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2.  Draft Genome Sequences of 21 Salmonella enterica Serovar Enteritidis Strains 
Journal of Bacteriology  2012;194(21):5994-5995.
Salmonella enterica subsp. enterica serovar Enteritidis is a common food-borne pathogen, often associated with shell eggs and poultry. Here, we report draft genomes of 21 S. Enteritidis strains associated with or related to the U.S.-wide 2010 shell egg recall. Eleven of these genomes were from environmental isolates associated with the egg outbreak, and 10 were reference isolates from previous years, unrelated to the outbreak. The whole-genome sequence data for these 21 human pathogen strains are being released in conjunction with the newly formed 100K Genome Project.
doi:10.1128/JB.01289-12
PMCID: PMC3486122  PMID: 23045502
3.  Fully Assembled Genome Sequence for Salmonella enterica subsp. enterica Serovar Javiana CFSAN001992 
Genome Announcements  2013;1(2):e00081-13.
We report a closed genome of Salmonella enterica subsp. enterica serovar Javiana (S. Javiana). This serotype is a common food-borne pathogen and is often associated with fresh-cut produce. Complete (finished) genome assemblies will support pilot studies testing the utility of next-generation sequencing (NGS) technologies in public health laboratories.
doi:10.1128/genomeA.00081-13
PMCID: PMC3622996  PMID: 23516208
4.  On the Evolutionary History, Population Genetics and Diversity among Isolates of Salmonella Enteritidis PFGE Pattern JEGX01.0004 
PLoS ONE  2013;8(1):e55254.
Facile laboratory tools are needed to augment identification in contamination events to trace the contamination back to the source (traceback) of Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis). Understanding the evolution and diversity within and among outbreak strains is the first step towards this goal. To this end, we collected 106 new S. Enteriditis isolates within S. Enteriditis Pulsed-Field Gel Electrophoresis (PFGE) pattern JEGX01.0004 and close relatives, and determined their genome sequences. Sources for these isolates spanned food, clinical and environmental farm sources collected during the 2010 S. Enteritidis shell egg outbreak in the United States along with closely related serovars, S. Dublin, S. Gallinarum biovar Pullorum and S. Gallinarum. Despite the highly homogeneous structure of this population, S. Enteritidis isolates examined in this study revealed thousands of SNP differences and numerous variable genes (n = 366). Twenty-one of these genes from the lineages leading to outbreak-associated samples had nonsynonymous (causing amino acid changes) changes and five genes are putatively involved in known Salmonella virulence pathways. While chromosome synteny and genome organization appeared to be stable among these isolates, genome size differences were observed due to variation in the presence or absence of several phages and plasmids, including phage RE-2010, phage P125109, plasmid pSEEE3072_19 (similar to pSENV), plasmid pOU1114 and two newly observed mobile plasmid elements pSEEE1729_15 and pSEEE0956_35. These differences produced modifications to the assembled bases for these draft genomes in the size range of approximately 4.6 to 4.8 mbp, with S. Dublin being larger (∼4.9 mbp) and S. Gallinarum smaller (4.55 mbp) when compared to S. Enteritidis. Finally, we identified variable S. Enteritidis genes associated with virulence pathways that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future outbreaks involving S. Enteritidis PFGE pattern JEGX01.0004.
doi:10.1371/journal.pone.0055254
PMCID: PMC3559427  PMID: 23383127
5.  Prediction System for Rapid Identification of Salmonella Serotypes Based on Pulsed-Field Gel Electrophoresis Fingerprints 
Journal of Clinical Microbiology  2012;50(5):1524-1532.
A classification model is presented for rapid identification of Salmonella serotypes based on pulsed-field gel electrophoresis (PFGE) fingerprints. The classification model was developed using random forest and support vector machine algorithms and was then applied to a database of 45,923 PFGE patterns, randomly selected from all submissions to CDC PulseNet from 2005 to 2010. The patterns selected included the top 20 most frequent serotypes and 12 less frequent serotypes from various sources. The prediction accuracies for the 32 serotypes ranged from 68.8% to 99.9%, with an overall accuracy of 96.0% for the random forest classification, and ranged from 67.8% to 100.0%, with an overall accuracy of 96.1% for the support vector machine classification. The prediction system improves reliability and accuracy and provides a new tool for early and fast screening and source tracking of outbreak isolates. It is especially useful to get serotype information before the conventional methods are done. Additionally, this system also works well for isolates that are serotyped as “unknown” by conventional methods, and it is useful for a laboratory where standard serotyping is not available.
doi:10.1128/JCM.00111-12
PMCID: PMC3347130  PMID: 22378901
6.  High resolution clustering of Salmonella enterica serovar Montevideo strains using a next-generation sequencing approach 
BMC Genomics  2012;13:32.
Background
Next-Generation Sequencing (NGS) is increasingly being used as a molecular epidemiologic tool for discerning ancestry and traceback of the most complicated, difficult to resolve bacterial pathogens. Making a linkage between possible food sources and clinical isolates requires distinguishing the suspected pathogen from an environmental background and placing the variation observed into the wider context of variation occurring within a serovar and among other closely related foodborne pathogens. Equally important is the need to validate these high resolution molecular tools for use in molecular epidemiologic traceback. Such efforts include the examination of strain cluster stability as well as the cumulative genetic effects of sub-culturing on these clusters. Numerous isolates of S. Montevideo were shot-gun sequenced including diverse lineage representatives as well as numerous replicate clones to determine how much variability is due to bias, sequencing error, and or the culturing of isolates. All new draft genomes were compared to 34 S. Montevideo isolates previously published during an NGS-based molecular epidemiological case study.
Results
Intraserovar lineages of S. Montevideo differ by thousands of SNPs, that are only slightly less than the number of SNPs observed between S. Montevideo and other distinct serovars. Much less variability was discovered within an individual S. Montevideo clade implicated in a recent foodborne outbreak as well as among individual NGS replicates. These findings were similar to previous reports documenting homopolymeric and deletion error rates with the Roche 454 GS Titanium technology. In no case, however, did variability associated with sequencing methods or sample preparations create inconsistencies with our current phylogenetic results or the subsequent molecular epidemiological evidence gleaned from these data.
Conclusions
Implementation of a validated pipeline for NGS data acquisition and analysis provides highly reproducible results that are stable and predictable for molecular epidemiological applications. When draft genomes are collected at 15×-20× coverage and passed through a quality filter as part of a data analysis pipeline, including sub-passaged replicates defined by a few SNPs, they can be accurately placed in a phylogenetic context. This reproducibility applies to all levels within and between serovars of Salmonella suggesting that investigators using these methods can have confidence in their conclusions.
doi:10.1186/1471-2164-13-32
PMCID: PMC3368722  PMID: 22260654
7.  Virulence Characterization of Shiga-Toxigenic Escherichia coli Isolates from Wholesale Produce▿  
The 13 Shiga-toxigenic Escherichia coli (STEC) strains isolated from wholesale spinach and lettuce consisted mostly of serotypes that have not been implicated in illness. Among these strains, however, were two O113:H21 that carried virulence genes common to this pathogenic serotype (stx2, ehxA, saa, and subAB), suggesting that their presence in ready-to-eat produce may be of health concern.
doi:10.1128/AEM.01872-10
PMCID: PMC3019700  PMID: 21057025
8.  Simultaneous Analysis of Multiple Enzymes Increases Accuracy of Pulsed-Field Gel Electrophoresis in Assigning Genetic Relationships among Homogeneous Salmonella Strains▿  
Due to a highly homogeneous genetic composition, the subtyping of Salmonella enterica serovar Enteritidis strains to an epidemiologically relevant level remains intangible for pulsed-field gel electrophoresis (PFGE). We reported previously on a highly discriminatory PFGE-based subtyping scheme for S. enterica serovar Enteritidis that relies on a single combined cluster analysis of multiple restriction enzymes. However, the ability of a subtyping method to correctly infer genetic relatedness among outbreak strains is also essential for effective molecular epidemiological traceback. In this study, genetic and phylogenetic analyses were performed to assess whether concatenated enzyme methods can cluster closely related salmonellae into epidemiologically relevant hierarchies. PFGE profiles were generated by use of six restriction enzymes (XbaI, BlnI, SpeI, SfiI, PacI, and NotI) for 74 strains each of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium. Correlation analysis of Dice similarity coefficients for all pairwise strain comparisons underscored the importance of combining multiple enzymes for the accurate assignment of genetic relatedness among Salmonella strains. The mean correlation increased from 81% and 41% for single-enzyme PFGE up to 99% and 96% for five-enzyme combined PFGE for S. enterica serovar Enteritidis and S. enterica serovar Typhimurium strains, respectively. Data regressions approached 100% correlation among Dice similarities for S. enterica serovar Enteritidis and S. enterica serovar Typhimurium strains when a minimum of six enzymes were concatenated. Phylogenetic congruence measures singled out XbaI, BlnI, SfiI, and PacI as most concordant for S. enterica serovar Enteritidis, while XbaI, BlnI, and SpeI were most concordant among S. enterica serovar Typhimurium strains. Together, these data indicate that PFGE coupled with sufficient enzyme numbers and combinations is capable of discerning accurate genetic relationships among Salmonella serovars comprising highly homogeneous strain complexes.
doi:10.1128/JCM.00120-10
PMCID: PMC3020462  PMID: 20980570
9.  PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species 
BMC Microbiology  2010;10:90.
Background
The genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species.
Results
In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well.
Conclusion
This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations providing information more quickly than other time-honoured methods traditionally used in these types of analyses.
doi:10.1186/1471-2180-10-90
PMCID: PMC2856557  PMID: 20331883
10.  Phenotypic and Genotypic Analyses of Neisseria gonorrhoeae Isolates That Express Frequently Recovered PorB PIA Variable Region Types Suggest that Certain P1a Porin Sequences Confer a Selective Advantage for Urogenital Tract Infection▿  
Infection and Immunity  2008;76(8):3700-3709.
Typing of the porB variable region (VR) is an epidemiological tool that classifies gonococcal strains based on sequence differences in regions of the porB gene that encode surface-exposed loops. The frequent isolation of certain porB VR types suggests that some porin sequences confer a selective advantage during infection and/or transmission. Alternatively, certain porin types may be markers of strains that are successful due to factors unrelated to porin. In support of the first hypothesis, here we show urogenital tract isolates representing the most common PIA VR types identified in an urban clinic in Baltimore, MD, over a 10-year period belonged to several different clonal types, as determined by pulsed-field gel electrophoresis (PFGE). Serum resistance, which was confirmed by factor H and C4b-binding protein binding studies, was more often associated with gonococcal the most common VR types. In contrast, three porin-independent phenotypes, namely, lactoferrin utilization, β-lactamase production, and multiple transferable resistance (Mtr), were segregated with the PFGE cluster and not with the VR type. Data combined with another PIA strain collection showed a strong correlation between serum resistance and the most common VR types. A comparison of VR typing hybridization patterns and nucleotide sequences of 12 porB1a genes suggests that certain porin loop 1, 3, 6, and/or 7 sequences may play a role in the serum resistance phenotype. We conclude that some PorB PIA sequences confer a survival or transmission advantage in the urogenital tract, perhaps via increased resistance to complement-mediated killing. The capacity of some porin types to evade a porin-specific adaptive immune response must also be considered.
doi:10.1128/IAI.00265-08
PMCID: PMC2493220  PMID: 18541655
11.  Enhanced Subtyping Scheme for Salmonella Enteritidis 
Emerging Infectious Diseases  2007;13(12):1932-1935.
To improve pulsed-field gel electrophoresis–based strain discrimination of 76 Salmonella Enteritidis strains, we evaluated 6 macro-restriction endonucleases, separately and in various combinations. One 3-enzyme subset, SfiI/PacI/NotI, was highly discriminatory. Five different indices, including the Simpson diversity index, supported this 3-enzyme combination for improved differentiation of S. Enteritidis.
doi:10.3201/eid1312.070185
PMCID: PMC2876743  PMID: 18258051
Salmonella Enteritidis; subtyping; differentiation; pulsed-field gel electrophoresis; molecular epidemiology; clone; restriction endonuclease; genetic diversity; dispatch
12.  Incidence and Tracking of Escherichia coli O157:H7 in a Major Produce Production Region in California 
PLoS ONE  2007;2(11):e1159.
Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995–2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human illness.
doi:10.1371/journal.pone.0001159
PMCID: PMC2174234  PMID: 18174909
13.  Genetic Diversity among Clonal Lineages within Escherichia coli O157:H7 Stepwise Evolutionary Model 
Emerging Infectious Diseases  2007;13(11):1701-1706.
Molecular characterization and subtyping show genetic diversities within clonal complexes.
Escherichia coli O157:H7 variants were examined for trait mutations and by molecular subtyping to better define clonal complexes postulated on the O157:H7 evolution model. Strains of β-glucuronidase–positive, sorbitol-negative O157:H7 isolated in United States and Japan were identical to A5 clonal strain and shared sequence type (ST)–65 by multilocus sequence typing (MLST); thus, they belong in A5. However, these strains exhibited pulsed-field gel electrophoresis (PFGE) profile differences that suggested genomic divergence between populations. Sorbitol-fermenting O157 (SFO157) strains from Finland, Scotland, and Germany were identical to A4 clonal strain and belong in A4. Some SFO157 strains, isolated years apart and from different countries, had identical PFGE profiles, suggesting a common origin. Despite similarities, some Finnish and Scottish and all of the German strains have ST-75 (“German clone”), whereas others have ST-76, a new variant (“Scottish clone”). MLST of strains in other clonal complexes also discriminated strains thought to be identical and showed that genetic differences will further distinguish clonal populations into subclones.
doi:10.3201/eid1311.070381
PMCID: PMC3375798  PMID: 18217554
Enterohemorrhagic E. coli O157:H7; clonal complexes; genetic diversity; molecular evolution; research
14.  Effect of Repeat Copy Number on Variable-Number Tandem Repeat Mutations in Escherichia coli O157:H7 
Journal of Bacteriology  2006;188(12):4253-4263.
Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 × 10−4 mutations/generation and a combined 28-locus rate of 6.4 × 10−4 mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2 = 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2 = 0.833, P < 0.0001) or excluded (r2 = 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.
doi:10.1128/JB.00001-06
PMCID: PMC1482962  PMID: 16740932
15.  Bacillus anthracis Bioterrorism Incident, Kameido, Tokyo, 1993 
Emerging Infectious Diseases  2004;10(1):117-120.
In July 1993, a liquid suspension of Bacillus anthracis was aerosolized from the roof of an eight-story building in Kameido, Tokyo, Japan, by the religious group Aum Shinrikyo. During 1999 to 2001, microbiologic tests were conducted on a liquid environmental sample originally collected during the 1993 incident. Nonencapsulated isolates of B. anthracis were cultured from the liquid. Multiple-locus, variable-number tandem repeat analysis found all isolates to be identical to a strain used in Japan to vaccinate animals against anthrax, which was consistent with the Aum Shinrikyo members’ testimony about the strain source. In 1999, a retrospective case-detection survey was conducted to identify potential human anthrax cases associated with the incident, but none were found. The use of an attenuated B. anthracis strain, low spore concentrations, ineffective dispersal, a clogged spray device, and inactivation of the spores by sunlight are all likely contributing factors to the lack of human cases.
doi:10.3201/eid1001.030238
PMCID: PMC3322761  PMID: 15112666
Bacillus anthracis; bioterrorism; anthrax; epidemiology; Aum Shinrikyo; Japan
16.  Molecular Investigation of the Aum Shinrikyo Anthrax Release in Kameido, Japan 
Journal of Clinical Microbiology  2001;39(12):4566-4567.
In 1993, the Aum Shinrikyo cult aerosolized Bacillus anthracis spores over Kameido, Japan. Spore samples were obtained from the release site, cultured, and characterized by molecular genetic typing. The isolates were consistent with strain Sterne 34F2, which is used in Japan for animal prophylaxis against anthrax.
doi:10.1128/JCM.39.12.4566-4567.2001
PMCID: PMC88589  PMID: 11724885

Results 1-16 (16)