Differentiation of Streptococcus pneumoniae Conjunctivitis Outbreak

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					                                       Differentiation of Streptococcus
                                       pneumoniae Conjunctivitis Outbreak
                                       Isolates by Matrix-Assisted Laser
                                       Desorption Ionization-Time of Flight Mass
                                       Yulanda M. Williamson, Hercules Moura, Adrian R. Woolfitt,
                                       James L. Pirkle, John R. Barr, Maria Da Gloria Carvalho,
                                       Edwin P. Ades, George M. Carlone and Jacquelyn S.
                                       Appl. Environ. Microbiol. 2008, 74(19):5891. DOI:

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                                       Published Ahead of Print 15 August 2008.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2008, p. 5891–5897                                                                  Vol. 74, No. 19
0099-2240/08/$08.00 0 doi:10.1128/AEM.00791-08

   Differentiation of Streptococcus pneumoniae Conjunctivitis Outbreak
    Isolates by Matrix-Assisted Laser Desorption Ionization–Time of
                         Flight Mass Spectrometry
  Yulanda M. Williamson,1,2 Hercules Moura,2 Adrian R. Woolfitt,2 James L. Pirkle,2 John R. Barr,2*
    Maria Da Gloria Carvalho,1 Edwin P. Ades,1 George M. Carlone,1 and Jacquelyn S. Sampson1
           National Center for Immunizations and Respiratory Diseases1 and National Center for Environmental Health,2
                                Centers for Disease Control and Prevention, Atlanta, Georgia 30333
                                               Received 7 April 2008/Accepted 6 August 2008

             Streptococcus pneumoniae (pneumococcus [Pnc]) is a causative agent of many infectious diseases, including

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          pneumonia, septicemia, otitis media, and conjunctivitis. There have been documented conjunctivitis outbreaks
          in which nontypeable (NT), nonencapsulated Pnc has been identified as the etiological agent. The use of mass
          spectrometry to comparatively and differentially analyze protein and peptide profiles of whole-cell microor-
          ganisms remains somewhat uncharted. In this report, we discuss a comparative proteomic analysis between NT
          S. pneumoniae conjunctivitis outbreak strains (cPnc) and other known typeable or NT pneumococcal and
          streptococcal isolates (including Pnc TIGR4 and R6, Streptococcus oralis, Streptococcus mitis, Streptococcus
          pseudopneumoniae, and Streptococcus pyogenes) and nonstreptococcal isolates (including Escherichia coli, En-
          terococcus faecalis, and Staphylococcus aureus) as controls. cPnc cells and controls were grown to mid-log phase,
          harvested, and subsequently treated with a 10% trifluoroacetic acid–sinapinic acid matrix mixture. Protein and
          peptide fragments of the whole-cell bacterial isolate-matrix combinations ranging in size from 2 to 14 kDa were
          evaluated by matrix-assisted laser desorption ionization–time of flight mass spectrometry. Additionally Ran-
          dom Forest analytical tools and dendrogramic representations (Genesis) suggested similarities and clustered
          the isolates into distinct clonal groups, respectively. Also, a peak list of protein and peptide masses was
          obtained and compared to a known Pnc protein mass library, in which a peptide common and unique to cPnc
          isolates was tentatively identified. Information gained from this study will lead to the identification and
          validation of proteins that are commonly and exclusively expressed in cPnc strains which could potentially be
          used as a biomarker in the rapid diagnosis of pneumococcal conjunctivitis.

   Streptococcus pneumoniae (pneumococcus [Pnc]) is a facul-                  shire, New Jersey, and Maine. Martin et al. (15) and Carvalho et
tative anaerobic bacterium that is an important human patho-                  al. (3) reported microbiological, biochemical or genetic evi-
gen worldwide. The microorganism is a causative agent of                      dence that all of the Pnc strains from these outbreaks lacked a
many infections, including community-acquired pneumonia,                      detectable polysaccharide capsule. Lack of a capsule, as well as
meningitis, septicemia, bacteremia, otitis media, and conjunc-                the insensitivity of pneumococcal culture and diagnostic assays,
tivitis (8, 10, 17). Pnc contains many virulence factors, includ-             presents a challenge to correctly diagnose pneumococcal con-
ing a polysaccharide capsule that is antiphagocytic, enabling                 junctivitis.
the organism to avoid being engulfed and thus escape immune                      Molecular and immunological technologies (real-time PCR
detection. Based on capsular polysaccharides, 91 serotypes of                 and enzyme-linked immunosorbent assays) detecting expres-
Pnc are known. However, there are strains that do not react                   sion of Pnc genes or antibodies in bodily fluids have been used
with Pnc typing antisera and thus are nontypeable (NT) or                     with a limited degree of sensitivity for detection and diagnosis
nonencapsulated (3), although they meet the identification cri-                of pneumococcal disease (4, 23). However, advances in the
teria (optochin sensitivity, bile solubility, and being GenProbe              field of proteomics and bioinformatics have now made it pos-
positive) as being Pnc. Moreover, many NT strains are actually                sible to identify novel diagnostic targets or biomarkers aimed
just variants of normally encapsulated strains.                               at improved detection. These expressed-gene or protein tar-
   Pneumococcal conjunctivitis, an infection of the conjunctiva,              gets could prove useful in differentiating infectious strains that
is of significant public health concern in highly populated en-                have been associated with previous conjunctivitis outbreaks
vironments such as college campuses, nursing homes, and day                   and could reduce transmission of this infection.
care centers. Through the years, there have been large out-                      Mass spectrometry (MS), a rapid, powerful, and sensitive
breaks of conjunctivitis that have occurred in various regions of             analytical tool has been used recently for the differentiation,
the United States, including New York, California, New Hamp-                  identification, and characterization of microbial pathogens. In
                                                                              particular, MS techniques such as matrix-assisted laser desorp-
                                                                              tion ionization–time of flight (MALDI-TOF) MS have been
   * Corresponding author. Mailing address: Centers for Disease Con-          used to analyze whole bacterial cells that have not been mod-
trol and Prevention, 4770 Buford Highway, Building 110, MS-F50,
Chamblee, GA 30341. Phone: (770) 488-7848. Fax: (770) 488-0509.
                                                                              ified chemically or by mechanical disruption (6). In recent
E-mail:                                                        years, MALDI-TOF MS has been used to differentiate signif-
     Published ahead of print on 15 August 2008.                              icant human pathogens such as Helicobacter pylori, Bacillus

5892       WILLIAMSON ET AL.                                                                                                            APPL. ENVIRON. MICROBIOL.

cereus, Escherichia coli, and Coxiella burnetii (1, 6, 9, 11–14, 16,                               TABLE 1. Bacterial strains used in this study
20, 21, 24, 25). Studies by Friedrich and colleagues employed                                       Straina                                      Source
MALDI-TOF MS for rapid identification of 10 different spe-
                                                                                     Sp   165  (1138-80)............................1980, New York conjunctivitis, NT
cies of viridans streptococci (7). Additionally, the MALDI                           Sp   166  (1139-80)............................1980, New York conjunctivitis, NT
technology has been used to identify Mycobacterium and more-                         Sp   168  (61-81)................................1981, California conjunctivitis, NT
over distinguish between multiple strains within a species (18).                     Sp   169  (62-81)................................1981, California conjunctivitis, NT
By use of high-throughput measures such as MALDI-TOF,                                Sp   170  (63-81)................................1981, California conjunctivitis, NT
                                                                                     Sp   245  (1852-02)............................2002, New Hampshire
protein/peptide fingerprints can be generated based on a pro-
                                                                                                                                               conjunctivitis, NT
teomic profile. These proteins or patterns could serve as                             Sp 246 (1853-02)............................2002, New Hampshire
uniquely expressed pathogen-specific peptide or protein bio-                                                                                    conjunctivitis, NT
markers that may prove useful for diagnostic purposes.                               Sp 247 (2136-02)............................2002, New Jersey conjunctivitis, NT
   In this report, we describe a differential proteomic analysis                     Sp 248 (2136-02)............................2002, New Jersey conjunctivitis, NT
                                                                                     Sp 263 (71-03)................................2003, Maine conjunctivitis, NT
using MALDI-TOF MS of representative Pnc conjunctival                                Sp 264 (72-03)................................2003, Maine conjunctivitis, NT
(cPnc) U.S. outbreak isolates. The unique cPnc outbreak iso-                         Sp 265 (73-03)................................2003, Maine conjunctivitis, NT
lates were compared with other nonconjunctival, pneumococ-                           Sp 266 (74-03)................................2003, Maine conjunctivitis, NT

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cal and streptococcal isolates and a limited number of non-                          R6 (BAA-228) ...............................Derivative of D39, nonencapsulated
                                                                                     TIGR4 (BAA-344) ........................Encapsulated, serotype 4
streptococcal strains and species. Additionally, statistical
                                                                                     (M) ..................................................S. mitis
algorithms as well as traditional cluster analysis were used to                      (3) SS1246/NCTC 10712...............S. mitis
identify similarities among these isolates, in particular the cPnc                   (35) 1165/Mitis 26 .........................S. mitis
isolates. A list of peptides/proteins found among the isolates                       (40) SS1059/JC67...........................S. mitis
was compiled in which at least one peptide/protein was com-                          (67) SS1303/NCTC 12261.............S. mitis
                                                                                     (O)...................................................S. oralis
mon and exclusively expressed in the cPnc isolates. These cPnc                       (6) SS1236/ATCC 35037...............S. oralis
proteomic signatures or biomarkers could ultimately be useful                        (7) SS900/ATCC 15914.................S. oralis
in the diagnosis of this infection.                                                  (21) SS911/ATCC 10557...............S. oralis
                                                                                     Sp 83 ...............................................Pnc serotype 4, encapsulated
                                                                                                                                               7-valent vaccine
                        MATERIALS AND METHODS
                                                                                     Sp 86 ...............................................Pnc serotype 6B, encapsulated
   Materials and reagents. All chemicals used during this study were purchased                                                                 7-valent vaccine
from Sigma-Aldrich (St. Louis, MO), except where indicated. Culture medium           Sp 95 ...............................................Pnc serotype 9V, encapsulated
(Todd-Hewitt broth) was obtained from the Scientific Resources Program at the                                                                   7-valent vaccine
Centers for Disease Control and Prevention (CDC).                                    Sp 105 .............................................Pnc serotype 14, encapsulated
   Bacterial strains. All strains were from the CDC Streptococcus Reference                                                                    7-valent vaccine
Laboratory. Study strains consisted of 13 cPnc outbreak isolates as well as          Sp 116 .............................................Pnc serotype 18C, encapsulated
controls Streptococcus pneumoniae serotype 4, Pnc TIGR4, and Streptococcus                                                                     7-valent vaccine
pneumoniae unencapsulated strain R6; other streptococcal species, including          Sp 117 .............................................Pnc serotype 19F, encapsulated
Streptococcus oralis, Streptococcus mitis, Streptococcus pseudopneumoniae, and                                                                 7-valent vaccine
Streptococcus pyogenes (group A); and strains from heterologous genera Esche-        Sp 125 .............................................Pnc serotype, 23F, encapsulated
richia coli (group B), Staphylococcus aureus (group C), and Enterococcus faecalis                                                              7-valent vaccine
(group D). In addition, pneumococcal serotypes contained within the 7-valent         (P)....................................................S. pseudopneumoniae
pneumococcal conjugate vaccine and NT pneumococcal sterile-site isolates were        ATCC BAA-960 (65)....................CDC-RC, S. pseudopneumoniae
also used in the study for comparison (Table 1). The controls used in the study      290-03 (72) .....................................CDC-RC, S. pseudopneumoniae
were not associated with the conjunctivitis outbreaks and were used to validate      288-03 (74) .....................................CDC-RC, S. pseudopneumoniae
the methods’ abilities to differentiate at the species and genus level. Groups A,    276-03 (77) .....................................CDC-RC, S. pseudopneumoniae
B, C, and D were included as outgroups for statistical purposes. The 13 cPnc         253-03 (83) .....................................CDC-RC, S. pseudopneumoniae
isolates described in this study are a limited sampling population and are con-      844-00..............................................Sterile site (blood), NT
sidered representatives of all the clinical conjunctival isolates from the afore-    5094-02............................................Sterile site (blood or cerebrospinal
mentioned U.S. outbreaks (New York in 1980, California in 1981, New Hamp-                                                                      fluid), NT
shire in 2002, New Jersey in 2002, and Maine in 2003).                               6024-01............................................Sterile site (blood or cerebrospinal
   Bacterial cell growth and harvest for MS analysis. Bacterial isolates stored at                                                             fluid), NT
   70°C were initially streaked on Trypticase soy agar (BBL, Becton Dickinson,       7232-99............................................Sterile site (blood or cerebrospinal
Franklin Lakes, NJ) with 5% defibrinated sheep’s blood plates and incubated                                                                     fluid), NT
overnight at 37°C with 5% CO2. After confluent growth, a full loop of bacteria        Streptococcus pyogenes...................Gram-positive, capsulated,
was inoculated in 10 ml of Todd-Hewitt broth (with 5% yeast extract) and grown                                                                 respiratory pathogen
to mid-log phase (optical density at 420 nm [OD420] of 0.4) at 37°C with 5%          Escherichia coli...............................Gram-negative intestinal pathogen
CO2 for 4 to 5 h. The bacterial suspension was centrifuged at 4,600 g for 10 min     Enterococcus faecalis .....................Gram-positive intestinal pathogen
at 4°C. The supernatant was decanted, and the pellet was washed twice in sterile     Staphylococcus aureus....................Gram-positive human pathogen
distilled water, followed by centrifugation at 10,000 g at room temperature for        a
10 min. The pellet ( 1012 cells) was resuspended in 50 l of water, aliquoted (2          The “SS” designations and the numbers and letters in parentheses are strain
                                                                                     identity codes from the CDC catalog for Streptococcus.
  l) in microcentrifuge tubes, and stored at 70°C until further use. To ensure
purity among the isolates, the resuspended bacterial inoculum was streaked on a
Trypticase soy agar blood plate and incubated overnight at 37°C with 5% CO2.
All strains were cultured and grown three separate times over a 3-day period.        well stainless steel MALDI target plate (Applied Biosystems [AB], Framingham,
The strains were grown to the same OD (mid-log phase at OD420 of 0.4) to             MA) was used in the study. The plates were washed with Milli-Q-grade water,
ensure consistency in growth.                                                        treated with methanol, and allowed to dry at room temperature. When dry, 0.5
   Preparing bacterial cell suspensions for MALDI-TOF analysis. The MALDI              l of premixed suspensions containing matrices and whole bacterial forms or
matrix consisted of saturated solutions (20 mg/ml) of 3,5-dimethoxy-4-hydroxy-       mass standards for calibration (Sequazyme peptide mass standards kit; AB) were
cinnaminic acid (sinapinic acid [SA]) (Sigma-Aldrich). SA was mixed with 50%         spotted in four separate wells to create quadruplicates of samples and controls.
acetonitrile and Milli-Q-grade water containing 10% trifluoroacetic acid. A 192-      In addition, 0.5 l of bovine cytochrome c (1 mM) was added to one well of each
VOL. 74, 2008                                                    MALDI-TOF MS ANALYSIS OF PNEUMOCOCCAL CONJUNCTIVITIS                                5893

sample and used as an internal standard. After air drying, the plates were                                       RESULTS
inserted into the instrument for MALDI-TOF MS analysis.
   MALDI-TOF MS analysis. Mass spectra were acquired using a MALDI-TOF/                 MALDI-TOF MS spectra of cPnc isolates. MALDI-TOF
TOF mass spectrometer (AB 4700 Proteomics Analyzer) equipped with a nitro-           MS fingerprinting revealed similarities among representative
gen laser (Nd:YAG) at 337 nm and a 200-Hz repetition rate. Analyses were
                                                                                     U.S. cPnc outbreak isolates. Summed, smoothed, and normal-
performed at least 3 different days in linear delayed-extraction positive-ion mode
at an accelerating voltage of 20 kV. The instrument was calibrated and checked       ized MALDI-TOF MS spectra from bacterial samples grown
before analysis with several calibration mixtures from either the peptide mass       on three separate occasions revealed that the outbreaks share
standards kit or the 4700 standard kit (AB), depending on the analysis mass          commonalities within the 2- to 14-kDa mass range. In partic-
range. Mass accuracy for each standard was within 0.05% of the corresponding
                                                                                     ular, 11 major ion signals were observed in the region between
average molecular weight. After initial manual laser intensity optimization and
baseline data acquisition, spectra were acquired in automatic control mode,          4,000 and 10,000 Da, including a peak at m/z 4,425 (Fig. 1). In
using uniform parameters to improve consistency and reproducibility. For opti-       this mass range, it is reasonable to assume that almost all
mum data quality of mass spectra in the m/z range of 2,000 to 14,000, SA was         signals originate from small proteins, and as is typical for
used as the matrix. The instrument was programmed to examine signals from at         MALDI-TOF spectra that in the absence of evidence to the
least 12 to a maximum of 100 randomly positioned nonoverlapping locations in
each sample well, and the signals from the first 10 acquisitions for each spot that
                                                                                     contrary, these are singly charged ([M H] forms). Among
met the acceptance criteria were accumulated into one final-profile mass spec-         the cPnc outbreak isolates themselves, there were also minor

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trum. A minimum of 11 individual spectra representing 10 accumulated subspec-        differences in which several of the isolates, including NH Sp
tra were obtained from each well. The acceptance criteria, based on 1,000 laser      246 (Fig. 2), lacked some protein peaks. Moreover, there are
shots per spot, were signal intensities between 2,000 and 55,000 counts and a
                                                                                     also visual differences among spectra found in conjunctival
signal/noise ratio of 10 or greater.
   Data processing. Mass spectra from three harvestings were processed in the        outbreak isolates that are not observed in the controls Pnc
following manner. Spectral data were exported as text format m/z-intensity           TIGR4 and R6. As would be expected, the E. coli, S. aureus, E.
lists with a unified m/z scale, using custom Microsoft Visual Basic for Appli-        faecalis, and S. pyogenes isolates are very different (Fig. 1).
cations (VBA) macros in Data Explorer, the AB viewing application. The text
                                                                                        A peak ion list (Table 2) of 16 peak masses, but not inclusive
data were further processed and viewed by use of a suite of custom Microsoft
Visual Basic .NET (VB.NET) programs. One custom program, MultiSpec                   of all 487 separate protein and peptide masses, generated from
Viewer, was designed to display hundreds of spectra at once in a number of           a manual visual peak comparison was obtained from all 45
formats, including a simulated gel view for visual analysis of the data set,         isolates and compared to a UniProt Pnc protein mass library.
which comprised several thousand individual spectra. Spectra failing to meet         Two percent (9/487) of the data queried resulted in similarities
the quality requirements (usually containing no recognizable peaks due to
failures of the automatic acquisition algorithms [approximately 10% of the
                                                                                     to known ribosomal proteins. A ribosomal spectrum overlay,
total]) were discarded. The remaining spectra were subjected to background           from the same Pnc database, using the MultiSpec Viewer, also
subtraction and then were summed by MALDI by well or by organism (to give            suggested the tentative identification of ribosomal proteins
   12 spectra or 1 representative high-quality spectrum, respectively); normal-      among the conjunctival outbreak isolates as well as among Pnc
ized to the base peak; smoothed using a 21-point, 2-pass Gaussian algorithm;
                                                                                     TIGR4 and R6. The overlay constituted 11 ion peaks, within
and finally standardized and denoised using a custom Fortran program (22).
The output of the standardizing and denoising programs was a set of profile           the mass range of 4,000 to 8,000 Da (Fig. 2).
spectra containing relative intensities of only the statistically significant peaks      Cluster analysis of cPnc outbreak isolates. The hierarchal
(22), with zeros at all other m/z values. Thus, these data sets were in an ideal     cluster analysis using the PAST program with a Jaccard simi-
format for further analysis by a range of commercial statistical and data-
                                                                                     larity coefficient indicated that 12 of the 13 conjunctival iso-
mining applications. To decrease the time required for statistical analyses, the
summed spectra were typically compressed by a factor of 20, reducing                 lates are clustered together and share 76 to 86% similarity
   18,000 points to 900 for a typical m/z 2,000 to 14,000 spectrum. We used          (Fig. 3), while cPnc NH Sp 245 exhibited only 70% similarity
PAST software v1.34 ( for hierar-          with respect to the other conjunctival isolates. In addition, the
chical cluster analysis, with the single summed spectra (one summed spec-            cPnc isolates displayed 58, 58, 45, and 45% similarity to Pnc
trum representing each organism) for input. We used a Fortran program,
Random Forest (RF) v 5.1 (2;
                                                                                     R6, Pnc TIGR4, NT Pnc sterile condition-isolated strains, and
/RandomForests/cc_home.htm) for classification and identification, in this             Pnc vaccine serotype strains, respectively. The dendrogram
case with 9 summed spectra from three harvestings of each organism as a              suggests that the conjunctival isolates are distantly related to S.
training set and 3 separate summed spectra as unknowns. Recompiling the              mitis, S. oralis, and S. pseudopneumoniae (45 to 48%), and
Fortran RF code for each experimental condition was automatically driven by
VB.NET programs, and custom viewing applications were developed to aid in
                                                                                     there was little relationship to S. aureus, E. coli, E. faecalis, and
interpreting the RF results.                                                         S. pyogenes (10 to 12%) (Fig. 3).
   Tentative peak matching and database searching. A tentative identification of         RF analysis of cPnc isolates. RF, a statistical algorithm that
prominent peaks was done using the Tag-ident proteomics tool or ExPASy               computes proximities between data sets, locates outliers, and
sequence retrieval system ( In addition, “MS DB Filter,” a
                                                                                     computes error rates by bootstrapping (2), was performed.
custom VB.NET algorithm, was used to construct a CDC-modified database
filtered from UniProt ( MS DB Filter         Initially, a total of 900 spectra from the 45 isolates or classes
excludes any Swiss-Prot and TrEMBL or UniProt entry described as a fragment,         were analyzed, with an overall error rate of 8.33%. Outlier and
strips out signal and prepeptide sequences, and applies a rule to add or remove      misclassified spectra were then identified by RF by running the
initial methionine as described by Pineda (19). The CDC-modified filtered da-          analysis 200 times using subsets of randomly selected spectra
tabase was used for data mining the deduced proteome from several bacterial
species used in this study which have had the whole genome sequenced. As of
                                                                                     (68% of each class); this number of repeats was chosen so as to
April 2008, information for TIGR4 and R6 species/isolates used in this study         give reliable statistics on each spectrum. Outliers (with an RF
could be found in the Swiss-Prot and TrEMBL databases (UniProt). Custom              outlier distance of 5 or above) and consistently misclassified
algorithms within MultiSpec Viewer were also used to generate peak lists from        spectra (incorrect identification rate of 25% or above) were
the acquired mass spectra. In addition a manual screen of an extensive Microsoft
Excel spreadsheet consisting of the 45 isolates from 2 to 14 kDa was used to
                                                                                     excluded, and the randomized RF analysis was repeated with
correlate generated peaks with the CDC-modified database in order to provide          the new data set a total of three times. A total of 125 spectra
tentative protein identifications.                                                    were excluded, and the overall error rate was reduced to 3.18%
5894      WILLIAMSON ET AL.                                                                                             APPL. ENVIRON. MICROBIOL.

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   FIG. 1. Differentiation of cPnc outbreak isolates and nonconjunctival bacterial controls by MALDI MS. The mass spectrum (A) and simulated-
gel (B) views were prepared using a custom program, MultiSpec Viewer. The peak masses (2,000 to 14,000) in the spectrum and simulated-gel views
are represented as m/z, and the relative intensity (0 to 100 [white to blue]) is expressed as a percentage. The three distinct colored lines along the
right y axis are illustrated to easily distinguish the three main groups in the study (cPnc isolates, red; pneumococcal and streptococcal control
isolates, green; control isolates for heterologous genera, blue). Lanes 1 to 13, cPnc outbreak isolates Sp 165, Sp 166, Sp 168, Sp 169, Sp 170, Sp
245, Sp 246, Sp 247, Sp 248, Sp 263, Sp 264, Sp 265, and Sp 266, respectively. Lanes 14 to 22, Pnc TIGR4, Pnc R6, S. mitis, S. oralis, S.
pseudopneumoniae, E. coli, S. pyogenes, S. aureus, and E. faecalis, respectively. Each trace is the sum of all individual spectra (typically 10 to 20)
for that organism, after background subtraction and smoothing.

among the individual classes. In essence, the RF clusters the                whole-organism MS fingerprinting coupled with high-perfor-
conjunctival isolates and controls into distinct clonal groups.              mance statistical algorithm is a promising tool capable of dis-
                                                                             tinguishing unique and sample-limited NT cPnc outbreak
                                                                             strains from other pneumococcal, streptococcal, and nonstrep-
                                                                             tococcal species.
   Pneumococcal conjunctivitis, usually a self-limiting infection               Previous studies using molecular techniques, such as pulse-
of the ocular mucosal surface, poses serious public health con-              field gel electrophoresis, multilocus sequence tagging, and
sequences if not diagnosed early. The ease with which the                    PCR, have revealed that the cPnc isolates are similar genotyp-
infection spreads among individuals warrants the need for                    ically (3, 15). Using MS, proteins are the most characteristic
more rapid and improved detection methodologies. The sim-                    macromolecule that can be assessed without extraction, sepa-
plicity and feasibility of generating mass spectra from whole-               ration, or amplification (6), as required by the aforementioned
cell bacteria, the reproducibility of the sample preparation,                technologies. In this proteomic study, albeit confirmatory with
and the ability to differentiate among genera, species, and                  previous genetics-based investigations (3, 15), MALDI-TOF
strains makes MALDI-TOF MS a powerful methodology to be                      MS analysis as evident by visual spectrum analyses and hier-
applied to the field of clinical diagnostics. MALDI-TOF                       archal cluster analysis also demonstrated that the cPnc out-
VOL. 74, 2008                                                       MALDI-TOF MS ANALYSIS OF PNEUMOCOCCAL CONJUNCTIVITIS                            5895

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   FIG. 2. Strain differentiation among cPnc isolates and identification of tentative ribosomal proteins present in cPnc isolates by MALDI MS.
The spectrum view was prepared using a custom program, MultiSpec Viewer. The peak masses (2,000 to 14,000) in the spectrum are represented
as m/z, and the relative intensity (0 to 100) is expressed as a percentage. Black arrows indicate the absence of ion peaks in isolate Sp 246. In
addition, an overlay representing ribosomal proteins, obtained from a UniProt Pnc protein mass library, is illustrated (orange lines). Lanes 1 to
8, cPnc outbreak isolates Sp 165, Sp 169, Sp 170, Sp 246, Sp 247, Sp 248, Sp 263, and Sp 265, respectively. Each trace is the sum of all individual
spectra (typically 10 to 20) for that organism, after smoothing.

                                                                                       break isolates are very similar. The conjunctival isolate clus-
                                                                                       tering is a reflection of unique strain characteristics of cPnc
                                                                                       within the subset of proteins being examined in this study.
TABLE 2. Tentative peak list (representatives) of conjunctival and                     Moreover, uniquely expressed genes that are identified will
                  nonconjunctival isolatesa
                                                                                       make ideal candidates for biomarker evaluation.
                                  Observed                                                Additionally, RF was able to separate the strains in this
         Strain(s)                mass (Da             Putative protein or peptide
                                approximate )b
                                                                                       study into groups at the genus, species, and, to a certain extent,
                                                                                       strain level (Sp 246) with minimal error. The low error rate of
Sp 165, Sp 166, Sp 168,              2,424         ?                                   3.13% among the cPnc isolates indicates that the RF algorithm
  Sp 169, Sp 170
Sp 169, Sp 170                       2,610         ?
                                                                                       is able to correctly identify and categorize mass spectra to the
Sp 165, Sp 166, Sp 168,          2,943–2,945       ?                                   given appropriate class (individual strains or isolates) or group
  Sp 169, Sp 170, Sp                                                                   (similar strains, i.e., specific cPnc outbreaks). The spectra that
  245, Sp 246, Sp 247,                                                                 are consistently being misclassified after successive screenings
  Sp 248, Sp 263, Sp                                                                   resulting in error rates may be due to low-quality spectra that
  264, Sp 265, Sp 266
Sp 166, Sp 168                       3,465         ?                                   were not filtered appropriately. Interestingly, from a biological
TIGR4                                4,003         ?                                   perspective, error rates may not necessarily be a negative. In
TIGR4                                4,218         ?                                   our case, mismatched spectra which resulted in low error rates
R6                                   4,741         ?                                   can simply imply that the cPnc isolates are biologically related
TIGR4, R6                        5,481–5483        50S ribosomal protein L33
Sp 245, Sp 247, Sp 263,          5,495–5,499       ?
                                                                                       and are too similar for the algorithm to distinguish.
  Sp 264, Sp 266                                                                          MALDI-TOF MS is a tool with great promise for the med-
TIGR4                                6,276         Ribosomal protein L30               ical, public health, and scientific communities. Mass spectral
R6                                   6,648         Ribosomal protein L32               fingerprinting using MALDI-MS has been used to detect bio-
Sp 165, Sp 166, Sp 168,          6,872–6,875       30S ribosomal protein S21           markers from whole unfractionated microorganisms, including
  Sp 169, Sp 170, Sp
  245, Sp 246, Sp 247,                                                                 viruses, prokaryotes, and a few unicellular eukaryotes (1, 6, 9,
  Sp 248, Sp 263, Sp                                                                   11–14, 16, 20, 21, 24, 25). These biomarkers have proven useful
  264, Sp 265, Sp 266                                                                  for rapidly identifying and differentiating microbial pathogens.
TIGR4, R6                            6,877         30S    ribosomal   protein   S21    For instance, small acid-soluble proteins have been used to
R6                                   7,998         50S    ribosomal   protein   L29
TIGR4                               10,414         30S    ribosomal   protein   S15
                                                                                       characterize Bacillus species (5). Additionally, Shaw et al. re-
Sp 165, Sp 248, Sp 265              11,001         50S    ribosomal   protein   L24    ported the identification of biomarkers in unfractionated C.
                                                                                       burnetii cells phase I purified from embryonic egg yolk sac
    Boldface indicates that the results are unique in all cPnc outbreak isolates.
    Observed masses are derived from peak tops of unresolved isotopic clusters 1 Da,   preparations (24). Furthermore, spectral markers in the mass
assuming all ions were M H .                                                           range of 2,000 to 8,000 Da were obtained from MALDI-TOF
5896     WILLIAMSON ET AL.                                                                                          APPL. ENVIRON. MICROBIOL.

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   FIG. 3. Hierarchal cluster analysis of cPnc outbreak isolates and nonconjunctival bacterial controls. The PAST program, using the Jaccard
similarity coefficient (expressed as a percentage), was used to assess the relatedness of the cPnc outbreak isolates and controls. A dendrogram of
cPnc outbreak isolates compared with pneumococcal, streptococcal, and nonstreptococcal species is presented. Input data had been summed (all
spectra for each organism), background subtracted, smoothed, standardized, and denoised. Shown are results for cPnc outbreak isolates (group
1), S. mitis (group 2), S. oralis (group 3), S. pseudopneumoniae (S. pseudopn. [group 4]), Pnc R6 and TIGR4 (group 5), Pnc sterile-site isolated
strains (group 6), and Pnc 7-valent vaccine serotypes (group 7) and heterologous genera, including, E. coli, S. pyogenes (SMIC), S. aureus,
and E. faecalis (group 8).

MS analysis of four human microsporidian isolates (16). Bio-               fied, one of which was common and exclusively expressed in
markers for Mycobacterium species have also been detected by               cPnc isolates. These cPnc proteomic signatures or biomarker
MALDI primarily in the 500- to 2,000-Da range, most likely                 candidates could ultimately be fruitful in the diagnosis of this
representing lipid molecules or small polypeptides (18).                   infection. These expressed biomarkers are advantageous com-
   Protein biomarkers identified by MALDI-TOF MS are often                  pared to genetic markers that would provide only information
basic, such as the highly conserved and abundant ribosomal                 based on their expressive potential. Conjunctival isolate pro-
protein families (19). In the present study, several ribosomal             tein biomarkers would be a true indication of the organisms’
proteins, as illustrated in Fig. 2, were tentatively identified in          ability to cause disease. Moreover, MALDI-TOF MS, with its
the range of 2,000 to 14,000 Da by database searching and                  high sensitivity, may also prove useful in gaining insight into
spectrum overlay. The tentative proteins appeared to be con-               the pathogenic mechanisms of disease, in particular mecha-
served, based on mass, among the cPnc isolates as well as in               nisms by which these NT cPnc strains cause large sporadic
other penumococcal strains. In addition, there was a peak at               outbreaks. For instance, cPnc surface proteins associated with
m/z 2,944 that was common to and uniquely expressed in the                 adherence or attachment to host cells that would subsequently
cPnc isolates relative to other strains tested. This biomarker             initiate infection could be used as biomarkers. Furthermore,
candidate will require amino acid sequencing for validation as             understanding how and why these cPnc strains cause disease
a clinical diagnostic marker.                                              can aid in the development of better treatments and even
   In conclusion, MALDI-TOF MS, a rapid and sensitive meth-                prophylactic measures to minimize the spread of infection dur-
odology, was successfully utilized for differentiating cPnc U.S.           ing future outbreaks.
outbreak isolates. Through statistical algorithms and hierar-
chal clustering, it was demonstrated that the cPnc outbreak
isolates from California and the northeastern United States                                      ACKNOWLEDGMENTS
are very similar. Based on their MALDI-TOF MS fingerprints,                  This work was supported in part by an Emerging Infectious Diseases
putative peptide/protein biomarkers were tentatively identi-               Research Fellowship sponsored by the Association of Public Health
VOL. 74, 2008                                                      MALDI-TOF MS ANALYSIS OF PNEUMOCOCCAL CONJUNCTIVITIS                                               5897

Laboratories and the National Center for Infectious Diseases at the                          K. L. Wahl. 2000. Extracting and visualizing matrix-assisted laser desorption/
Centers for Disease Control and Prevention.                                                  ionization time–of-flight mass spectral fingerprints. Rapid Commun. Mass
  We thank Rickard Facklam for insight.                                                      Spectrom. 13:1586–1594.
  The findings and conclusions in this report are those of the authors                  13.   Krader, P., and D. Emerson. 2004. Identification of archaea and some
                                                                                             extremophilic bacteria using matrix-assisted laser desorption/ionization
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                                                                                             time-of-flight (MALDI-TOF) mass spectrometry. Extremophiles 8:259–268.
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