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
Spectrometry
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.
Sampson
Appl. Environ. Microbiol. 2008, 74(19):5891. DOI:
10.1128/AEM.00791-08.

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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: jbarr@cdc.gov. 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

5891
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 (http://folk.uio.no/ohammer/past/doc1.html) 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; http://www.stat.berkeley.edu/users/breiman
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 (http://us.expasy.org). 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 (http://www.ebi.ac.uk/uniprot/index.html). 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-
DISCUSSION
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.
a
burnetii cells phase I puriﬁed from embryonic egg yolk sac
Boldface indicates that the results are unique in all cPnc outbreak isolates.
b
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.
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