 1 log to 107 spores per
gram of soil while the detection limits for the other kits remained the same. In the
beginning of the study the pre-treatment step appeared to help produce amplicons in the
PCR assays. Using the DNA extracted with the MoBio UltraClean™ Soil DNA Isolation
Kit, PCR could detect a lower concentration of spores in the pretreated Florida and Texas
soils than was detected in the untreated samples. However, an examination of the table
shows that the step is not always necessary when using the other extraction methods.

       A paper describing the work with the different DNA extraction kits for soil is
being prepared now to be submitted to a peer review journal.
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 Table 1: Soils and Corresponding GPS Locations


Soil Number         Latitude                  Longitude


TX-A3           N 32deg 7.404 min        W 102deg 27.776 min
FL-51           N 28 deg 3.475 min       W 82 deg 25.203 min
Com-A              not applicable           not applicable

TX-E2          N 33 deg   31.209 min    W 101 deg   46.392 min
TX-E3          N 33 deg   31.209 min    W 101 deg   45.897 min
TX-E4          N 33 deg   31.211 min    W 101 deg   45.297 min
TX-F4          N 34 deg   18.914 min    W 102 deg   18.271 min
TX-F5          N 34 deg   19.145 min    W 102 deg   18.267 min
TX-G2          N 33 deg   43.761 min    W 101 deg   43.817 min
TX-G3          N 33 deg   43.730 min    W 101 deg   43.813 min
TX-G4          N 33 deg   43.723 min    W 101 deg   43.870 min
TX-H3          N 34 deg   49.419 min    W 102 deg   31.710 min
TX-H5          N 34 deg   50.330 min    W 102 deg   35.094 min
TX-H6          N 34 deg   50.333 min    W 102 deg   35.242 min
TX-N2          N 33 deg   43.805 min    W 101 deg   43.940 min
TX-N4          N 33 deg   43.798 min    W 101 deg   43.923 min
TX-N5          N 33 deg   43.798 min    W 101 deg   43.860 min
                                                                                                                                           10



   Table 2:       The lowest concentration of B. anthracis sporesa detected by PCRb for capC gene in DNA extracted from three
                  soils with and without enrichment step in PLET
                                                                               Soils


                                 Florida                                      Texas                                       Garden
                                          PLET                                        PLET                                        PLET
 Kit/Method       Untreatedc Pretreated enrichment            Untreatedc Pretreated enrichment            Untreatedc Pretreated enrichment

Epicentre
SoilMaster™           106          107            102             106           105           101              Nd            N                  102

MoBio
UltraClean™           N            106            102             105           104           102              106           N                  102

Q-BIOgene Fast
DNA® SPIN             N             N             102             107            N            101              107          107                 102

MagNaPure®LC          N             N             N                N             N             N               N             N                  N

Qiagen®
BioRobot M48
Workstation           105          106            102             104           103           102              N             N                  101


   a. Average numbers of spores added to the different soils were: 1.33 ± 0.32 x 107; 1.58 ± 0.95 x 106; 2.5 ± 1.84 x 105; 1.84 ± 1.31 x
   104; and 1.6 ± 0.31 x 103.
   b. PCR assays for the 16S target were consistently positive for the three soils and all five methods of DNA extraction.
                                                                                                                                11



c. “Untreated” soils were directly added to the components of the manufacturer’s DNA extraction kits as directed by the
manufacturer; “Pretreated” soils were treated to remove orgnanic and humic acids before DNA extraction following manufacturer
directions. “PLET” soils were added to PLET broth and incubated overnight before being pelleted and then used in the DNA
extraction kits.
d. “N” = denotes “not detected”. The B. anthracis target genes were not detected in the sample after addition of 107 spores.
                                                                                        12



      Enhancing PLET medium for use in soil cultures

To prove linkage between an environmental sample and an anthrax case, there must be
isolates obtained from both that can be compared. Although Bacillus anthracis is easily
isolated from powder samples, isolating it from soil is difficult because of the high
bacterial count in it. Formulations of PLET were prepared, inoculated with B. anthracis,
B. cereus and B. thuringiensis and examined for growth. Two hundred eighty-three
isolates including 23 B. anthracis were placed onto one formulation while MICs against
trimethoprim-sulfamethoxazole were determined. The media supported B. anthracis
growth at 30°C and inhibited almost all other bacterial growth, including closely-related
species. Sensitivity for B. anthracis and selectivity against other Bacillus and against
non-Bacillus were 96.8 %, 100 % and 97.2 % respectively. Isolates that grew had MICs
>4 and >76 µg/ml-1 against trimethoprim and sulfamethoxazole, respectively. Soils
spiked with 102 B. anthracis spores and suspended in PLET broth yielded a 6-7 log10
increase in B. anthracis. Other growth was inhibited. PLET supplemented with
sulfamethoxazole (38 µg mL-1), trimethoprim (2 µg mL-1), polymyxin B (15,000 U L-1),
and lysozyme (150,000 U L-1) can successfully select for B. anthracis and will facilitate
agricultural, environmental and forensic investigations of B. anthracis isolates.

A paper was submitted and accepted.
Luna, Gulledge, Cannons, and Amuso. 2009. Journal of Microbiol Methods



      Testing efficacy of two novel copper solutions against USA300 MRSA isolates
       (Vicki Luna)

After discussions with CBD, Dr. Tony Hall at Remedy, Inc based in London, UK
requested that CBD test a new decon agent that is copper-based against USA300 MRSA
isolates presently in our collection. We have agreed to do this with the full intention of
obtaining data for a paper that will be written in concert with Dr. Hall. We have already
tested the USA300s against several drugs and agreed to perform both MIC and MBC
determinations and to try time-kill studies on their solutions. Dr. Hall sent us two
solutions both having copper as the antimicrobial agent. Other researchers have claimed
that the solutions are effective against a small sample of various staphylococci and
against both Clostridium difficile and Haemophilus influenzae. Yet it is unknown if the
solutions are truly effective at killing MRSA.

Methods: MICs were performed using 96-well microtiter plates. Prior to testing, the S.
aureus isolates strains were taken out of the -80°C freezer and subcultured twice on
trypticase soy agar (TSA) plates containing 5% sheep blood (BA) (BD BioSciences,
Sparks, Md.). Incubation temperatures were 35oC. On the day of testing, the 96-well
microtiter plates were prepared with two fold dilutions of AL42 or WB50 (the two copper
solutions) in 150mL volumes. The dilutions could not contain any bacterial media (i.e.
Mueller Hinton or TSA or BHI) or hard water. Therefore we used deionized water that
                                                                                       13


had been sterilized and measured 259 mV in conductance. Overnight growth (4-5
colonies) are taken from BA and resuspended in demineralized water and adjusted to a
0.5 MacFarland. To make the inoculum, ten µL of the bacteria-water suspension is
inoculated into 9.99 mL RMPI 1640 media and mixed vigorously. Ten µL are taken
from the RMPI-bacteria mixture and used to inoculate a 9.99 mL of BHI broth (TREK
Diagnostics, Cleveland OH). From the BHI tube, 100 µL are used to inoculate a colony
counting plate (inoculum count plate) used for the MBC determinations later.
Subsequently, 20 l of the RMPI- bacteria mixture was added to each well (~ 1 x 105
cells/ml) and plates were sealed with non-removeable plastic seals in ambient air at 35°C
for 24 hours. S. aureus ATCC 29213 was the control organism for the tests. Plates were
assessed visually for growth. After the 24 hour reading, all wells with no visual growth
were subcultured (10 µL) onto BA plates and incubated overnight at 35°C. The colonies
that grew on these subcultures were counted and compared to the inoculum count. Those
wells that produced colony counts that exceeded the inoculum count were considered
having growth, while those wells with colony counts less than the inoculum counts were
considered as “no growth”. The MBC is defined as the lowest concentration of drug that
reduces the initial inoculum by ≥ 3 log10.
        For comparison, benzalkonium chloride (BZC) (Sigma-Aldrich) was prepared and
diluted in distilled water for MIC and MBC assays (test range of 0.025 to 2160 ppm).
Time-kill curve studies with BZC were performed at 5 ppm, 50 ppm, 150 ppm and 300
ppm.

Results: The MIC range, MIC50 and MIC90 (0.59 – 18.75, 4.69, and 4.69 ppm,
respectively) and the MBC range, MBC50 and MBC90 (1.17- 18.75, 4.69, and 9.38 ppm)
for CuAL42 were identical with the values obtained with CuWB50 except that the MBC
range for CuWB50 was wider (0.59 – 37.5 ppm), Table 4. The MIC range for
benzalkonium chloride was 0.10 to 40 ppm while the MIC50 and MIC90 were 10 ppm and
40 ppm, respectively. The MBC values were identical to the MIC values, Table 3. In
the time-kill studies, Table 4, total kill of 6 log10 CFU with CuAL42 was achieved within
1 h with 150 ppm and 0.5 h with 300 ppm. With CuWB50, the complete killing of cells
took 1.5 h with 150ppm and 0.75 h with 300 ppm. BZC at 150 ppm and 300 ppm
needed 1 h and < 0.25h , respectively, to accomplish the total kill of 6 log10 CFU.
                                                                                                                                                  14



  Table 3:          The distribution of MIC and MBC values for 169 isolates of USA 300 MRSA.

                                                                    Biocide Concentration (ppm)

         Biocide        <0.15      0.29        0.59     1.17       2.34         4.69          9.38    18.75    37.5         75   150        300

         CuAL42            0        0           1           2       54          103            8           1    0           0     0          0
MIC
         CuWB50            0        0           2           19      38          97            12           1    0           0     0          0

         CuAL42            0        0           0           3       35          69            58           4    0           0     0          0
MBC
         CuWB50            0        0           2           6       33          69            54           4    1           0     0          0


  Table 4:          MIC and MBC ranges, 50, and 90 percentiles and total kill times for CuAL42 and CuWB50.

                                  MIC (ppm)                                     MBC (ppm)                        Time to complete kill

      Biocide      Range                50 %          90 %       Range                 50 %          90 %      150 ppm           300 ppm

      CuAL42       0.59 - 18.75         4.69          4.69       1.17 - 18.75          4.69          9.38           1h            0.5 h

      CuWB50       0.59 - 18.75         4.69          4.69       0.59 - 37.5           4.69          9.38           1.5 h         0.75 h

      BZCa         0.10 - 40              10           40        0.10 - 40              10            40            1h           < 0.25 h
        a. “BZC” denotes benzalkonium chloride.
                                                                                      15



      Testing soils from USGS for presence of B. anthracis (Vicki Luna, Jenny
       Gulledge, and Debbie King)

Dr. Dale Griffin at USGS has sent to CBD different soils that were positive by their PCR
assays for B. anthracis. We have used DNA-DNA hybridization and PCR to either rule
out or confirm the presence of B. anthracis in these soils. Fourteen soils were received
and tested for Dr. Griffin. Another nineteen have been recently received and will be
processed for the next report.

Methods: 0.25 -0.5 g of soil was used with the Epicentre SoilMaster™ DNA Extraction
Kit following the manufacturer’s directions. No enrichment in PLET media was done
prior to the extraction. Dot blots containing 355 – 1294 ng of DNA from the extracted
soil DNA were made using Immobilon-nYplus nylon membrane (Millipore, Bedford,
MA) or Roche nylon membrane (Roche Diagnostics, Indianapolis, IN) and prepared
following standard protocols. The DNA was bound to the membrane by UV irradiation
using the Spectrolinker XL1000 (Spectronics Corporation, Westbury, NY).
Oligonucleotide probes specific for internal portions of the targeted genes were designed
with LaserGene (DNAStar) and labeled with digoxigenin using the DIG
Oligonucleotide Tailing Kit (Roche) as per the manufacturer’s instructions. The different
probes detected as low as 2 ng of positive DNA. Positive (B. anthracis Pasteur CBD 63
and B. anthracis Sterne BB001) and negative controls (B. cereus CBD 58) were used
with each hybridization.
        Primers for capC were previously designed using LaserGene
(DNAStar,Madison, WI) and B. anthracis pX02 sequence obtained from GenBank
(accession number NC_ 002146) (Table 5) (Luna et al 2006). This produced a 1535 bp
amplicon. Primers for lef and pag genes are noted on the following table.
        Using MegAlign® (DNAStar®), an alignment of the 16S sequences of different
Bacillus species, including B. atrophaeus (X60607), B. anthracis (AE017334 and
AE017225), B. cereus (X55060 and X55063), B mycoides (X55061), B. subtilis
(EF422864) and B. thuringiensis (X55062) was used to derive primers that would work
with various Bacillus species. The resulting 16S assay 403 bp amplicon was used as a
control to demonstrate that the PCR chemistry was not inhibited.
        PCR reactions of 10 L [1.5 mM MgCl2; 1X buffer; 2 M dNTP; 1.3 M
primers; 0.025U Taq DNA polymerase (Takara, Madison, WI)] were carried out using
0.5 to 2 ng of extracted DNA as template in a T1 Thermocycler (Biometra, Horsham,
PA). For the 16S, pag and lef assays, the following parameters were followed: initial
heating for 2 minutes at 94C, followed by 40 cycles of 20 seconds at 94C, 20 seconds
at 58C, and 3 minutes at 72C, with a final extension at 72C for 5 minutes. For the
capC assay, the annealing temperature was raised to 68C while the denaturing and
annealing times were lengthened to 1 minute and the final extension time to 7 minutes.
PCR reactions were performed in triplicate. Positive and negative controls were used for
all assays (B. anthracis Pasteur CBD 63 (+); B. anthracis CBD 131(+); B. anthracis
Sterne BB001 (+), B. cereus CBD 58 (-) and / or dH2O (-). In addition, positive control
DNA was mixed with an equal amount of the extracted DNA obtained by each of the
                                                                                                    16


       methods and used as template to show that the PCR reactions worked in that particular
       soil.
              The PCR products and the “DNA” from the soils were electrophoresed on a 1 %
       agarose gel (0.5X TBE with 0.05 g/mL ethidium bromide) for 50 minutes at 80v
       constant voltage. Staining with additional ethidium bromide and destaining were not
       necessary. The DNA bands were visualized with ultraviolet light and photographs were
       made using the GelDoc (Bio-Rad, Hercules, CA).


       Table 5:       Oligonucleotides a Used for PCR assays and DNA-DNA hybridizations
                      for 16S rDNAand capC, lef and pag genes

         Primer/                                                                 Size     GenBank
Gene      Probe                         Sequence (5’ → 3’)                       (bp)     number          organism

16S     16SBac F           CGT GGG GAG CGA ACA GGA TTA GAT A                     400       X55060         B. cereus
        16SBac R           GTT TGT CAC CGC GAG TCA CCT TAG AG                              X55060         B. cereus

capC     capB 2F          GGT CTT CCC AGA TAA TGC ATC GCT TG                     1535    NC_002146       B. anthracis
         capA 1R        AGT TGT TGT CTC CAC TGA TAC TTG ATT TTC                          NC_002146       B. anthracis
         capA1F       CAA CAT TTG CAA TCA TGA ATA TTT ATT ACT TAT
                                                                                         NC_002146       B. anthracis

capC      capCF             GGA AGA ACA AAT TGG CAA AAA GC                       635     NC_002146       B. anthracis
          capCR          CGC AGC TAT TAA TAT AAC TGC GAT AAG                             NC_002146       B. anthracis
         capB2R     TTC TTT CTG TAA AAA TAA GGC TCA GTG TAA CTC CT
                                                                                         NC_001496       B. anthracis
 lef      LF1F          TTA GAT AAT GAG CGT TTG AAA TGG AGA A                    432     NC_001496       B. anthracis
          LF1R         TAG GGA GAG TAA TAT CGG TAA AAA CAA ATC                           NC_001496       B. anthracis
           LF5          TTA GAT AAT GAG CGT TTG AAA TGG AGA A                            NC_001496       B. anthracis

pag       PA1F               AGT GCA TGC GTC GTT CTT TGA TA                      529     NC_001496       B. anthracis
          PA1R              GAA TTT GCG GTA ACA CTT CAC TCC                              NC_001496       B. anthracis
           PA6              AGG GGA AAG AAC TTG GGC TGA AA                               NC_001496       B. anthracis
           PA7          CCT TAG CTT TAA TTG TCG CGA GTG TTT GAT                          NC_001496       B. anthracis



               Oligonucleotides were used as PCR primers and / or probes (*) for either
       Southerns of PCR amplicons or whole cell DNA dot blots. Oligonucleotide probes were
       internal to, and specific for, the target sequence as determined by N-blast with NCBI


       Results: Nine of the fourteen soils gave positive results by either PCR or DNA-DNA
       hybridization for the pX01 and pX02 plasmids of B. anthracis, Table 6. The pre-
       enrichment step using PLET media has been used with these soils and may in fact aid in
       the PCR detection of the genes in the soil. It is interesting that not all soils are positive
       for both pag and lef genes on the pX01 plasmid although the rpoB and cap assays gave
       positive results. This most likely reflects a laboratory false negative in that assay. This
                                                                                         17


emphasizes the need to test for several genes on a plasmid before calling it negative.
We will culture the positive soils, using the PLET media, in an attempt to isolate B.
anthracis strains.

Table 6:       Results of testing for B. anthracis in different soil samples from USGS

               PCR                   DNA hybridization
                                                          pX01
           Chromosome            pX02 plasmid            plasmid
   Soil        rpoB            cap genes or dep         pag   lef
    827         pos                  pos                pos   neg
   5000         pos                  pos                pos   pos
   5275         pos                  neg                neg   neg
   5691         pos                  pos                pos   pos
  10203         pos                  neg                neg   neg
  12027         pos                  pos                pos   pos
     96         pos                  neg                neg   neg
    591         pos                  neg                neg   neg
  13008         pos                  pos                pos   pos
  12176         pos                  pos                pos   pos
   9963         pos                  pos                pos   neg
   1680         pos                  pos                pos   pos
   9104         pos                  pos                neg   pos
   1232         pos                  neg                neg   neg
                                                                                          18



      Staphylococcal Cassette Chromosome mec (SCCmec) Typing Using a
       Multiplex Bead-based Suspension Array (Jill Roberts).

Methicillin-resistant Staphylococcus aureus contains a large mobile genetic element
known as SCCmec which includes the mecA gene which is central to methicillin
resistance. The combination of mecA and a variety of other genes is known as the
SCCmec type and several types have been described. The type of SCCmec present in an
isolate is clinical relevant as it is related to treatment options. Traditional SCCmec typing
involves the amplification by multiplex PCR of 10 products, electrophoresis of these
products, and comparison to known controls. The disadvantage of this technique is
human error due to misinterpretation of the banding pattern and the requirement that at
least five controls are run on every gel. The purpose of this project is to eliminate the gel
electrophoresis step, the fingerprint pattern comparison step, and to increase the number
of samples that can be simultaneously assayed. SCCmec typing will require bead
coupling of 10 different PCR products to 10 different beads and assaying the resulting
products on the Bio-plex system. One of the 10 products, dcs, has been successfully
bound using the following four steps:

1. Coupling of Target-specific Oligonucleotide to the Surface of a Fluorescent
   Micro-sphere:

Preparation:
   1. Allow all reagents to warm to room temperature.

    2. Resuspend the amine-substituted oligonucleotide to 1.0 mM in sterile, deionized
       water.

Procedure:
   1. Disperse the pellet with sonication, and vortex the container for 20 seconds.

    2. Transfer 5.0 × 106 of the stock microspheres into a 1.5 mL microcentrifuge tube

    3. Microcentrifuge the xMAP microspheres at ≥ 8,000 × g for 1 to 2 minutes.

    4. Aspirate the supernatant, being careful not to disturb the pellet. Resuspend the
       microspheres in 50 μL of 0.1 M MES, pH 4.5. Vortex and sonicate.

    5. Prepare a 1:10 dilution of the 1 mM capture oligo in ddH2O. Add 2 μL (0.2
       nanomole) of the 1:10 diluted capture oligo to the resuspended microspheres.
       Vortex briefly

    6. Immediately before use, prepare a 10 mg/mL solution of EDC powder. Vortex
       until dissolved.
                                                                                        19


     7. Add 2.5 μL of the fresh EDC solution to the xMAP microspheres. Vortex
        immediately.

     8. Incubate for 30 minutes at room temperature in the dark.

     9. Repeat steps 6-8 with fresh EDC (for a total of two EDC additions).

     10. Add 1.0 mL of Tween 20 (0.02% v/v). Vortex.

     11. Microcentrifuge xMAP microspheres at ≥ 8,000 × g for 1 to 2 minutes.

     12. Aspirate the supernatant, being careful not to disturb the pellet.

     13. Add 1.0 mL of SDS (0.1% w/v). Vortex.

     14. Microcentrifuge the xMAP microspheres at ≥ 8,000 × g for 1 to 2 minutes.

     15. Remove the supernatant, being careful not to disturb the pellet.

     16. Resuspend the xMAP microspheres in 100 μL of TE Buffer (10mM Tris, 1mM
         EDTA, pH 8.0). Vortex and sonicate for about 20 seconds.

2.    Label Target to Allow Fluorescent Detection and Quantitation
     1. Biotinylate primers.
     2. Amplify the target product using the SCCmec multiplex PCR protocol:
        denature
        94 ºC 4 min
        amplify (30 cycles)
        94ºC 30 sec
        53ºC 30 sec
        72ºC 60 sec
        final extend
        72ºC 240 sec

3.      Hybridize Bead-coupled Probe to Biotinylated Product

     1. Select appropriate oligonucleotide-coupled microsphere sets.

     2. Resuspend microspheres by vortex and sonication for approximately 20 seconds.

     3. Prepare a Working Microsphere Mixture by diluting coupled microsphere stocks to
         150 microspheres of each set/μL in 1.5X TMAC Hybridization Solution. (Note:
         33 μL of Working Microsphere Mixture is required for each reaction.)

     4. Mix the Working Microsphere Mixture by vortex and sonication for approximately
         20 seconds.
                                                                                          20



   5. To each sample or background well, add 33 μL of Working Microsphere Mixture.

   6. To each background well, add 17 μL TE, pH 8.

   7. To each sample well, add biotinylated complementary oligonucleotide (5 to 200
       femtomoles) and TE, pH 8.0 to a total volume of 17 μL.

   8. Mix reaction wells gently by pipetting up and down several times.

   9. Cover the reaction plate to prevent evaporation and incubate at 95-100°C for 1 to 3
       minutes to denature any secondary structure in the sample oligonucleotides. *

   10. Incubate the reaction plate at hybridization temperature for 15 minutes. *

   11. Prepare fresh Reporter Mix by diluting streptavidin-R-phycoerythrin to 10μg/mL
       in 1X TMAC Hybridization Solution. (Note: 25 μL of Reporter Mix is required
       for each reaction.)

   12. Add 25 μL of Reporter Mix to each well and mix gently by pipetting up and down
       several times.

   13. Incubate the reaction plate at hybridization temperature for 5 minutes.

   14. Analyze 50 μL at hybridization temperature on the Luminex analyzer
       according to the system manual.
   * These steps can be combined with the use of a thermal cycler programmed as
   follows –
   Hold at 95°C, 1 (to 3) minutes
   Hold at hybridization temperature, FOREVER

4.      Assay Microspheres Using Flow Cytometry.
This step is performed by the Luminex system. Two signals are assayed: the unique
fluorescent signal that identifies the probe and the fluorescent signal that indicates that
hybridization has occurred. Each bead is labeled with a specific fluorescent dye
combination which uniquely identifies that bead allowing multiple targets to be assayed
in a single well. Relative fluorescence intensity of the hybridization signal is used to
quantitate the target.
Sample                  MFI        NTC         MFIPN
                                                                                     21


 1 µL – 280 fm         3325.5    221.5        15.01
 1 µL – 28 fm          1100.5    221.5        4.97
 1 µL – 2.8 fm         230.25    221.5        1.04
 17 µL – 280 fm        7717.8    221.5        34.85
 17 µL – 28 fm         3606.5    221.5        16.28
 17 µL – 2.8 fm        981       221.5        4.43
 1 µL – 200 fm         2107      221.5        9.51
 1 µL – 20 fm          755.5     221.5        3.41
 1 µL – 2.0 fm         335.5     221.5        1.51
 17 µL – 200 fm        4811.5    221.5        21.72
 17 µL – 20 fm         2209.5    221.5        9.98
 17 µL – 2.0 fm        842       221.5        3.80

Table 6:      Mean Fluorescence Intensity (MFI) of dcs coupled beads.

These data demonstrate that fluorescence as high as 34 times background were achieved
for the dcs probe indicating that labeling, coupling, and hybridization were successful
(Table 6). The next six products have now been biotinylated (Figure 1) and bead
coupling of the probe is in progress. In addition, a control strain for SCCmec type V
strains has now been acquired from NARSA (Network on Antimicrobial Resistance in
Staphylococcus aureus). Preliminary work has included purchasing primers for SCCmec
PCR and testing on this control strain (Figure 2).
                                                           22




Figure 1:   Biotinylated PCR products for SCCmec typing.
                                                                                        23




Figure 2:      PCR of SCCmecV control strain (CBD1375)

Future work will include biotinylation of the remaining targets, designing primer and
probe sets for the newly acquired type V strain, and testing of combinations of bead sets
as follows:
    a. CIF2 + dcs + mecA = SCCmec type I
    b. dcs + ccrB2 + kdp + mecI + mecA = SCCmec type II
    c. RIF5 + SCCmecIII + mecI + mecA = SCCmec type III
    d. dcs + ccrB2 + mecA = SCCmec type IVa
    e. ccrC + SCCmecV + mecA = SCCmec type V
    f. dsc + mecA = SCCmec type VI



      Molecular Characterization of USA300 and USA400 Methicillin-Sensitive
       Staphylococcus aureus (Jill Roberts)


Few studies have reported the contribution of methicillin-sensitive S. aureus (MSSA)
USA300 to the ongoing S. aureus pandemic, despite MSSA rates in the community
increasing at an alarming rate. USA300 MSSA are thought to differ from the epidemic
MRSA due to mutation or deletion of mobile genetic elements (SCCmec) but these
hypotheses remain untested. Studies in our laboratory have infrequently identified
isolates which are identical in pulsotype to epidemic MRSA but are in fact MSSA. The
purpose of this study was first to describe these isolates using PFGE and spa typing,
second to determine why these isolates are methicillin-sensitive using three methods of
SCCmec characterization, and finally to compare the pathogenic potential of these
isolates using PCR detection and sequence confirmation of virulence genes (29 loci).

Isolates for this study were selected based on pulsotypes similar to USA300 and USA400
as determined previously by PFGE. PFGE was performed as follows:

Protocol – grow cells overnight in TSA broth at 37C with gentle shaking. Pellet cells in
centrifuge for 10 minutes at 4100 rpm. Resuspend pellet in PIV solution (Tris, NaCl).
                                                                                      24


Prepare plugs by mixing 300 l of cells with 300 l of incert agarose. Pipet mixture into
plug molds and allow to cool for 15 minutes at 4C. Two large size plugs are prepared for
each S. aureus isolate. Plugs are then placed in 15 ml conical tubes containing 8 ml of
EC-lysis solution (Tris, NaCl, detergents, lysozyme, lysostaphin) and incubated for five
hours at 37C with gentle shaking. After five hours, the EC-lysis solution is decanted
and replaced with ESP solution (EDTA, detergent, proteinase K) and incubated overnight
at 50C with gentle shaking. The plugs are then washed three times (30-45 minutes for
each wash) with TE (Tris, EDTA) and stored in fresh TE at 4C until needed.

Plugs were digested overnight using SmaI enzyme at room temperature. PFGE was run
for 20 hours at 200 volts, in 0.5x TBE using switch times from 5.2 to 34.9s. Isolates
were visualized using the GelDoc system and analyzed using BioNumerics (Figure 3).




Figure 3:     Dendrogram of USA300 and USA400 MSSA showing variation in the
              patterns which may be related to the compliment of SCCmec
              components.

PFGE was then performed again on these isolates to compare directly to the USA300 and
USA400 controls (Figure 4).
                                                                                            25




Figure 4:      PFGE of USA300 and USA400 MSSA. Isolates are run on same gel
               for direct comparison to the controls strains.

This gel with higher resolution seems to indicate that CBD0860 and CBD1124 are
contaminated due to the presence of ghost bands (i.e. DNA at lower concentrations
causing additional bands). To test this hypothesis both isolates were reconstituted from
the culture collection and streaked for isolation. The isolation streak on both indicated
the presence of both a large and small colony. Small colony variants (SCVs) are
common with S. aureus and do not necessarily indicate contamination. Four colonies
from each plate, two large and two small, were isolated by streaking to fresh media and
PFGE was performed on these eight colonies (Figure 5).




Figure 5:      PFGE of CBD0860 and CBD1124 for detection of ghost bands.
                                                                                       26



The results of this gel demonstrate that CBD1124 has distinct ghost bands and is likely a
mixed culture. Further confirmation was attempted using spa PCR on the eight colonies
to determine if the products are the same size. Mixtures should in most cases result in
spa products of different sizes. The spa PCR was performed using the conditions listed
below for SCCmec typing (Figure 6).




Figure 6:      spa PCR of CBD0860 and CBD1124.

The results of this gel do not indicate any contamination in either CBD0860 or
CBD1124.

SCCmec typing was next performed on all of the isolates using the protocol as described
by Milheirico et al. (AAC 2007 Vol. 51(9);3374-7). In that protocol the PCR is
performed as a multiplex using the following 10 sets of primers:
    1. CIF – Type I, J1 region
    2. ccrC – Type V, ccr complex
    3. RIF5 – Type III, J3 region
    4. SCCmec V JI – Type V, J1 region
    5. Dcs – Types I, II, IV, and VI, J3 region
    6. ccrB2 – Types II and IV, ccr complex
    7. kdp – Type II, J1 region
    8. SCCmec III J1 – Type III, J1 region
    9. mecI – Types II and III, mec complex
    10. mecA – Internal positive control

In our study PCR was again performed using individual sets of primers if anything was
detected in the multiplex in an effort to detect and define any components of the SCCmec
complex in the methicillin-resistant isolates. PCR was performed using the same
conditions in the multiplex and in the individual reactions:
        denature
        94 ºC 4 min
        amplify (30 cycles)
                                                                                       27


       94ºC 30 sec
       53ºC 30 sec
       72ºC 60 sec
       final extend
       72ºC 240 sec

The initial PCR identified no SCCmec fragments in CBD860, CBD1124, CBD1228 or
CBD1275. Fragments were present in CBD0476, CBD0535, and CBD0796. PCR for
individual segments of SCCmec was then performed on all of the isolates. PCR for
individual fragments required a large number of controls to confirm fragments observed
were of expected size. For example, amplification products were seen in CBD0796 for
mecA, dcs, and mecI (Figure 7) but the mecI product was not the correct size.




Figure 7:     Individual SCCmec PCR for CBD0796 illustrating the large number
              of controls required for SCCmec typing. Lanes 2-6 are USA100
              control PCR reactions and lanes 7,8, and 10 are USA300 control PCR
              reactions. Lanes 11-15 are PCR results for CBD0796 indicated that
              this MSSA is positive for the dcs segment.

PCR was performed for all 10 possible SCCmec products and representative results for
some of strains used in this study are shown below (figures 8-9).
                                                                                       28




Figure 8:      Individual SCCmec PCR for CIF, ccrC, RIF, and mecV in CBD0479,
               CBD1124, and CBD1275. Note: only one positive control CBD0802 is
               shown as the type V control strain was not available. The control
               strain was acquired 11/5/09 and these results were confirmed using
               the controls (not shown).




Figure 9:      Individual SCCmec PCR for dcs, ccrB2, kdp, and mecIII (left) and
               mecI and mecA (right) for CBD0479, CBD1124, and CBD1275.
               Positive controls for the PCR are indicated by (+). Only CBD0479 was
               positive for any of the fragments tested.

Detection of SCCmec fragments was then attempted using another method which can
detect different portions of SCCmec as described by Boye et al. (Clin. Micro. and Infect.
Vol.13(7); 725-46. This method uses 4 primer sets:
    1. α/β – Types II and IV, ccrA2-B
    2. ccrC3 – Types III and V, ccrC
    3. 1272 – Types I and IV, IS1272
    4. 5RmecA – Type V, mecA-IS431

PCR was performed as follows:
     denature
                                                                                         29


       94 ºC 4 min
       amplify (30 cycles)
       94ºC 30 sec
       55ºC 30 sec
       72ºC 60 sec
       final extend
       72ºC 4 min


PCR was performed for the 4 possible SCCmec fragments using this protocol and
representative results are shown (Figure 10). The control strain for 5Rmec was not yet
available when this experiment was performed but was later confirmed (not shown).




Figure 10:    SCCmec typing using an alternative protocol and isolates CBD0476
              and CBD0535. Only the control isolates were positive for SCCmec
              segments using this method for these two isolates.

Finally detection of SCCmec elements was attempted using a third protocol specifically
designed to detect type IV isolates as all isolates in this study have USA300 and 400
pulsotypes and should contain type IV if any SCCmec elements. This protocol described
by Milheirico et al. (JAC 2007 Vol. 60; 42-8) involves PCR for the following 7
fragments:
    1. ccrB2 – ccrB2 internal positive control
    2. J IVa – Type IVa
    3. J IVb – Type IVb and IVF
    4. J IVc – Type IVc and IVE
    5. J IVd – Type IVd
    6. J IVg – Type IVg
    7. J IVh – Type IVh

PCR was performed as follows:
                                                                                        30


       denature
       94 ºC 4 min
       amplify (35 cycles)
       94ºC 30 sec
       48ºC 30 sec
       72ºC 2 min
       final extend
       72ºC 4 min

PCR using this technique was not really reliable as positive controls were only available
for type IVa and type IVb strains (Figure 11) therefore results would not have been
reported for other types. However, type IVa was the only product detected in the strains
of interest.




Figure 11:     SCCmec type IV subtyping for CBD0860 and CBD1228. These data
               demonstrate the positive controls for type IVa and type IVb and
               multiple products for type IVc and IVg.

All SCCmec fragments detected using the three techniques were purified using the
Promega Wizard PCR DNA Purification kit according to manufacturer’s protocol and
sequenced as follows:

Preparation of the DNA sequencing reaction:
DH20                        Volume to 20L
DNA Template                5L
                                                                                          31


Primer (3.2pmol)              3L
DTCS                          8L

Thermal Cycling Program:
96 C                         20 sec
50 C                         20 sec
60 C                         4 min
run for 30 cycles.

96-Well Ethanol Precipitation:
   1. Add 4L of stop solution (equal volumes of 1.5M NaOAc pH5.2 and 50mM
       EDTA pH 8.0) and add 1L of glycogen (20mg/ml) to a labeled, sterile 0.5 mL
       microfuge tube.
   2. Transfer the sequencing reaction to the appropriately labeled tube and mix.
   3. Add 60L of 95% ethanol (cold) and mix.
   4. Centrifuge at 14000 RPM at 4C for 15 min.
   5. Carefully remove the supernatant with a micropipette.
   6. Rinse pellet with 200L of 70% ethanol (cold).
   7. Centrifuge at 14,000 RPM at 4C for 10 min.
   8. Carefully remove the supernatant with a micropipette.
   9. Repeat steps 6 to 8.
   10. Dry in concentrator for 20 minutes.
   11. Resuspend in 40L of sample loading solution.
   12. Transfer samples to 96-well sequencing plate.
   13. Add 1 drop of mineral oil to each well.
   14. Load sequencer and run appropriate program.

The resulting sequences were aligned using the Seqman program of the DNASTAR
analysis software package. Identities were confirmed by BLAST and are reported below.
A summary of all SCCmec fragments detected using the three protocols for the isolates of
interest is as follows:
    1. CBD0476 (USA400 pulsotype) – dcs (+)
    2. CBD0535 (USA400 pulsotype) – dcs (+)
    3. CBD0860 (USA400 pulsotype) – no mec elements
    4. CBD1124 (USA400 pulsotype) – no mec elements
    5. CBD0796 (USA300 pulsotype) – dcs, IVa, 5Rmec, 1272 (+)
    6. CBD1228 (USA300 pulsotype) – no mec elements
    7. CBD1275 (USA300 pulsotype) – no mec elements

The next step in this project was to determine the presence of virulence factors in the
seven isolates of interest including exotoxins, adhesions, and mobile genetic elements.
PCR was performed for each of the exotoxins as follows:
        denature
        94 ºC 4 min
        amplify (30 cycles)
                                                                                     32


        94ºC 30 sec
        50ºC 30 sec
        72ºC 2 min
        72ºC 4 min
Isolates were screened for a total of 16 exotoxins (Figure 12).




Figure 12:     Exotoxin screen. Control isolates listed as (+) indicate exotoxin tested
               on each gel.
                                                                                  33




Figure 12 (continued). Exotoxin screen. Control isolates listed as (+) indicate
exotoxin tested on each gel.
                                                                                        34




Figure 12 (continued). PCR for S. aureus exotoxins. Seven USA300 and USA400
MSSA were screened for the presence of 16 exotoxins. The controls strains indicate
the exotoxin tested.

These results demonstrate the presence of 13 exotoxins in the strains of interest (Table 1)
and demonstrate that each isolate has at least one exotoxin. The identification of each
exotoxin was confirmed by sequencing and BLAST analysis using the methods described
above. Significantly, two USA400 pulsotype isolates contained the same exotoxin
complement (CBD0476 and CBD0535) (Table 7).




Toxin      Control    CBD476 CBD535 CBD796 CBD860 CBD1124 CBD1228 CBD1275
sea        CBD1128    Positive   Positive   Negative   Negative   Negative   Negative   Negative
seb        CBD0007    Negative   Negative   Negative   Negative   Positive   Negative   Negative
sec        CBD1130    Positive   Positive   Negative   Negative   Negative   Negative   Negative
sed        CBD0471    Negative   Negative   Negative   Negative   Negative   Negative   Negative
see        CBD1128    Negative   Negative   Negative   Negative   Negative   Negative   Negative
seg        CBD1130    Negative   Negative   Negative   Positive   Positive   Negative   Negative
seh        CBD0798    Positive   Positive   Negative   Negative   Negative   Negative   Negative
sei        CBD0019    Negative   Negative   Negative   Positive   Positive   Negative   Negative
sej        CBD1064    Negative   Negative   Negative   Negative   Negative   Negative   Negative
sek        CBD799     Positive   Positive   Positive   Negative   Negative   Positive   Negative
sel        CBD1067    Positive   Positive   Negative   Negative   Negative   Negative   Negative
sem        CBD1064    Negative   Negative   Negative   Positive   Positive   Negative   Negative
sen        CBD1064    Negative   Negative   Negative   Positive   Positive   Negative   Negative
seo        CBD1064    Negative   Negative   Negative   Positive   Positive   Negative   Negative
sep        CBD0797    Negative   Negative   Negative   Negative   Negative   Negative   Positive
seq        CBD0799    Positive   Positive   Positive   Negative   Negative   Positive   Negative
                                                                                       35


tst        CBD1335   Negative   Negative   Negative   Negative   Negative   Negative   Negative
acme       CBD1066   Negative   Negative   Positive   Negative   Negative   Negative   Negative
pvl        CBD1066   Negative   Negative   Positive   Negative   Negative   Positive   Negative
opp-3      CBD1066   Negative   Negative   Positive   Negative   Negative   Negative   Negative
mecA       CBD1066   Negative   Negative   Negative   Negative   Negative   Negative   Negative
bsa        CBD1067   Positive   Positive   Positive   Negative   Negative   Positive   Positive
lukE       CBD1067   Positive   Positive   Positive   Positive   Positive   Positive   Positive
hla        CBD1067   Positive   Positive   Positive   Positive   Positive   Positive   Positive
hlb        CBD1067   Negative   Negative   Positive   Negative   Positive   Positive   Positive
hlg        CBD0835   Negative   Negative   Negative   Negative   Negative   Negative   Negative
eta        CBD1135   Negative   Negative   Negative   Negative   Negative   Negative   Negative
etb        CBD1135   Negative   Negative   Negative   Negative   Negative   Negative   Negative
psm        CBD1066   Positive   Positive   Positive   Positive   Positive   Positive   Positive
Table 7:      Summary of PCR screening for S. aureus virulence factors in
              USA300/400 MSSA.

PCR was then performed for virulence factors commonly associated with USA300 and
USA400 including Panton-Valentine Leukocidin (PVL), oligopeptide permease (opp-3),
the arginine catabolic mobile element (ACME) and mecA (Figure 13).




Figure 13:    PCR for USA300 and USA400 virulence factors in USA300/400
              MSSA. The controls strains indicate the exotoxin tested.
                                                                                        36



These results demonstrate the presence of classical community-associated S. aureus
virulence factors in some of the isolates but also demonstrate that none of the MSSA
have the mecA gene (Table 1). This is in contrast to hypotheses suggesting these isolates
possess point mutations in the mecA gene which results in their MSSA status. All CA
virulence factors were confirmed by sequencing and BLAST analysis (not shown).

PCR was then performed to screen the isolates for additional known virulence factors
including toxic shock toxin (tst), bacteriocin (bsa), phage encoded toxin (lukE),
hemolysins (hla, hlb, hlg), exfoliative toxins (eta, etb), and phenol soluble modulin (psm)
(Figure 14). These results were also confirmed by sequencing (not shown).
                                                                                       37




Figure 14:     PCR for S. aureus virulence factors in USA300 and USA400 MSSA.
               The controls strains indicate the exotoxin tested.

PCR confirmed the presence of psm, lukE, and hla in all of the isolates tested (Table 7).
The USA300 and USA400 MSSA isolates were then characterized using spa Typing.
Staphylococcal protein A (spa) gene typing has been reported as an alternative technique
for typing S. aureus that results in data that is easily shared between laboratories. The
USA300 and USA400 type strains have been shown to possess spa type 8 and the
purpose of this experiment was to determine if the MSSA are also spa type 8.

Genomic DNA from the seven isolates was isolated as follows:
Bacterial isolates from freshly sub-cultured plates were grown in 4 ml of Trypticase Soy
Broth for 16 hours at 37°C. One ml of the TSB culture was centrifuged at 8000 X g for
10 minutes and 900 µl of the supernatant was discarded. The pellet was resuspended in
130 µl of bacterial lysis buffer followed by the addition of 20 µl of Proteinase K. Cells
were incubated at 65 °C for 10 minutes and at 95 °C for 10 minutes prior to automated
extraction of genomic DNA. The genomic DNA was diluted 1:20 prior to PCR
amplification.

The spa gene was amplified by PCR as follows:
95 C                        10 min
95 C                        30 sec
60 C                        30 sec
72 C                        45 sec*
*repeat above for 30 cycles.

72 C                         10 min
4 C                          hold
                                                                                          38


The spa PCR was first performed with the positive control USA300 DNA and four
different spa primer sets to determine the optimum conditions for this experiment (Figure
15).




Figure 15:     Test of published spa primer sets. spa*FR resulted in a single
               amplified product.

As shown in Figure 15, spa*FR primer set produced a reliable product and was used to
amplify spa from the seven strains of interest (Figure 16). The sizes of the resulting
products indicate that the isolates differ in the number of spa repeats and will therefore be
classified separately.




Figure 16:     spa PCR of USA300 and USA400 MSSA isolates.

The PCR products were purified using the Wizard prep kit, precipitated, and sequenced
as described above for the SCCmec typing. The resulting sequences were aligned using
the Seqman program of the DNASTAR analysis software package. The resulting repeats
were then compared to those listed in an online database
(http://www.ridom.de/spaserver/) resulting in a pattern of repeats as shown in Figure 17.
                                                                                      39




Figure 17:    Repeat pattern and spa type assignment for CBD1275.

As expected based on the size of the spa fragment (Figure 16), CBD1275 contains a
small number of repeats. Results for all seven isolates are given in Table 8.

  Strain               Repeats               spa Type
     476   R7-23-21-16-34-33-13                    127
     535   R7-23-21-16-34-33-13                    127
     796   R11-19-12-21-17-34-24-34-22-25             8
     860   R7-23-21-16-34-33-13                    127
    1124   R4-12-17                                287
    1228   R11-19-12-21-17-34-24-34-22-25             8
    1275   R11-10-34-22-25                         104
Table 8:      spa typing repeat and ST assignments.

The purpose of this study was first to describe these isolates using PFGE and spa typing,
second to determine why these isolates are methicillin-sensitive using three methods of
SCCmec characterization, and finally to compare the pathogenic potential of these
isolates using PCR detection and sequence confirmation of virulence genes (29 loci).
PFGE confirmed the identification of both USA300 and 400 among the few MSSA
isolates available to our laboratory. Three of the USA400 MSSA isolates; CBD476, 535,
and 860 were identical to the control strain pulsotype, despite the lack of nearly all
detectable SCCmec elements. A fourth USA400 isolate (CBD1124) differed from the
control strain by a single band, possessed a short spa sequence, and no SCCmec
elements. Virulence gene complement was identical in both CBD476 and 535 and highly
consistent with known USA400 data. However, despite different pulsotypes and spa
types, CBD860 and 1124 also contained nearly identical virulence genes. CBD796 was
consistent with USA300 in all of the tests performed except it lacks the mecA gene.
CBD1228 and 1275 have identical pulsotypes, one band variation from the USA300
control, and no SCCmec components, but they possess different spa types and different
virulence genes. These data demonstrate that despite proposed hypotheses the lack of
SCCmec will not necessarily change the pulsotype, point mutations in mecA did not
                                                                                          40


contribute to MSSA status, all epidemic MSSA are not identical, and USA400 MSSA
also occurs in the United States. Furthermore, the presence of key virulence factors in
these MSSA demonstrates the pathogenic potential of these isolates in the ongoing S.
aureus pandemic.


      Characterization of Methicillin-Resistant Staphylococcus aureus Epidemic
       Clone USA100 Isolates Identified Among Strains Collected for Community-
       associated Staphylococcus Studies (Jill Roberts).


Staphylococcus aureus is a well-known cause of both community- and hospital-
associated infections. Community-associated MRSA (CA-MRSA) differ from hospital-
associated MRSA (HA-MRSA) in their antibiotic resistance profiles, virulence
determinants, and ability to cause disease in patients without risk factors. While CA-
MRSA is well researched, few studies have characterized HA-MRSA within the
community environment. Our Center described 100 USA100 isolates collected as part of
a community S. aureus study by the type of disease present, molecular typing using both
pulsed-field gel electrophoresis (PFGE) and SCCmec typing, and determined the
frequency of significant virulence genes in these isolates. Work on this project is
continuing with analysis using spa typing and comparison of this data with that obtained
using other typing methods. Epidemiologically, the most common source of USA100
isolates was nasal swabs (47%), followed by wound (27%) and blood (13%) samples.
Previously we reported the PFGE, PCR, and SCCmec typing data for these isolates and
that data is not shown here. Recently, due to the optimization of spa primers reported
above for the USA300/400 MSSA project, we have resumed spa typing of USA100
isolates using this new primer set. The PCR for spa typing was performed as listed
above. Isolates which previously yielded poor PCR results were better resolved with this
new primer set as shown in Figure 18. PCR amplicons were then purified using the
Promega Wizard prep kit and sequenced as described above. Sequence alignment and
spa assignment for these isolates is ongoing.
                                                                              41




Figure 18:   spa PCR of USA100 isolates. Isolates with * were not processed
             further.
                                                                                          42


      Broad Range PCR for Detection of BT Agents (Jenny Gulledge)

Broad range PCR is a technique that can be used to detect any bacteria and possibly new
bacteria that have avoided cultivation from a normally sterile clinical sample. It is also a
powerful technique to assess bacterial diversity in a complex non-sterile clinical or
environmental sample. Basically, broad range PCR utilizes primers to conserved regions
of the bacterial 16S rRNA gene to amplify the gene as two products of about 700 bp from
virtually any eubacterium. The amplicons are then cloned and sequenced and compared
to extensive 16S rRNA sequences in the microbial databases. Alternatively, if only one
bacterial 16S rRNA gene is present and the amplicon is pure, it can be sequenced
directly. We intend to explore the potential of this technique as a preliminary screening
tool for determining the presence/absence of pathogens in a sample. This technique will
prove useful for the detection of non culturable pathogens in the complex clinical and
environmental samples, which can often prove difficult to work with.


Methods & Materials
Bacillus strains used for 16S sequencing last quarter were: CBD 63 Bacillus anthracis
Pasteur from the CBD Culture Collection and BB005 B. anthracis Davis and BB0006 B.
anthracis Kruger from the BSL-3 Collection. Since then three other B. anthracis strains:
BB0001 Sterne, BB0004 Ames and BB0007 were sequenced. A Bacillus pumilus (CBD
400) was also sequenced along with DNA extracted from 22 soils received from U.S.
Geological Survey.

B. anthracis DNA was isolated by growing up the cultures overnight at 35°C and using
the Spore-Free protocol developed by Debbie King. The DNA was extracted using the
MagNaPure Compact (Roche Diagnostics). A total of 10ul out of 100ul from each DNA
extraction is plated on a TSA plate and incubated for 7 days at 35°C. If there are no
colonies after 7 days the DNA samples can be taken out of the BSL-3 and used in the
detection lab for sequencing.

The soil DNA was extracted with either the Epicentre SoilMaster™ DNA Extraction Kit
(SM02050) or MO-BIO PowerSoil® DNA Extraction Kit (12888-50). Prior to extracting
the DNA from the soil, the soil was processed using PLET (polymyxin lysozyme
disodium ethylenediaminetetraacetate (EDTA) with thallium acetate (Knisely 1966). The
PLET mixture was sonicated for 30 minutes. The mixture was then vigorously vortexed
and heated at 65°C for 30 minutes and after it cools to room temperature has
polymyxinB, lysozyme, sulfamethoxazole and trimethoprim added. The mixture was
placed into a 30°C shaking water bath overnight for 24hours at 125 RPM. The next day,
the mixture was vortexed hard and then sat for 10 minutes for large soil particles to settle.
PLET was poured off and centrifuged at high speed for 1 hour to pellet as many of the
cells as possible. The pellet is then reconstituted in water and centrifuged again for 1 hour
and then processed with the DNA extraction kits.
                                                                                      43



Primers for the 16S gene were designed with a Bacillus thuringiensis sequence from
GenBank (AY461761.2). The Bacillus species have a high level of similarity (>99%).The
Pyrosequencing Assay Design Software was used for primer design since 16S forward
primer for 16S was already designed (Table 9). PCR assays of 30 L [25mM MgCl2;
10X buffer; 25uM dNTP; 1.0 uM primers; 5U/ul Taq DNA polymerase (Takara,
Madison, WI)] were carried out using 3.0 ng of extracted DNA as template in a T1
Thermocycler (Biometra, Horsham, PA). For the 16S assay the following parameters
were followed: initial heating for 2 minutes at 94C, followed by 40 cycles of 20 seconds
at 94C, 20 seconds at 58C, and 3 minutes at 72C, with a final extension at 72C for 5
minutes. A total of 5ul out of the 30ul amplified DNA samples were electrophoresed on a
1 % agarose gel (0.5X TBE with 0.05 g/mL ethidium bromide) for 50 minutes at 80v.
A 1200-bp fragment was viewed for the three Bacillus samples.

Table 9: 16S Primers
forward   Biotin-5’-ACG ATG CGT AGC CGA CCT – 3
reverse   rev 5’-ACT TCA CCC CAA TCA TCT GTC C -3’



The amplified DNA was then cleaned with the Promega Wizard PCR Preps DNA
Purification System and eluted to 30ul with nanopure water. The DNA was prepared for
the CEQ 8000 Beckman Coulter sequencer by using the sequencing reaction as per
Beckman Coulter protocol:


              DNA Sequencing – PCR Cycle

              DTCS (Dye Terminator Cycle Sequencing)        8ul
              Primer (forward or reverse) (25 pmol total)   3ul
              DNA                                           6ul
              Water                                         3ul
                                                            20ul total


After the PCR cycle, three ethanol precipitations are performed before the samples are
loaded onto the CEQ 8000. The completed sequences are added to SeqMan Pro from
DNAStar Lasergene and assembled along with the GenBank sequence.

Results
The three Bacillus anthracis strains (BB0001, BB0004 and BB0007) were sequenced and
were nearly identical. Initially the soil PCR assays had 9 out of the 22 (41%) soils
positive for 16S primers (1365 base pairs). When the DNA was sequenced using 2, 4 or 8
ul of DNA, none of the sequences came up positive. Further purification of the soil DNA
may be needed to improve the sequencing. Further work will be done extracting DNA
from other BSL-3 B. anthracis strains and soil DNA.
                                                                                     44


      Pyrosequencing of Bacillus anthracis (Jenny Gulledge)

Pyrosequencing has the potential advantages of accuracy and rapid analyze of specific
genetic sequences with high reproducibility. The rapid analyzes will help in microbial
identification. The Pyrosequencer is a DNA sequencing technique that is based on the
detection of the release of pyrophosphate (PPi) during DNA synthesis. Designed primers
for Bacillus anthracis DNA will enhance the detection and distinguish between virulent
and avirulent species.


Methods, Assumptions and Procedures

The 908 capsule and gerXA primers for the detection of B. anthracis in soil samples were
tested. The encapsulation genes (908 capsule primers) are located in the pXO2 plasmid
(95 kb). Primers for the capsule gene were designed in close proximity to primers
currently used in pcr for the detection of B. anthracis Pasteur (CBD 63). The gerXA gene
is responsible for spore germination. It is located between the pagA and atxA genes on
the pXO1 plasmid.

The Pyrosequencing Assay Design Software was used for primer design 908 cap and
gerXA (Table 10). The selected primers made Pyrosequencing fragments that were less
than 200 bp. The PCR primers were normally 15-24 bp in length with approximately the
same GC-content as the fragment as a whole. The organisms used from the CBD Culture
Collection and BSL-3 Collection are listed on Table 11. DNA extracted from B.
anthracis strains Sterne and Pasteur and the soil were cultured and processed the same
way as the Bacillus for the broad range PCR sequencing project.

PCR protocol

20mM Tris-HCL pH9.0,50 mM KCl
2 mM MgCl2
0.2 mM of each dNTP
10 umoles of each PCR primer (formally 10 pmoles)
1 U Taq polymerase
20 ng bacterial DNA (formally 10ng)
MilliQ water to 50 ul

The typical PCR program for the16S gene fragments were : 95°C 5min, 35x(95°C 30s,
57°C 30s, 72°C 30s), 72°C 7min, 4°C hold. A 1.5% agarose gel was run on all samples
before being loaded onto the Pyrosequencer. PCR samples with single bands were used in
the PyroMark ID. The pcr samples were processed and loaded onto the Pyrosequencer
(Biotage PyroMark ID using Pyro Gold reagents) as follows:


    1. In a 96-well plate add each of the following to each well:
           a. 20 l PCR product
                                                                                           45


             b. 40 l binding buffer
             c. 17 l water
             d. 3 l strepavidin sepharose beads
   2.     Shake product plate for 10 min, 1400 rpm.
   3.     Add 40 l of 0.4M sequencing primer diluted in annealing buffer to each well.
   4.     Load both plates on the Vacuum Prep Tool and perform the following steps:
             a. Attach beads
             b. Rinse with 70% ethanol
             c. Denature with 0.2M sodium hydroxide
             d. Rinse with Wash Buffer
             e. Release beads in primer plate
   5.     Anneal primer for 2 mins at 80C.
   6.     Run PyroMark ID according to software.

Table 10:        Pyrosequencing primers

Source or       Primer                   Seq. (5'→3')                Length    G/C %       Plasmid
 Name                                                                 (bp)
                forward   GTC AGG GCG GCA ATT CAT AA                   20        50
908 Cap         Reverse   Biotio-CGG ATC CAG GAG CAA TGA GAA           21       52.3        pXO2
                seq
                Reverse   ACG TAA TGT TGA TGA GGG AT                   20        25

                forward   Biotin-TGA TGG TTC ACC GAC TGC A             19       52.6
gerXA           Reverse   AAT GCG TAG GCC TGC TTG TC                   20        55         pXO1
                seq
                Reverse   GGT AAT CAT AAT CAT CCC C                    19       42.1



Table 11:     Bacillus species used with Pyrosequencer
  CBD species         Strain Designation
 accn#
 BB001 anthracis Sterne
   63     anthracis Pasteur CDC BC3132




Results and Discussion
The pcr protocol for the capsule and gerXA gene yielded positive results for the Sterne
(BB001) and Pasteur CBD63 DNA positive controls and negative for all 27 soil samples.
This has been an ongoing problem when working with soil DNA. Soil DNA might be
purified with Promega Wizard PCR Preps DNA Purification System that is used now for
the CEQ Sequencer or a new kit from MO-BIO.

New primers and probes for Yersinia pestis plasmid pMT1 ( caf1 gene) and 16S have
been designed and will be used for upcoming Pyrosequencing projects.
                                                                                                               46


      Pulsed Field Gel Electrophoresis of S. Typhi with Spe-1 (Aparna Tatavarthy)

In the last reports, the PFGE profiling of the 17 S. Typhi strains with the enzyme Xba-1
and 11 S. Typhi strains with Spe-1 was reported. This quarter the profiling of the Typhi
isolates with Spe-1 has been completed. Thirteen pulsotypes at 40% or more similarity
were observed (Figure 19). The antibiotic profiling is also represented in the figure. Four
isolates CBD 1355, 1356, 1358, and 1359 showed high identity to each other (Figure 19).
Two strains CBD 1267 and CBD 1346 showed 100% identity to each other. Their
antibiotic resistance profiles also are identical. One of the strains CBD 1353 shows
limited similarity with the rest of the strains (<40%).




       Dice (Tol 1.0%-1.0%) (H>0.0% S>0.0%) [0.0%-100.0%]
       PFGE Spe-1                                                      PFGE Spe-1
                                                                 100
       40




                 50




                           60




                                      70




                                                80




                                                            90




                                                                                    CBD 1266
                                                                                    .          . Typhi
                                                                                               S.        S
                                                                                                         .
                                                                                    CBD 1350
                                                                                    .          S.
                                                                                               . Typhi   AmpChStrCot
                                                                                    .
                                                                                    CBD 1349   S.
                                                                                               . Typhi   AugAmpChStrCot
                                                                                                         .
                                                                                    CBD 1348
                                                                                    .          .
                                                                                               S.Typhi   Nal
                                                                                                         .
                                                                                    CBD 1351
                                                                                    .          S.
                                                                                               . Typhi   .
                                                                                                         S
                                                                                    CBD 1352
                                                                                    .          S.Typhi
                                                                                               .         Nal
                                                                                                         .
                                                                                    .
                                                                                    CBD 1265   S.
                                                                                               . Typhi   S
                                                                                                         .
                                                                                    CBD 1355
                                                                                    .          .
                                                                                               S.Typhi   S
                                                                                                         .
                                                                                    CBD 1356
                                                                                    .          S.Typhi
                                                                                               .         S
                                                                                                         .
                                                                                    .
                                                                                    CBD 1358   S.Typhi
                                                                                               .         S
                                                                                                         .
                                                                                    CBD 1359
                                                                                    .          .
                                                                                               S.Typhi   S
                                                                                                         .
                                                                                    CBD 1357
                                                                                    .          S.Typhi
                                                                                               .         .
                                                                                                         S
                                                                                    CBD 1354
                                                                                    .          S.Typhi
                                                                                               .         S
                                                                                                         .
                                                                                    .
                                                                                    CBD 1267   S.
                                                                                               . Typhi   AmpChNalStrCot
                                                                                                         .
                                                                                    CBD 1346
                                                                                    .          . Typhi
                                                                                               S.        AmpChNalStrCot
                                                                                                         .
                                                                                    CBD 1347
                                                                                    .          S.
                                                                                               . Typhi   .
                                                                                                         ChStrCot
                                                                                    CBD 1353
                                                                                    .          S.Typhi
                                                                                               .         S
                                                                                                         .



Figure 19:            The isolates were digested by Spe-1 enzyme and the strains were
                      analyzed in BioNumerics using Dice coefficient. The phylogenetic
                      relationship between isolates was studied by dendrograms constructed
                      with UPGMA (Unweighted Pair GroupMethod with Arithmetic averages),
                      with 1% position tolerance. Resistance to various drugs tested is also
                      shown. amoxicillin/clavulanic Acid (Aug), ampicillin (Amp),
                      chloramphenicol (Ch), nalidixic acid (Nal), streptomycin (Str),
                      tetracycline (Tet), trimethoprim/sulfamethaxazole (Cot)
                                                                                         47



      Isolation of E. coli O157:H7 from artificially contaminated lettuce (Aparna
       Tatavarthy and William Veguilla)

In the last quarter, we reported that diluting the bead-bacteria complex and plating the
dilutions on Rainbow agar resulted in good recovery of E. coli O157:H7 from the high
microbial load lettuce. In this quarter we completed the “detection and isolation of E. coli
from lettuce” project and initiated the IMS work on Shigella sonnei.

Four samples of ready to eat salad containing iceberg lettuce were artificially
contaminated with E. coli O157:H7 in sterile filter bags (3 CFU/25g, Table 12). An
unspiked negative control was set up in parallel with each set of experiments. 225 ml of
buffered peptone water (BPW) was added to each spiked/unspiked 25 g sample. The
samples were gently massaged for 1 min before homogenization in a stomacher. The
filtrate was then aseptically transferred into sterile 500 ml flasks. The samples were
incubated for 6 hr at 35oC. Pre-enriched samples were selective enriched by IMS. Twenty
l of anti-E. coli O157:H7 antibody-coated magnetic beads were added to 1 ml of each of
the spiked and unspiked pre-enriched sample. After an adsorption period, the beads and
bound E. coli were alternately pelleted over a magnet then re-suspended in wash buffer to
remove non-E. coli O157:H7. Bead-bound E. coli cells were plated on the selective and
differential medium, Rainbow agar. The 6h enriched sample as well as the bacteria-bead
suspension after IMS was further diluted before the final plating onto Rainbow agar to
eliminate the background flora (see below for the detailed method).

       Plating after BPW enrichment for high microbial load foods

       i) 10 -4 dilution – Transfer 1 ml of 6h pre-enriched broth into 99 ml of BPBW
            and mix well by shaking thoroughly. Plate 100 l of this onto SMac/Rainbow
            agar plate with a lazy L- spreader
       ii) 0.5x10 -4 dilution – Plate 50 l of the 10 -4 dilution onto SMac/Rainbow agar
            plate with a lazy L- spreader
       iii) 0.25x10 -4 dilution - Plate 25 l of the 10 -4 dilution onto SMac/Rainbow agar
            plate with a lazy L- spreader

       Selective concentration by IMS (high microbial load foods)

               1) After 6 h incubation at 35 oC, move the flasks into the BSC.
               2) Place one ml of each pre-enriched BPW sample into a sterile, 2-ml
                  microcentrifuge tube
               3) Add 20 l of anti- Salmonella Dynabeads (vortex before use) to each
                  sample, followed with brief vortex mixing.
               4) Place safety cap covers on each sample tube prior to loading onto the
                  Dynal sample mixer for 30 min at 23 RPM.
               5) Place the tubes on the Dynal-magnet for 3-5 min with gentle rocking
                  of the unit to concentrate the beads along the back of the tube adjacent
                  to the magnet.
                                                                                                   48


               6) Tip the magnet rack forward and remove the liquid from each tube
                   with a 1 mL pipettor without disturbing the bead pellet.
               7) Wash the beads by adding 400 l of sterile PBST (0.05%Tween),
                   followed by gently inverting the tube to resuspend the beads in the
                   wash fluid.
               8) Repeat steps 4 thru 6 thrice for a total for 4 washes.
               9) Resuspend the beads for each sample in 1000 l of sterile PBS.
               10) Plate the resuspended bead suspensions for each sample in 100, 50, 25,
                   12.5 & 5 l volumes to SMac/Rainbow agar plates; 12.5 & 5 l
                   volumes/sample to TSA plates in order to monitor the total of
                   background flora being carried over with the IMS beads.
               11) Note: Add 100 l sterile BPW to any plate not receiving 100 l
                   volume to promote spreading.
               12) Incubate plates for 22-24 h @ 35 oC.

The plates were incubated at 35oC for approximately 18 hr and were counted for the
number of E. coli O157:H7 cells. The background of the lettuce was estimated by aerobic
plate counts (APC) following the BAM protocol. DNA was extracted using ABI
PrepMan reagent from the 6h and the 22h enriched BPW. This DNA was used for real-
time PCR targeting the stx-2 gene. If probable E. coli O157:H7 colonies were achieved
from the IMS method, they were subjected to colony PCR and/or the latex agglutination
test (RIM). For the colony PCR, one to several isolated/ mixed probable E. coli O157:H7
were suspended in molecular biology grade water and boiled for 10 min at 95oC. The
boiled prep was used as a template for the real-time PCR. For the latex agglutination test,
apparent E. coli colonies were re-isolated on blood agar plates and incubated for 12-18h.
The colonies were subjected to RIM latex test by following the manufacturer’s
instructions (RIM™ E. coli O157:H7 Latex Test).

Grayish colonies resembling E. coli O157:H7 were achieved from all the spiked samples
(VS16, VS17, VS18, and VS19) after IMS. Isolated colonies were observed in two out of
the four spiked samples after the six h BPW enrichment even before the IMS (Table 12).
For all the spiked samples VS 16, 17, 18, and 19 both the colony PCR (stx-2 gene) and
the RIM test were also positive (Table 13).

Sample         Spike level   No. of CFU           No. of CFU- Post IMS (dilutions)          Background        RIM
               (CFU/25g)      BPW: Pre-                                                     flora (APC)       Test
                                    IMS
                                          100l         50l     25l   12.5l    5l
VS 16         3.3            0            3             2         1       1          1      8.4x105       +
VS17                         4            10            4         1       1          1                    +
VS18                         0            1             0         0       0          0                    +
VS-19                        5            4             3         3       5          0                    +
VS-                          0            0             0         0       0          0                    -
Unspiked
                                                                                          49


Table 12:      Number of colonies isolated from artificially contaminated lettuce on lab day
               2 using the short pre-enrichment and immunomagnetic separation. The
               results show the colonies from pre-IMS and post-IMS dilutions on rainbow agar.
               APC- aerobic plate count, VS- veggie salad (lettuce)

Table 13 shows the real time results of all the lettuce/veggie salad experiments conducted
to date. The mean Ct values of duplicate reactions are represented in the table. The
template DNA was extracted after 6h and 22h of enrichment/incubation. Out of the 19
samples tested only three were positive for stx-2 gene after 6h enrichment (~15%). On
the contrary, 17/19 samples (89%) were positive for stx-2 gene after 22h of enrichment.
Clearly, with a low spike (<5 CFU/25g) 6h enrichment is not sufficient to generate a
positive PCR reaction for E. coli O157:H7. The colony real-time PCR was positive for E.
coli O157:H7 in 10/13 spiked samples tested. RIM test was positive in 11/13 spiked
sample tested. In some of the unspiked and spiked samples there were no colonies
resembling E. coli O157:H7, therefore colony PCR and RIM test was not performed on
those samples. The advantage of colony PCR is that, even though the culture is mixed or
there is only one isolated colony, PCR still works.


Sample        Spike        Ct Value- 6 h    Ct Value -22 h    Colony PCR        RIM Test
              (CFU/g)      Enrichment       Enrichment
VS1           7.6          UN               +,27.44           ND                ND
VS2                        UN               +,25.00           ND                ND
VS3                        UN               +,23.24           ND                ND
VS-                        UN                                 ND                ND
Unspiked
VS 4          1.6          UN               +, 36.66          ND                ND
VS 5                       UN               +,35.47           ND                ND
VS 6                       UN               +,29.75           ND                ND
VS-                        UN               UN                ND                ND
unspiked
VS 7          1.3          UN               UN                UN                -
VS 8                       UN               +, 33.13          UN                -
VS 9                       UN               +, 23.85          +                 +
VS-                        UN               UN                UN                ND
unspiked
VS-10         2.3          UN               +,34.77           +                 +
VS-11                      UN               +,34.14           +                 +
VS-12                      UN               UN                UN                +
VS-                        UN               UN                UN                -
unspiked
VS-13         4.6          UN               +,35.26           +                 +
VS-14                      UN               +,37.27           +                 +
VS-15                      +,39.3           +,26.21           +                 +
VS-                        UN               UN                UN                ND
unspiked
                                                                                        50


VS-16         3.3         UN               +,34.22           +,20.54          +
VS-17                     +,38.0           +,33.78           +,21.15          +
VS-18                     UN               +,39.26           +,22.97          Weakly +
VS-19                     +,39.1           +,35.54           +,25.52          +
VS-                       UN               UN                UN               -
unspiked


Table 13:      Results of real-time PCR after 6h and 22h enrichment, colony PCR
               and RIM latex agglutination test for E. coli O157:H7. Ct- mean cycle
               threshold value from two reactions, + = positive reaction, UN=
               undetected, ND=not determined. Grey shading indicates this quarter’s data

In conclusion, 6h enrichment is not adequate for the detection of E. coli O157:H7 from
lettuce samples by real-time PCR. On the contrary, 22 +/- 2h enrichment is better for the
detection of E. coli O157:H7 using the stx-2 gene.


      Isolation of Shigella sonnei by immunomagnetic separation (Aparna
       Tatavarthy and William Veguilla)

We developed anti-Shigella antibody beads for the rapid isolation of Shigella species
from artificially contaminated foods. The polyclonal antibodies were ordered from Novus
Biologicals. These antibodies presumably cover all the Shigella species including Sh.
sonnei, Sh. boydii, Sh. flexneri, and Sh. dysenteriae. We coupled the anti-Shigella
antibodies to the tosylactivated Dynabeads following the manufacturer’s protocol
(Invitrogen). Six samples of artificially contaminated BPW and three samples of infant
formula were tested using the newly coupled antibody beads.

Ten ml of BPW was contaminated with Shigella sonnei (Table 14) and incubated for 22h
at 35oC. After the incubation, three 1ml samples were subjected to IMS. In an attempt to
optimize the volume of the antibody beads required for IMS, we performed a
concentration gradient for the beads. Samples numbers 1, 2, and 3 received 20 l, 10 l,
and 5l of the antibody beads respectively. The IMS method was performed as
previously described. The bead bacteria complex was then plated on MacConkey agar
(Mac) and TSA plates. The 22h enriched sample was also plated on Mac agar before
performing the IMS. This experiment was performed twice to confirm that the antibody
was successfully coupled to the tosylactivated paramagnetic beads.

Three baby formula samples (25 ml each) were spiked with Sh. sonnei (Table 15). Two
hundred twenty five ml of BPW was added to each sample and incubated for 6h a 35 oC.
After the 6h incubation, dilutions of each sample were plated on Mac agar. One ml of the
sample was subjected to IMS with 10 l of Dynabeads as previously described and plated
on Mac and TSA plates. Real-time PCR targeting the ipaH gene was performed on the 6h
and 22 h enriched samples. DNA extraction on these samples was done as previously
described using the ABI PrepMan reagent.
                                                                                          51



With a spike level of 25 or 15 CFU/ 10ml, Sh. sonnei colonies were isolated on Mac agar
from both pre-IMS and post-IMS BPW samples (Table3). These colonies were
morphologically identical to the control. Based on the IMS results, there was no
significant difference between amounts of beads used (20l, 10l, and 5l). Addition of
5l worked as well as the 20 l in concentrating the sample for the isolation of Sh.
sonnei.


   Sample           Spike      Volume        Pre-IMS           Post-IMS plating(CFU)
                    (CFU)      of beads      plating (50 l-
                               for IMS       Mac) (CFU)
                               (l)                            50 l           25 l
   BPW-1            25         20            TNTC              400          280
   BPW-2            25         10            TNTC              TNTC         104
   BPW-3            25         5             TNTC              141          158
   BPW-4            15         20            TNTC              200          99
   BPW-5            15         10            TNTC              TNTC         139
   BPW-6            15         5             TNTC              TNTC         200

Table 14:      Number of colonies isolated from artificially contaminated BPW using
               the immunomagnetic separation. The results show the colonies from
               pre-IMS and post-IMS platings on MacConkey agar.

The results of isolation of Sh. sonnei from baby formula are shown in Table 15. Isolated
Shigella colonies resembling the control were isolated from all the three BF samples in
the pre-IMS BPW enrichments. However, Shigella colonies were not isolated from any of
the BF samples after IMS. The formula almost had no background flora, so interfering
microbial load can be ruled out. If the food particles are interfering with the bead-bacteria
interaction is to be determined. A longer pre-enrichment (22-24h) may be required for the
isolation of Shigella by IMS.


Sample            Spike            No. of CFU BPW :       No. of CFU : Post-        Background Flora
                                   Pre-IMS                IMS                       (APC)
BF1               17                                 26                         0
                  CFU/200l
BF2                                                  24                         0
BF3                                                  18                         0
BF-Unspiked                                           0                         0
                                                                                    <100 CFU/ml

Table 15:      Number of presumptive Shigella sonnei colonies isolated from
               artificially contaminated baby formula on lab day 2 using the short
               pre-enrichment and immunomagnetic separation. The results show the
                                                                                      52


                number of isolated colonies from pre-IMS and post-IMS platings on Mac
                agar. APC- aerobic plate count, BF- baby formula

Real-time PCR was successful in identifying the target gene from both the 6h and the 22h
enriched BF samples (Table 16).

Sample          Spike      Ct Value- 6 h   Ct Value -22 h
                (CFU/g)    Enrichment      Enrichment
BF1             17         +,35.85         +,17.2
BF2                        +,39.24         +,15.99
BF3                        +,37.59         +,15.37
BF-                        UN              UN
Unspiked
Table 16:       Results of real-time PCR after 6h and 22h enrichment targeting the
                ipaH gene. Ct value- mean cycle threshold value from two reactions, + =
                positive reaction, UN= undetected, BF=baby formula


         FERN Proficiency Test- Listeria monocytogenes testing (Aparna Tatavarthy)

CBD was a part of the proficiency testing by the Food Emergency Response Network
(FERN). Milk samples were sent as a part of the Listeria PT exercise. The samples were
blindfolded and some of them were artificially contaminated with several species of
Listeria and organisms belonging to other genera. The samples were homogenized by
Dave Wingfield of Florida Dept. of Health. DNA was extracted from the 6h enriched
samples as well as overnight enrichments for molecular detection by real-time PCR.
After 6h incubation one ml aliquot each of the five enriched samples was saved for later
use. The samples were placed in cryovials and stored at -80oC for approximately 12h
before use. The overnight samples were taken at approximately after 22h incubation and
were processed immediately for DNA extraction. DNA was extracted from the 6h and the
22h samples following the manufacturer’s protocol. Briefly, one ml of each of the sample
was centrifuged at high speed for three min. The supernatant was discarded and 200 l of
ABI PrepMan (Applied Biosystems) reagent was added and the samples were boiled for
10 min at 100oC. The boiled prep was centrifuged at high speed for 3 min and the
supernatant was used for PCR detection. The real-time PCR was performed on the ABI
7500 fast system using default parameters. The primers targeted the listeriolysin O
(hlyA). The probe was labeled with the reporter dye, 6 – carboxyfluorescein (FAM) on
the 5’ end and the quencher dye, Black Hole Quencher (BHC) on the 3’ end. A 20 l
reaction was set up with 2X master mix, 18 pmol of each primer, 5 pmol of the probe and
2 l of the template DNA. Each sample was run in duplicate and the mean Cycle
Threshold (CT) value was calculated.

Results

Sample - 6h enrichment                             Mean CT value (2 reactions)
                                                                                          53


FERN 1                                               UN
FERN 2                                               UN
FERN 3                                               UN
FERN 4                                               UN
FERN 5                                               UN
Sample - 22h enrichment                              Mean CT value (4 reactions)
FERN 1                                               +, 30.69
FERN 2                                               +, 36.38
FERN 3                                               +,32.11
FERN 4                                               UN
FERN 5                                               UN
Sample - Colony PCR from Oxford Agar                 Mean CT value (2 reactions)
FERN-1                                               +, 20.29
FERN 2                                               +, 21.62
FERN 3                                               +, 20.33
FERN 4                                               UN
FERN 5                                               NA
Sample – Controls (pure DNA)                         Mean CT value (2 reactions)
L. monocytogenes (ATCC 9525)                         +, 17.39
L. monocytogenes (ATCC 19115)                        +,15.84
L. monocytogenes (ATCC 19111)                        +, 18.21
L. ivanovii (ATCC 700402)- colony PCR                UN

Table 17:      + = presence of hly gene, UN = undetected, NA- not applicable, CT =
               Cycle Threshold value based on the reactions specified


Based on the results of Table 17, samples 1, 2, and 3 have the listeriolysin O gene (hlyA),
therefore potentially contain Listeria monocytogenes. Although, the primers and probe
designed are specific to Listeria monocytogenes (Nogva et al, AEM 2000) a later study
suggests the presence of hly gene (Volokhov et al JCM, 2002) in other species of Listeria
including L. ivanovii, and L. seeligeri. Our control L. ivanovvi was negative for this gene.
Although we are very confident that samples 1, 2, and 3 are positive for L.
monocytogenes; we cannot completely rule out the possibility of L. ivanovii, or L.
seeligeri in these samples. The results correlated with the culture results obtained by
Dave Wingfield and Gail Scilabro of FLDOH. The results were later confirmed to be
accurate by the FERN team.
                                                                                           54


                 Bead validation for Detection of Pathogens on the Luminex System (Andy
                  Mackley)

Last report improved validation procedures and the use of new Streptavadin
Phycoerythrin (SAPE) helped to obtain final validation of one bead set, bead 50-uid.
This report covers the validation of the remaining bead sets 70-stx, 42-ipaH, and 44-uidA
and testing versus PCR product.

The bead 50-uid, bead 70-stx, bead 42-ipaH bead sets all had good P:N ratios, with
maximums greater than 300, Figure 20 a-c). This is good for proceeding to testing on
PCR products.

Figure 20:                      P:N rations of validation of bead sets
A: 50-uid
                                      bead 50-uid Validation
                                             11-25-09
                 350
                 300
                 250
    P:N ratio




                 200
                 150
                 100
                  50
                   0
                       1              10          100             1000   10000
                                           Template Conc (nM)



B: 70-stx

                                    bead 70-stx Validation

                 1000

                  800
     P:N Ratio




                  600

                  400

                  200
                       0
                           10              100              1000         10000
                                           Tem plate Conc. (nM)
                                                                                   55


                              bead 42-ipaH Validation

               1000

               800
   P:N Ratio




               600

               400

               200

                 0
                      10            100               1000        10000
                                     Tem plate Conc. (nM)


C: 42-ipaH

The improved coupling procedures and new SAPE provided excellent results for all but
one of the bead sets, bead 44-uidA, Figure 21.

Figure 21:                 P:N ratio for bead 44-uidA with SAPE

                              bead 44-uidA Validation

               1000

               800
   P:N Ratio




               600

               400

               200

                 0
                      10            100               1000        10000
                                     Tem plate Conc. (nM)




PCR Products

The uid amplification probes have worked well with both E. coli K-12 and E. coli
O157:H7, Figure 22

Figure 22:                 uid PCR amplification from E.coli
                                                                                              56



The PCR probes for use with the stx and ipaH bead sets have not worked as well, or at
all.

The PCR probes used for uidA were designed in house using sequence data from
Genbank for E. coli K-12 strains and O157:H7 strains

uidA
uidA K-12 F 5’ Biotin – ATATTCCCGTGCACCTTG
uidA K-12 R 5’ Biotin – TATAAGGGCACGTGGAAC
uidA K-12 Capture - 5’-/5AmMC12/CGC TAG TGC CTT GTC CAG TTG CAA C -3'

uid
uid1-F - 5’- /5Biosg/GGC TTC TGT CAA CGC TGT TT -3’
uid1-R - 5’- ACA GTT TTC GCG ATC CAG AC -3’
uid1 cap - 5’- /5AmMC12/GCG ATC TAT ATC ACG CTG TGG GTA TTG CAG -3’

The probes for ipaH and stx were obtained from a multiplex PCR paper.

ipaH
ipaH-F – 5’- CCT TGA CCG CCT TTC CGA TA -3’
ipaH-R – 5’- /5Biosg/AAT CAG TTT TCC CGA TGC AG -3’
ipaH capture - 5’-/5AmMC12/TTC GAC AGC AGT CTT TCG CTG TTG CT -3’

stx
stx F – 5’- TGG TTG CGA AGG AAT TTA CC -3’
stx R – 5’- /5Biosg/ CGC CCT TCC TCT GGA TCT AT -3’
stx capture - 5’-/5AmMC12/AGA CTT CTC GAC CGC AAA GAC GTA TG -3’

Though they worked fine for the authors on their strains perhaps there are sufficient
interstrain differences that I need to either obtain some of the exact strains they used or
more likely I’ll design some for the strains in our culture collection.
                                                                                         57


Section 2:     Support Activities

      CBD BSL-2 Stock Culture Collection and Database (Kealy Peak and William
       Veguilla)

The BSL-2 and BSL-3 Stock Culture Collections underpin all laboratory research at the
Center for Biological Defense (CBD). Authentic, documented and preserved strains are
critical to the accuracy and reproducibility of decontamination studies, equipment
validation testing, antimicrobial resistance profiling, strain typing and molecular-based
detection methods in development at the CBD.

The BSL-2 Culture Collection of 1377 validated strains is the linchpin supporting
virtually all laboratory research at CBD. In the last quarter, four Staphylococcus spp. and
a Shigella strain added to the collection. These strains have been accessioned,
characterized for differential traits and cryostocked at -85 ºC and in liquid nitrogen.

An Access database documents the collected strains. Organized in 35 tables, hundreds of
fields of data record salient attributes for each authenticated strain as well as details of
strain history and the precise location of each vial in the freezers.

Methods: After phenotypic characterization from a single, representative colony, each
CBD strain was cryostocked using a standardized cryostorage protocol (Figure 23). The
progenitor colony was inoculated to 100 ml of tryptic soy broth (TSB) and incubated at
optimum temperature for approximately 15 h in a waterbath shaking at 125 rpm. Thirty
ml of overnight culture were pelleted at 3571 x g for 10 min at RT, resuspended in 25 ml
of TSB with 10 % glycerol and aliquoted as 1 ml into 24 cryovials. Two of the vials are
film-sealed for storage in LN2. After 30 min of equilibration at RT with the
cryoprotectant, all cryovials were placed in NUNC® controlled freezing units (Nalgene,
Rochester, NY) with isopropanol according to manufacturer’s instructions and held at -85
ºC for 75 min to foster cooling of approximately -1 ºC/min to near -30 ºC. Immediately
thereafter, cryovials were removed from the units and held at -85 ºC until stabilized at
that temperature. Vials are transferred to long term storage in -85 ºC freezers or in liquid
nitrogen storage tanks.

After one week of cryostorage, during which most cell death occurs, a single cryovial of
each CBD strain was quick-thawed at 35 ºC. Viable counts were obtained from spread
plates of a 1:10 dilution series in order to document viability and purity. Of the 1377
strains cryostocked in the collection, over 1,300 have undergone the quality control
procedure in which a working-stock is thawed after one year of storage at –85 ºC and a
count performed to re-access viable cell numbers. Since Jan 2009, over 400 strains have
had this quality control procedure repeated. After up to seven years in cryostorage,
almost all vials retain the approximate viable cell density originally stocked.

Growth of Collection and Highlighted New Strains: The collection grew by five
strains, one Shigella flexneri and two Staphylococcus aureus and two Staphylococcus
epidermidis strains between October and Dec 2009 (Table 18). The Shigella flexneri
                                                                                         58


strain was provided by the Florida Dept. of Health, Tampa (FDOH-TBM) for Dr. Aparna
Tatavarthy’s research to enhance rapid detection and isolation of Shigella species from
foods. We intend to expand the number of Shigella spp. stocked in the CBD BSL-2
Collection. Dr. Tatavarthy will be soliciting clinical isolates of Shigella from other
FLDOH Branch labs, from local diagnostic labs and from hospital microbiology sections.

Database Documentation of Strains: Comprised of 35 tables, the Access database
documents the salient features and strain history of each authenticated strain as well as
the precise location of each vial in the cryostorage.
List of Representative Tables & Representative Fields:
     Accession & Identity: Twelve data fields including Genus & Species names;
        Serovar; Type Strain; Strain Designation; Source/Depositor; Deposit Date.
     Strain History: Eleven fields including Previous Holder; Other Holders; Isolation
        Source & Date; Synonyms; Strain Designations; Publications; Applications.
     General Characterization: Seven fields including Media; Growth Parameters; Cell
        & Colony Morphology; Gram Reaction; Critical Strain Traits.
     Frozen Working Stocks: Fifteen fields that inventory, locate, and document the
        viability of the working stocks of the strains in the collection.
     Staph Strain Typing Data: Twenty fields including methicillin-resistance,
        community-acquired, SCCmec type, spa Type, MLST type, and allele for 7 genes
        used in MLST typing.
     Salmonella Strain Typing Data: Ten fields including results of PCR testing for
        gene targets such the spvA gene for non-Typhi, Group I Salmonella and ompF,
        outer membrane protein F for subspecies I, II, IIIa, IV and V.

      A new Table has been developed to document all strains provided to researchers
       outside the CBD.

The strains of the BSL-2 collection represent transferable resources that are periodically
deposited into national repository and bioforensic collections as well as made available to
Department of Defense biodefense researchers. Sub-collections or groups of strains in
the BSL-2 collection are available to be transferred, along with documentation of
phenotypic and molecular-based strain typing data, to research labs within the
Department of Defense. The CBD culture collections will continue to grow with the
procurement of strains required for continuing and future research objectives. Such
objectives, including methods of molecular strain typing and antibiotic resistance
detection require substantial numbers of strains for validation studies. The perpetuation
of this research culture collection, the exchange of strains among research labs and the
deposit of published strains into national repository collections are all means of providing
the sin qua non for microbial research -- authentic, documented and preserved strains.
                                                                                59


 Figure 23:     Flowchart of CBD Culture Collection methods
              Flowchart of CBD Culture Collection Methods
                    Receipt of strain


                     Single Colony Isolation


                    Pure Culture


                    Assign Unique CBD Accession Number


                    Determine Growth Requirements & Strain Traits


                    Phenotypic Characterization: Authentification


                    Record-keeping: Access Database


                    Growth from Single Colony for Stocking



                    Freeze at -85oC in 10% glycerol


Re-Stock            Seed Stocks           Working Stocks
Depleted
Vials
              LN2 Safety Stocks          Sacrifice Vial for Viability, Purity
                                                & Identity Check

                                                Release for Research Use
                                                                                       60

Table 18: Overview of BSL-2 Culture Collection of the Center for Biological Defense
                                                         Strains Accessioned, Characterized and
                                                                         Preserved
                 Total All Genera                                           1377
         Genus             species
        Shigella                                                             20
                           dysenteriae                                        4
                           boydii                                             3
                           sonnei                                             7
                           flexneri                                           5
        Bacillus                                                            244
                           cereus                                            58
                           thuringiensis                                     19
                           mycoides/pseudomycoides                           16
                           anthracis                                          2
     Staphylococcus                                                         469
                           aureus                                           451
                                                                          29 NARSA control strains
                                                                           12 ATCC control strains
                                                                          382
                          MRSA                                                   357 clinical isolates
                                                                                  24 control strains
        Listeria          monocytogenes & others                           10
      Escherichia         coli                                            129
                                                                          104
                                     Serovar O157:H7                                50 CDC strains
                                                                                 48 Clinical isolates
       Salmonella         enterica                                        165
                                       Serovar Typhi                       17
       Yersinia           enterocolitica                                    3
     Campylobacter                                                        188
        Vibrio                                                             54
                          parahemolyticus                                  37
                                                                          5 ATCC Pandemic strains
                          cholerae                                          8
                          vulnificus                                        6
Acinetobacter             baumannii & calcoaceticus                        20
Burkholderia              cepacia                                           4
Clostridium               difficile                             1, type strain (derived)
                                                                              61


DOH Sub-Contract Input & Collaboration:

Roger Sanderson and Gail Scilabro were instrumental for the epidemiological
information on the S. Typhi study
                                                                                          62


Manuscripts published, in press or reviewed this quarter:

Manuscripts Published

Luna, V., Gulledge, J., Cannons, A.C., Amuso, P.T. (2009) Improvement of a selective
media for the isolation of B. anthracis from soils. Journal of Microbiological Methods.
79, 301–306.

King, D., Luna, V., Cannons, A., Amuso, P, (2009) Procurement of spore-free Bacillus
anthracis for molecular typing outside of BSL3 environment. Journal of Applied
Microbiology. ISSN 1364-5072

Manuscripts in press/review

The manuscript titled “Real –Time PCR Detection of Salmonella Species Using a Novel
Target: The Outer Membrane Porin F Gene (ompF)” was submitted to Letter in Applied
Microbiology. This manuscript has come back for revision.


Abstracts (for ASM, General Meeting, 2010)
a. Molecular typing and antibiotic resistance analysis of Salmonella enterica serotype
Typhi
Aparna Tatavarthy, Kealy Peak, William Veguilla, Roger Sanderson, Gail Scilabro, Phil
Amuso, and Andrew Cannons

b. Rapid detection and isolation of E. coli O157:H7 from artificially contaminated ready-
to eat- foods
Aparna Tatavarthy, William Veguilla, Kealy Peak, and Andrew Cannons

c. Molecular Characterization of USA300 and USA400 Methicillin-Sensitive
    Staphylococcus aureus
Jill Roberts, Andrew Cannons and Philip Amuso
                                                                                        63


Item c. Results Obtained Related to Previously-Identified Problem Areas
No problem areas have been identified at this point in time.

Item d. Any Significant Changes to Contractor’s Organization or Method of Operation,
to Project Management Network, or to Milestone Chart
None.

Item e. Problem Areas Affecting Technical or Scheduling Elements
No problems areas have been identified.

Item f. Problem Areas Affecting Cost Elements
None.

Item g. Cost Curves Showing Actual and Projected Conditions Throughout the Contract
Financial cost curves and charts have been provided in a separate unaudited report titled:
9th Quarter – Contract W911SR-07-C-0084- Financial Reports contract for Task 1 and 2
of the subtasks on Task 2.
Item h. Any Cost incurred for the Reporting Period and Total Contractual Expenditures
as of Reporting Date
Financial cost curves and charts have been provided in a separate unaudited report titled:
9th Quarter – Contract W911SR-07-C-0084- Financial Reports contract for Task 1 and 2
of the subtasks on Task 2.
Item i. Person-hours Expended for the Reporting Period and Cumulatively for the
Contract
                          9th Quarter Manpower Hours       YTD Manpower Hours
 Andy Cannons                                        459                        3668
 Phil Amuso                                          112                          784
 Vicky Luna                                          560                        3920
 Andy Mackley                                         56                        2912
 Kealy Peak                                           56                        2912
 Jenny Gulledge                                      560                        3920
 Debbie King                                         560                        3920
 William Veguilla                                    560                        3920
 Aparna Tatavarthy                                   280                        2120
 Jill Roberts                                        560                        3920
** These un-audited calculations were done based upon the following formula: % effort
X 40 hours per week X 14 weeks of pay (1 October 2009-1 January 2010)

Item j. Any Trips and Significant Results
No trips and results were taken.

Item k. Record of all Significant Telephone Calls
None
                                                                                      64


Item l. Summary of Engineering Change Proposal (ECP) Status
None

Item m. Contract Schedule Status
None.

Item n. Plans for Activities During the Following Reporting Period
Vicki Luna, Jenny Gulledge, Debbie King
        Continue work on pX02 plasmid
        Continued work on Bacillus spp. on Riboprinter and PFGE
        Continue characterization (typing, antimicrobial susceptibility testing) of the
           MRSA
        Continued analysis of soil samples for Bacillus
        Continue pyrosequencer project for detection on unknown pathogens
        Continued testing of copper solutions on MRSAs
Kealy Peak and William Veguilla
         Continued stocking of culture collection
         Continued typing of Acinetobacter
Jill Roberts
        Continued studies on Salmonella pathoadaptation
        Continued analysis of PFGE of Bacillus sp.
        S. aureus exotoxin typing
        S. aureus typing for public health labs.
Andy Mackley
        PCR product sequence data for the genes of interest for DNA multiarray
           design
        UV purification of water
Aparna Tatavarthy
         Typing Salmonella Typhi using Spe-1 enzyme will be continued.
         Isolation of E. coli from spiked lettuce sample will be completed and Shigella
           isolation work will be initiated

Item o. Name and telephone Number of Preparer of the Report
The overall report was compiled by: Dr. Andrew Cannons, (813) 974-1478, with
contributions by:
       Dr. Vicki Luna, Research Associate – Section 1
       Debbie King, Associate in Research – Section 1
       Jenny Gulledge, Associate in Research –Section 1
       Kealy Peak, Associate in Research – Section 2
       William Veguilla, Biological Scientist –Section 1 & 2
       Dr. Jill Roberts, Postdoctoral Fellow– Section 1
       Andy Mackley, Associate in Research – Section 1
       Dr Aparna Tatavarthy, Postdoctoral Fellow – Section 1
Item p. Appendices for Tables, References, Photographs, Illustrations and Charts
n/a

				
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