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					3. Methods                                                                                       15


3. Methods

3.1 Polyphasic taxonomy
By using polyphasic taxonomy, the clinical isolates from Hamburg can be identified. The
following phenotypic, genotypic and phylogenetic tests are accomplished.


3.1.1 Phenotypic tests
In order to make these analyses, bacteria are cultivated on blood agar plates (BAP) and
cultured for 24 hours at 37° C.


3.1.1.1 Catalase test

The Catalase test differentiates Staphylococci and Micrococci which are catalase-positive
from enterococci and streptococci that are catalase-negative. This test is based on the
possibility of certain bacteria to produce the enzyme catalase which decomposes hydrogen
peroxide into water and oxygen:


2H2O2     Katalase       2H2O + O2


Staphylococcus haemolythicus JCSC1435 (51.81) and Staphylococcus aureus 833 are
positive controls.
One bacterial colony is placed on a microscope slide by using an inoculation loop. One
drop of hydrogen peroxide is spotted onto the colony. If the bacteria are catalase positive,
gas production would be clearly visible (Figure 2A). Catalase-negative bacteria do not
reveal gas production (Figure 2B).


                 A                              B




Figure 2. Gas production indicates that the bacteria are catalase positive (A) and no gas production
catalase-negative strains (B) [Ruhr University Bochum, http://memiserf.medmikro.ruhr-uni-
bochum.de/praktikum/praktikum.html, 25.01.2009]
3. Methods                                                                                  16


The following possibilities could lead to false positive or negative results:
If the colonies are older than 24 hours, false-negative results can be observed. The test is
regarded as negative when weak gas production occurs in a delayed fashion. Blood cells
are catalase positive. Therefore the test will be meaningless if it is performed on a blood
agar plate [University of Tromsø, Institute of Medical Microbiology, ―mikrobiologiske
metoder; katalase-test‖].


3.1.1.2 Coagulase test
Coagulase can be accounted for by means of Staphaurex plus® that is a rapid latex
agglutination test. It identifies Staphylococci that bind coagulase protein A and/or surface
antigens that characterize Staphylococccus aureus. Staphaurex plus® contains latex
particles that are coated with fibrinogen and S.aureus specific rabbit-immunoglobulin G
(IgG).


A rapid agglutination will occur through the interaction of fibrinogen and bound coagulase,
IgG and protein A or specific IgG and surface antigens of the cell.
Control latex is used to identify and evaluate non-specific reactions. Thus a positive result
shows agglutination with ―Test Latex‖ and no agglutination with ―Control Latex‖.
Negative results show no reactions with both of the reagents (Figure 3).




Figure 3. Circle A shows a positive test and the circles B-E negative tests [modified O`Connor,
http://www.medicine.uiowa.edu/cme/clia/modules.asp?testID=11#Figure01, 26.01.2009]



The reagents are warmed to room temperature and shaken well. One drop of Test Latex
and one of Control Latex is placed on the agglutination plate, and some colonies are mixed
with both reagents. The plates are moved back and forth carefully for 30 seconds and
3. Methods                                                                                         17


observed for agglutination. Staphylococcus aureus, which is coagulase-positive, is used as
a control.


The following possibilities could lead to false positive or negative results:
S.capitis can give false-positive reactions. The reagents must not be frozen or overdue.
Contaminated aggregation plates can also lead to a false result. Agglutinations that occur
later than 30 seconds after adding the latex are regarded as negative [University of Tromsø,
Institute of Medical Microbiology, ―mikrobiologiske metoder; påvisning av koagulase ved
Staphaurex plus‖].


3.1.1.3 Gram staining
This staining approach is used to classify bacteria into two major groups: the gram-postive
and gram-negative bacteria that show different staining characertistics as a result of their
differences in cell-wall structure. The cell wall of gram-positive bacteria consist of 90%
peptidoglygan that is arranged in a layer of several sheets, one upon another. This layer can
have up to 25 sheets and many gram-positive bacteria have additionally teichoic acids
covalently bonded to the peptidogycan layer. The cell wall of gram-negative bacteria
consists of only 10% peptidoglycan. Most of the cell wall is made up of the outer
membrane that is a second lipid bilayer binding lipids and polysaccharides on its outer
layer. This complex is also called lipopolysaccharide layer (LPS) (Figure 4) [Madigan et
al., 2008].




Figure 4. The structure of the cell wall of gram-positive and gram-negative bacteria. Gram-positive
bacteria contain up to 25 sheets of peptidoglycan against what gram-negative bacteria contain a lean
layer but an outer membrane with LPS. [modified Mountain Empire Community College
http://water.me.vccs.edu/courses/ENV108/Lesson5_print.htm,26.1.2009]
3. Methods                                                                                   18


The glass slide has to be held over a flame in order to remove fat particles.Then, one drop
of saltwater is placed on the glass slide. One colony is mixed with the saltwater by using
an inoculation loop and afterward spread across the glass slide. The sample has to be air-
dried.
For the purpose of fixing the preparate, the glass slide is passed slowly through a flame
(3x). It is important that the sample is not oriented towards the flame. By this heat fix,
proteins denature and the cell wall gets permeable for the dye. Furthermore, the sample
will be stabilized onto the slide.
After fixation, Gram I (Crystal Violet) is added and incubated for 30 seconds. Then, Gram
II (Iodine-Potassium Iodide) is added and also incubated for 30 seconds. Crystal Violet and
Iodine-Potassium Iodide build a blue-violet dye complex that is water-insoluble and a
larger molecule than Crystal Violet or Iodine.All cells are now blue-violet.
The samples are decolorized by the adding of Gram III (etanol) as far as the dye is washed
away. Then, the sample is carefully rinsed with water. The etanol dehydrates the
peptidoglycan layer. The thick, tightened peptidoglycan layer of gram-positive bacteria
becomes impermeable for the large crystal-violet-iodine complex and stays therefore blue
violet. In contrast, the outer membrane of gram-negative bacteria is destroyed, and the thin
peptidoglycan layer cannot retain the dye resulting in colorless cells.
The sample is stained with the water-soluble Gram IV (safranin) and incubated for 1
minute. Then, it is washed with water and dried with a filter paper. Gram-positive bacteria
stay blue-violet because safranin is lighter than the crystal-violet-iodine complex so that it
is thus not able to superimpose the blue-violet color. Gram-negative bacteria are therefore
stained red (Figure 5 and 6).
The sample is analysed by fluorescence microscopy.


The following possibilities could lead to false results:
The results depend on several factors such as the age of the strain, the cell culture medium,
antibiotic treatment and the staining method. The staining results could give poor results if
the time intervals between the different reagents are too long or to short. Especially if the
decolorization with etanol is too little time, the sample will reveal false blue-staining. The
result can also be influenced by the quality of the reagents.
[University of Tromsø, Institute of Medical Microbiology ―mikrobiologiske metoder,
gram- farging‖; Madigan et al., 2008; Bruckner
3. Methods                                                                                19


http://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html,
25.01.2009].




                                                         Gram negative bacteria




Figure 5. Gram staining
[University of South Carolina, School of Medicine,
http://pathmicro.med.sc.edu/fox/culture.htm;
26.1.2009]
                                                         Figure 6. Gram positive and
                                                         negative bacteria [Vrije University
                                                         Amsterdam,
                                                         http://www.bio.vu.nl/geomicrob/proto
                                                         cols/General/Gram-Kleuring.pdf,
                                                         26.01.2009]



3.1.2 Genotypic tests- The PCR amplicon sequencing of the rpoB gene
The phenotypic methods are supported and confirmed by the genotypic approach ―the PCR
amplicon sequencing of the ―rpoB gene‖


The rpoB gene is a conserved gene suitable to use in species identification. In order to
determine the base sequences of the rpoB gene, a PCR based sequencing method is used.
The Polymerase Chain Reaction (PCR) is used to amplify DNA segments in vitro. It is thus
possible to amplify stretches of the template of up to a few thousand base pairs. This
method comprises two primers that are oligonucleotides of 17–30 nucleotides. These
primers flank the target sequence that is to be amplified. One of the primers is
complementary to the “sense strand” (or ―target strand‖) while the other is complementary
to the “antisense strand”. The primers will therefore bind to single-stranded target DNA
through complementary base pairing. The PCR-cycle contains the following four steps:
In order to achieve single-stranded DNA strands, the target DNA is heated (most often
around 94° C). This step is also called “Denaturation”, which is a completely reversible
3. Methods                                                                              20


process. When the mixture cools down, the two primers will bind to their complementary
stretch of the target DNA resulting in double-stranded hybrid DNA (Annealing). The
temperature depends on the length and the sequence of the primer and ranges from 45° to
60° C.
The DNA polymerase is then extending the primers: It binds to the 3`ends of the primers
and synthesize a new DNA strand using the original DNA strands as templates (Extension).
The polymerase that is normally used is that of Thermus aquaticus. This Taq DNA
Polymerase has a temperature optimum of 72° C and is resistant to even higher
temepratures so that it will not be deactivated by the ―Denaturation” step.
The first cycle results in two double-stranded DNA strands containing an original DNA
target strand and a shorter, new synthesized strand. The newly synthesized strands will
have a defined 5`ends but no defined 3`ends. Denauturation, Annealing and Extension is
repeated, and at the end of the second cycle, the two newly synthesized strands will both
have defined 5` and 3` ends. It is thus generated a primer flanking sequence that is to be
amplified. By repeating these steps several times (usually 20–30 times) an amplification of
the designated DNA sequence of up to a billionfold is reached.
After the final cycle, a final extension step (“Elongation”—72° C, 5 minutes) is performed
to be sure that all the single-stranded DNA has been replicated (Figure 7) [Reece, 2004;
Madigan et al., 2009]




 1.cycle



 From the
 2.cycle



Figure 7. Polymerase chain reaction [modified Cowrie Genetic Database Project (CGDP),
http://www.flmnh.ufl.edu/cowries/amplify.html, 9.02.2009]



These PCR products are then the starting point for the sequencing of the desired gene. This
method is called Chain termination sequencing.
3. Methods                                                                                    21


The DNA sequencing is based on dideoxynucleotides that are labeled with terminator dyes
(Big DyeTM terminators) having different emission properities.
By adding DNA polymerase, a primer and dNTP (deoxynucleotides), DNA replication can
be initialised. It is also inserted a little amount of ddNTP (didesoxynucleotides) that lacks a
3`hydroxyl group. If it is incorporated in the extending DNA strand, a further nucleotide
would not be able to bind to the 3`end, resulting in the termination of DNA synthesis.The
resulting DNA fragments have an identical 5`end but vary in sequence length and therefore
in a specific 3`end (Figures 8 and 9 ).
The products are then separated by capillary electrophoresis, and the sequence of the DNA
can be read: The DNA fragments migrate through a capillary, comprising a polymer and a
laser that excite the fluorescein donor dye of the labeled didesoxynucleotide. The labeled
ddNTPs at the end of each DNA fragment will therefore fluoresce at different wavelenghts
and intensity that can be analysed by a fluorescence detector. The information is then sent
to a computer whose software is able to convert all the information into a sequence [The
University Hospital of North Norway, Institute of Medical Genetics; Reece, 2004].




Figure 8. ddNTP and dNTP [ Herveg, http://home.scarlet.be/~tsk05520/biomolespa/Enzimas/ADN-
POL.html, 25.02.2009]
3. Methods                                                                                          22




A                                          B

Figure 9. The priciple of gel electrophoresis
A: Chromatogram: Results of gel electrophoresis. The colours represent the 4 bases Thymin Adenen,
Guanin and Cytosin [DNA Sequencing Service, http://dnasequencing.wordpress.com/2007/10/26/chain-
termination-methods/, 25.02.2009]
B: The principle of “Chain termination sequencing”. [The University Hospital of North Norway, Institute
of Medical Genetics]



3.1.2.1 Isolation and amplification of DNA
In this study, Staphylococcus haemolyticus JCSC 1435 (51.81) is used as a reference strain.
8–10 colonies, which have been cultured on a blood-agar plate, are transferred to an
ependorftube containing 1ml TE-buffer. The suspension is centrifuged at 5000 rpm for 5
minutes. The supernatant is removed and the pellet resuspended in 100 μl TE – buffer. The
suspension is boiled for 10 minutes at 100° C. A little hole is made in the lid of the
ependorftube to prevent the bursting of the tube. Then, the suspension is centrifuged once
again at 5000 rpm for 5 minutes. The supernatant is transferred to a new eppendorftube.
It has to be worked on ice. The transferred supernatant is then stored at -20° C
[University of Tromsø, Departement of Microbiology and Virology, ―Rapid template
isolation for PCR‖].
3. Methods                                                                              23


3 μl of the template is mixed with 22 μl mastermix containing 12.5 μl ReddymixTM, 3.0 μl
MgCl, 2.0 μl primer F, 2.0 μl Primer R and 2.5 μl dH2O [AMV mikrobiology, University
of Tromsø, Departement of Microbiology and Virology].
The tubes are transferred to the thermal cycler and the following rpoB sequencing program
is started [Drancourt and Raoult, 2002]:


Denauturation:        95° C 2 min


Denauturation:        94° C 30 sec
Annealing:            52° C 30 sec    35 x
Extension:            72° C 60 sec


Elongation:           72° C 5 min


                      4° C   ∞


3.1.2.2 Gel electrophoresis and preparation of the sample for sequencing
In order to check the presence and size of amplicons, the PCR products are run on a gel:
0.6 g agarose is dissolved in 60 ml TBE by boiling it. The 1% agarose suspension is then
mixed with 3 μl bromophenol blue.
The comb is placed on the casting stand so that wells are formed inside the gel when the
dissolved agarose is poured into the casting stand and cooled down for 30 minutes.
5 μl of the PCR products are applied on the gel and run at 120 V for 30 minutes using a gel
electrophorese chamber from BioRad with 0.5x TBE. Two kb+ are used as a molecular
weight marker.
The gel is transferred to an UV transilluminater to visualize the stained fragments [Bowen,
http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html,06.02.2009;
Sambrook and Russel, 2000]
When the results of the gelelectrophorese shows that the primer works appropriate and that
the samples are not contaminated, the extant suspension is treated with ExoSAP-IT®. The
extant 20 μl suspension is mixed with 4 μl ExoSAP-IT® and placed into a thermal cycler
(according to manufacturers’ description).
3. Methods                                                                                  24


The following ExoSAP - program is started:


37° C 1 h
85° C 15 min
 4° C ∞


In order to sequence the amplified gene, two suspensions have to be prepared: 1.5 μl PCR
suspension is diluted in 9 μl water. 5 μl of this diluted suspension is then mixed with 1 μl
Big Dye, 2.5 μl buffer, 8.9 μl dH2O and 19.2 μl primer F or primer R [AMV mikrobiology,
University of Tromsø, Departement of Microbiology and Virology].


3.1.2.3 Sequencing
The PCR amplicon sequencing of the rpoB gene is performed by the Department of
Medical Genetics at the University of Tromsø. The used sequencing apparatus is 3130 xl
Genetic Analyser from ―Applied Biosystem‖.


3.1.3 Phylogenetic trees (based on the rpoB gene)
To assess the results, the sequences of several close related gram-positive species are
included in the analysis. The sequences are found in ―BioCyc Database Collection‖ and the
―National Center for Biotecnology Information (NCBI)‖.
To align the sequences, they are inserted into the ―Alignment Explorer‖ of Mega.
ClustalW is a multiple sequence alignment program that is implemented in Mega. This
program is used for the multiple alignment of the sequences.
Phylogenetic trees are then generated: Mega is choosen to build Neighbour Joining- (NJ),
Minimum Evolution- (ME), Maximum Parsimony-, and UPGMA-based phylogenetic
trees. It is respectively built trees that are based on nucleotides and trees that are based on
the amino acid sequences.
By accomplishing bootstrapping (1000 datasets) that is a statistical method, the results are
supported. The choosen evolutionary models are Kimura 2 parameter (nucleotide-based
tree) and PAM 001 matrix (amino acid- based tree) [Tamura and Dudley].


3.2 Pulsed field gel electrophoresis (PFGE)
PFGE is used as a significant, epidemiological approach to study bacteria. It determines
the relatedness of strains. This method is based on the separation of DNA fragments from
3. Methods                                                                                25


100 bp to 10 Mbp by electrophoresis. It is thus possible to separate the whole genome of a
microorganism.
The separation relies on the fact that smaller molecules move faster through the agarose gel
than larger molecules so that the fragments are seperated according to their size. By the use
of ―conventional‖ gelelectrophoresis approach, it is not possible to separate molecules that
are larger than 15 kb, because the large molecules with an extended configuration would
get stuck in the gel.
The pulsed field gel electrophorese apparatus comprises several electrodes at different
angles. These electrodes are switched on and off for defined time periods that result in the
pole change of the electricity. This pole change leads to the realignment of the molecules,
and therewith to the further moving of them. How long these molecules can move through
the gel depends on the duration of the intervals.
The smaller a molecule is, the faster the relaxation of the conformational changing
molecule in the electric field. This means that smaller molecules realign and carry on
moving through the gel faster than larger molecules resulting in the separation appropriate
to the size of the molecules [Graw, 2005; University of Tromsø, Departement of
Microbiology and Virology, ― Pulsed field gel electrophoresis‖].


3.2.1 Preparation of agarose enclosing the DNA of the bacteria:
Bacteria colonies are inoculated in a BHI-medium and cultured overnight at 37° C.
Staphylococcus haemolyticus JCSC 1435 (51.81) is used as the reference strain.
In order to wash the culture, 0.7 ml of the overnight culture is transferred to an
eppendorftube and spinned at 7000 rpm for minutes. The supernatant is then removed, and
the pellet dissolved by adding 0.3 ml lysisbuffer. The cells are lysed by adding 4 μl
lysostaphin. The suspension has to be vortexed well and equilibrated at 50° C in a water
bath. A 2% ―Low Melting Point‖ (LMP) agarose is dissolved in lysisbuffer by boiling it.
The dissolved agarose is equilibrated at 50° C as well.
The DNA is embedded in agarose by mixing 300 μl of the cell suspension with 300 μl of
the dissolved agarose (this results in 1% agarose concentration). This mixture is transferred
to a plug moulds. It is important to work quickly and accurately because the warm agarose
is cooling down very quickly and solidly so that it becomes impossible to empty the pipet.
Try to avoid air bubbles inside the plugs.
The solidified plugs are transmitted into tubes comprising 2 ml lysisbuffer. In order to lyse
the plugs, they are inoculated overnight at 37° C.
3. Methods                                                                                   26


To wash the plugs, the lysisbuffer is removed carefully without destroying the susceptible
plugs, and then, 3 ml TE- buffer is added to the tube. And incubated for 1 hour at 55° C .
The TE-buffer is removed and 3 ml new TE-buffer is added to wash the plugs. This is done
two times with an interval of 15 minutes where the suspension is shaken slowly at room
temperature. The TE-buffer is replaced with 1ml new TE-buffer and placed at 4° C.


3.2.2 Restriction digestion of plugs:
2 mm of a plug are cut and transferred to an eppendorftube. To be sure to get a suitable
plug, it is prepared several pieces. 125 μl total restriction enzyme-mix containing 20 U
SmaI is added to the plugs and incubated with slow shaking at room temperature overnight.


3.2.3 Gel electrophoresis:
The plug piece is carefully transferred onto the comb. With the aid of a filter paper, the
fluid around the plug is removed. Elide the fields on the two outer edges where DNA
ladders are placed later.
A 1% agarose is dissolved in 100 ml 0.5x TBE-solution by boiling it. The dissolved
agarose is cooled down to 50°C. Then, the comb with the plugs is carefully placed so that
wells containing the plugs are formed inside the gel when the dissolved agarose is poured
into the casting stand. Two DNA ladders (Lambda Low Range) are placed inside the
unused wells on the outer edges. The gel solidifies for 30 minutes at room temperature.
Two litres of 0.5 x TBE are poured into the chamber of the PFGE-apparatus and the Chef
III, the cooling module, and the pump is turned on.
The run parameters are the following:


Switch time:           1-35s
Run time:              24h
Voltage gradient:      6V/cm
Angle:                 120°
Temperature:           12-14°C


The solidified agarose is placed in the frame of the chamber and the programm is started.
3. Methods                                                                                  27


3.2.4 Staining the gel:
The gel is transferred from the chamber to an ethidiumbromide solution in water
containing 50 μl ethidiumbromide and 500 ml MQ H2O. After 10 minutes, the gel is
transferred to 500 ml MQ H2O to destain it. 30 minutes later, the DNA can be visualized
by placing the gel on a UV transilluminater [University of Tromsø, Departement of
Microbiology and Virology, ―Pulsed field gel electrophoresis‖].


3.3 Modified Christensen method
To determine the biofilm capacity of the isolates of S. haemolyticus, a modified
Christensen method is used. The strains S. haemolyticus 51–03 and S. epidermidis 42–77
are used as a negative and a positive control respectively.
A single colony is inoculated in 5 ml TSB and cultured in a shaker at 37°C overnight. The
overnight culture is diluted 1:100 in TSB with 1% glucose.
150 μl of the overnight suspension is transferred to one column of a 96-well polystyrene
tissue culture plate. All in all, three columns of three different plates are inoculated.
It is also included a positive and a negative control. The assay is incubated for 24 hours at
37°C. The plates are washed by pouring away the liquid and washing with PBS. The wash
step is repeated three times. In order to fix the biofilm-forming bacteria, the plates are
incubated for 1 hour at 55°C. To stain the biofilm, 200 μl crystal violet (0.4 %) is added to
each well and incubated for 5 minutes. Then, the plates are washed by using tap water and
a Pasteur pipett.
200 μl ethanol/acetone (70/30) are added to each well to dissolve biofilm attached to the
walls of the wells. Afterward, the optical density is measured in an ELISA reader at 570
nm and single wavelength.
In the studies of Fredheim et al., bacteria were declared as ―biofilm positive‖ when they
had an optical density (OD) higher than 0.25 [University of Tromsø, Departement of
Microbiology and Virology, ―mikrobiologiske metoder; Modified Christensen method‖].


3.4 Observing the capacity of Biofilm formation by using confocal microscopy
A single colony is inoculated in 5 ml TSB and cultured in a shaker at 37°C overnight. The
strains that already show biofilm formation in the test tube above the liquid, are cultured
overnight on coverslips and chambered coverglass. Then, after having washed the biofilms
by removing the liquid and adding 200 μl PBS, the biofilms are stained with ―live-dead–
3. Methods                                                                                  28


staining‖ (3 μl; that is, 1.5 μl of each reagent is added to 1 ml suspension). The amount of
biofilm formation can be analysed by using confocal microscopy.
The biofilms are examined with Leica TCS SP5 confocal laser scanning microscope (Leica
Microsystems CMS Gmbh, Mannheim, Germany). To analyse and export the patterns, a
Leica LAS AF version 1.8.2 is employed. To identify SYTO9 (green channel), a 488 nm
line of the argon laser with a detection bandwidth of 495–515 nm is applied. For P1 (red
channel), a 561 nm line with a bandwidth of 615–660 nm is used. The fluorescent signals
are accumulated at 400Hz. To obtain images, a 63 x 1.2 NA HCX PL APO water
immersion is used [Flemming et al., 2009].


3.5 Epsilometer test (E-test)
The E-test is used to analyse the susceptibility to Oxacillin, Daptomycin, Fusidic acid and
Mupirocin of the strains. In order to make this analyse, bacterials are cultivated on blood
agar plates (BAP) and cultured for 24 hours at 37°C.
Single colonies are transferred to 2 ml 0.9% saltwater until the innoculate achieves 0.5
McFarland.
A small amount of the suspension is consistently placed on a Mueller Hinton agar plate by
using a cotton swab. Plates that are used to determine the susceptibilitiy of Oxacillin have
to contain additionally 2% NaCl.
The plate is left to dry for several minutes before application of the E-test.
The E-test strip is placed with forceps on the centre of the agar plate. Strips containing
Fusidic acid and Mupirocin are placed on the same agar plate, but in opposite directions.
When the strip has touched the agar, it must not be moved or lifted. The strip contains of a
gradient of antibiotic, and when it touches the agar, the antibiotic will diffuse into the agar
immediatley. Air bubbles are removed by carefully depressing the strip.
Small air bubbles will not disturb the result.
The susceptibility is determined after 24 hours. All antibiotics that are used are bactericide.
The MIC-value is found where complete repression of growth is observed, whereas single
colonies or microcolonies are no evidence for complete repression (Figure 10) [University
of Tromsø, Departement of Microbiology and Virology, ―Generell oppskrift for Etest‖,
―Stapylokokker-Etest‖, ―Staphylokokker-Etest for Oxacillin‖]
3. Methods                                                     29




Figure 10 reading of the MIC value (bactericide antibiotics)
[Tapia et al. 2003]