Ingested Salmonella enterica, Cronobacter sakazakii, Escherichia coli O157:H7, and Listeria monocytogenes: transmission dynamics from adult house flies to their eggs and first filial (F1) generation adults.

BACKGROUND: The mechanical transmission of pathogenic bacteria by synanthropic filth flies is widely recognized. While many studies report the fate and the temporospatial distribution of ingested foodborne bacteria by filth flies, there is little evidence about the transmission dynamics of ingested foodborne bacteria by adult house flies (Musca domestica) to their progeny. In this study, we fed parental house fly adults with food contaminated with low, medium, and high concentrations of Salmonella enterica, Cronobacter sakazakii, Escherichia coli O157:H7, and Listeria monocytogenes and evaluated the probability of transmission of these pathogens to house fly eggs and the surface and the alimentary canal of their first filial (F1) generation adults. RESULTS: All foodborne pathogens were present in samples containing pooled house fly eggs. The probability of transmission was higher after parental house flies ingested food containing medium bacterial loads. Cronobacter sakazakii was 16, 6, and 3 times more likely to be transmitted to house fly eggs than S. enterica, E. coli O157:H7, and L. monocytogenes, respectively. Only S. enterica and C. sakazakii were transmitted to F1 generation adults and their presence was 2.4 times more likely on their body surfaces than in their alimentary canals. The highest probabilities of finding S. enterica (60 %) and C. sakazakii (28 %) on newly emerged F1 adults were observed after parental house flies ingested food containing medium and high levels of these pathogens, respectively. CONCLUSION: Our study demonstrates that adult house flies that fed from food contaminated with various levels of foodborne bacteria were able to transmit those pathogens to their eggs and some were further transmitted to newly emerged F1 generation adults, enhancing the vector potential of these insects. Understanding the type of associations that synanthropic filth flies establish with foodborne pathogens will help to elucidate transmission mechanisms and possible ways to mitigate the spread of foodborne pathogens.

Ecological prevalence, genetic diversity, and epidemiological aspects of Salmonella isolated from tomato agricultural regions of the Virginia Eastern Shore.

Virginia is the third largest producer of fresh-market tomatoes in the United States. Tomatoes grown along the eastern shore of Virginia are implicated almost yearly in Salmonella illnesses. Traceback implicates contamination occurring in the pre-harvest environment. To get a better understanding of the ecological niches of Salmonella in the tomato agricultural environment, a 2-year study was undertaken at a regional agricultural research farm in Virginia. Environmental samples, including tomato (fruit, blossoms, and leaves), irrigation water, surface water and sediment, were collected over the growing season. These samples were analyzed for the presence of Salmonella using modified FDA-BAM methods. Molecular assays were used to screen the samples. Over 1500 samples were tested. Seventy-five samples tested positive for Salmonella yielding over 230 isolates. The most commonly isolated serovars were S. Newport and S. Javiana with pulsed-field gel electrophoresis yielding 39 different patterns. Genetic diversity was further underscored among many other serotypes, which showed multiple PFGE subtypes. Whole genome sequencing (WGS) of several S. Newport isolates collected in 2010 compared to clinical isolates associated with tomato consumption showed very few single nucleotide differences between environmental isolates and clinical isolates suggesting a source link to Salmonella contaminated tomatoes. Nearly all isolates collected during two growing seasons of surveillance were obtained from surface water and sediment sources pointing to these sites as long-term reservoirs for persistent and endemic contamination of this environment.

Fully assembled genome sequence for Salmonella enterica subsp. enterica Serovar Javiana CFSAN001992.

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.

Analysis of pulsed field gel electrophoresis profiles using multiple enzymes for predicting potential source reservoirs for strains of Salmonella Enteritidis and Salmonella Typhimurium isolated from humans.

We reported previously on a highly discriminatory pulsed field gel electrophoresis-based (PFGE) subtyping scheme for Salmonella enterica serovar Enteritidis (SE) and Salmonella Typhimurium (ST) that relies on combined cluster analysis of up to six restriction enzymes. This approach allowed for the high-resolution separation of numerous poultry-derived SE and ST isolates into several distinct clusters that sorted along several geographical and host-linked boundaries. In this study, 101 SE and 151 ST strains isolated from poultry, swine, beef, mouse, and produce origins were combined with 62 human SE and ST isolates of unknown sources. PFGE profiles were generated across six restriction enzymes (XbaI, BlnI, SpeI, SfiI, PacI, and NotI) for human SE and ST isolates. The combined six-enzyme UPGMA trees of SE and ST revealed six separate origins of North American human SE isolates including one association with a "cosmopolitan" cluster of SEs from poultry originating in Scotland, Mexico, and China. In the case of ST, human isolates assorted readily along host lines rather than geographical partitions with the majority of human STs clustering in a larger group of STs of potential porcine origin. Such observations may underscore the ecological importance of poultry and pork reservoirs for SE and ST transmission to humans, respectively. In an examination of the relationship between enzyme diversity and congruence among enzymes, pairwise genetic diversity ranged from 6.5% to 9.7% for SE isolates and, more widely, from 17.5% to 27.4% for ST isolates. Phylogenetic congruence measures singled out XbaI, BlnI, and SfiI as most concordant for SE while XbaI and SfiI were most concordant among ST strains. Thus, these data provide the first proof of principal for concatenated PFGE, when coupled with sufficient enzyme numbers and combinations, as one effective means for predicting geographical and food source reservoirs for human isolates of these two highly prevalent Salmonella serovars.

On the evolutionary history, population genetics and diversity among isolates of Salmonella Enteritidis PFGE pattern JEGX01.0004.

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.

Draft genome sequences of 21 Salmonella enterica serovar enteritidis strains.

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.

High resolution clustering of Salmonella enterica serovar Montevideo strains using a next-generation sequencing approach.

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.

Capsular serotype of Staphylococcus aureus in the era of community-acquired MRSA.

Capsular polysaccharide (CP) plays an important role in the pathogenicity and immunogenicity of Staphylococcus aureus, yet the common serotypes of S. aureus isolated from US pediatric patients have not been reported. We investigated capsular serotype as well as methicillin susceptibility, presence of Panton-Valentine leukocidin (PVL), and clonal relatedness of pediatric S. aureus isolates. Clinical isolates were tested for methicillin susceptibility, presence of mecA, lukS-PV and lukF-PV, cap5 and cap8 genes by PCR, and for capsular or surface polysaccharide expression (CP5, CP8, or 336 polysaccharide) by agglutination. Genetic relatedness was determined by pulsed-field gel electrophoresis. All S. aureus isolates encoded cap5 or cap8. Sixty-nine percent of 2004-2005 isolates were methicillin-susceptible (MSSA) and most expressed a detectable capsule. The majority of MRSA isolates (82%) were unencapsulated, exposing an expressed cell wall techoic acid antigen 336. Pulsed-field type USA300 were MRSA, PVL-positive, unencapsulated strains that were associated with deep skin infections and recurrent disease. Over half (58%) of all isolates from invasive pediatric dermatologic infections were USA300. All pediatric isolates contained either capsule type 5 or capsule type 8 genes, and roughly half of the S. aureus clinical disease isolates from our population were diverse MSSA-encapsulated strains. The majority of the remaining pediatric clinical disease isolates were unencapsulated serotype 336 strains of the PVL(+) USA300 community-associated-MRSA clone.

Evaluation of the taxonomic utility of six-enzyme pulsed-field gel electrophoresis in reconstructing Salmonella subspecies phylogeny.

Pulsed-field gel electrophoresis (PFGE) remains an important tool in the molecular epidemiological evaluation of strains emerging from disease outbreak clusters. Recent studies of Escherichia coli O157:H7 and Salmonella Enteritidis have noted marked improvements in the discriminatory power of PFGE when combining band profiles from up to six restriction enzyme datasets into a single concatenated analysis. This approach has provided more accurate assignments of genetic relationships among closely related strains and allowed effective phylogenetic inference of host and geographical reservoirs. Although this approach enhances epidemiological congruence among closely related strains, it remains unclear to what extent six-enzyme PFGE pattern similarity reiterates evolutionary relatedness among more distantly related Salmonella strains (i.e., serovar or subspecies levels). Here, taxonomic accuracy of six-enzyme PFGE is tested phylogenetically across two distinct Salmonella enterica populations-Salmonella reference collection B (SARB), representing the breadth of taxonomic diversity of S. enterica subspecies I only, and Salmonella reference collection C (SARC), comprising the seven disparate subspecies of S. enterica plus S. bongori. Cladistic analysis of SAR strains revealed substantial polyphyly between the two strain collections such that numerous SARB strains clustered more closely with diverged SARC subspecies rather than with other members of subspecies I. Also, in several cases, SARC sibling strains from the same subspecies were evolutionary obscured-broken into distant locales on the most parsimonious six-enzyme trees. Genetic diversity among SARB and SARC strains was comparable at 45% and 47%, respectively, while congruence testing revealed discordance among individual enzyme datasets. While six-enzyme PFGE is effective in ascertaining accurate genetic relationships for more closely related strains (e.g., strains within the same serovar), reconstitution of evolutionarily meaningful strain groupings may be elusive for Salmonella at the serovar level and above. Thus, caution is warranted when applying PFGE with a limited number of enzymes as the primary phylogenetic marker in these instances.

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.

PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species.

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.

Differentiation of Enterobacter sakazakii from closely related Enterobacter and Citrobacter species using fatty acid profiles.

Capillary gas chromatography with flame ionization detection (GC-FID) was used to determine the cellular fatty acid (CFA) profiles of 134 Enterobacter sakazakii strains, and these were compared to the CFA profiles of other closely related Enterobacter and Citrobacter species. For GC-FID analysis, whole cell fatty acid methyl esters (FAMEs) from cells cultured on brain heart infusion (BHI) agar at 37 degrees C for 24 h were obtained by saponification, methylation, and extraction into hexane/methyl tert-butyl ether. A database for E. sakazakii was prepared using fatty acid profiles from the 134 strains. Major fatty acids of E. sakazakii strains evaluated in this study were straight-chain 12:0, 14:0, and 16:0, unsaturated 18:1 omega7c, and 17:0 omegacyclo 7-8. Principal component analysis (PCA) based on CFA profiles for E. sakazakii strains shows separation of E. sakazakii subgroups A and B. The CFA profiles for E. sakazakii and Enterobacter cloacae show that there are several fatty acids, 14:0, 17:0 omegacyclo 7-8, 18:1 omega7c, and summed 16:1 omega6c/16:1 omega7c, that differ significantly between these two species. A PCA model based on CFA profiles for E. sakazakii strains clearly shows separation of E. sakazakii from closely related Enterobacter and Citrobacter species. Analysis of FAMEs from E. sakazakii strains grown on BHI agar by a rapid GC-FID method can provide a sensitive procedure for the identification of this organism, and this analytical method provides a confirmatory procedure for the differentiation of E. sakazakii strains from closely related Enterobacter and Citrobacter species.

Enhanced subtyping scheme for Salmonella enteritidis.

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.

Mutations, mutation rates, and evolution at the hypervariable VNTR loci of Yersinia pestis.

VNTRs are able to discriminate among closely related isolates of recently emerged clonal pathogens, including Yersinia pestis the etiologic agent of plague, because of their great diversity. Diversity is driven largely by mutation but little is known about VNTR mutation rates, factors affecting mutation rates, or the mutational mechanisms. The molecular epidemiological utility of VNTRs will be greatly enhanced when this foundational knowledge is available. Here, we measure mutation rates for 43 VNTR loci in Y. pestis using an in vitro generated population encompassing approximately 96,000 generations. We estimate the combined 43-locus rate and individual rates for 14 loci. A comparison of Y. pestis and Escherichia coli O157:H7 VNTR mutation rates and products revealed a similar relationship between diversity and mutation rate in these two species. Likewise, the relationship between repeat copy number and mutation rate is nearly identical between these species, suggesting a generalized relationship that may be applicable to other species. The single- versus multiple-repeat mutation ratios and the insertion versus deletion mutation ratios were also similar, providing support for a general model for the mutations associated with VNTRs. Finally, we use two small sets of Y. pestis isolates to show how this general model and our estimated mutation rates can be used to compare alternate phylogenies, and to evaluate the significance of genotype matches, near-matches, and mismatches found in empirical comparisons with a reference database.