Proceedings of the National Academy of Sciences of the United States of America, 2004
Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilizat... more Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilization. Y. pestis is a successful global pathogen, with active foci on all continents except Australia and Antarctica. Because the Y. pestis genome is highly monomorphic, previous attempts to characterize the population genetic structure within a single focus have been largely unsuccessful. Here we report that highly mutable marker loci allow determination of Y. pestis population genetic structure and tracking of transmission patterns at two spatial scales within a single focus. In addition, we found that in vitro mutation rates for these loci are similar to those observed in vivo, which allowed us to develop a mutation-rate-based model to examine transmission mechanisms. Our model suggests there are two primary components of plague ecology: a rapid expansion phase for population growth and dispersal followed by a slower persistence phase. This pattern seems consistent across local, regiona...
Virginia is the third largest producer of fresh-market tomatoes in the United States. Tomatoes gr... more 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.
The study objective was to characterize and analyse the distribution of enterotoxins and genes en... more The study objective was to characterize and analyse the distribution of enterotoxins and genes encoding enterotoxins in Staphylococcus aureus strains recovered from the 601 environment and ingredient samples obtained during multiple inspections of a bakery implicated in two separate staphylococcal food poisoning incidents. Staphylococcus aureus isolates were evaluated using serological assays for identification of classical staphylococcal enterotoxins (SEs) SEA-SEE and polymerase chain reaction for the detection of newly described SE and SE-like enterotoxin genes seg-seu. Pulsed field gel electrophoresis identified thirteen pattern types. During these investigations, a total of 585 environmental swabs and 16 raw ingredient samples were collected by investigators, 85 of which were confirmed to contain Staph. aureus; of those isolates, 95·3% (81/85) harboured enterotoxin genes and 4·7% (4/85) carried newly described SE and SE-like enterotoxin genes in the absence of classical enteroto...
Imported foods must meet the same U.S. Food and Drug Administration (FDA) standards as domestic f... more Imported foods must meet the same U.S. Food and Drug Administration (FDA) standards as domestic foods. The FDA determines whether an imported food is in compliance with the Federal Food, Drug, and Cosmetic Act. Pursuant to its regulatory activities, the FDA conducts compliance surveillance on imported foods offered for entry into the U.S. commerce. The National PulseNet Database is the molecular surveillance network for foodborne infections and is widely used to provide real-time subtyping support to epidemiologic investigations of foodborne diseases. FDA laboratories use pulsed-field gel electrophoresis to subtype foodborne pathogens recovered from imported foods and submit the molecular patterns to the National PulseNet Database at the Centers for Disease Control and Prevention. There were 60 isolates of Listeria monocytogenes in the FDA Field Accomplishment and Compliance Tracking System from 2001 to 2008 due to cheese imported from the following countries: Mexico (n=21 isolates)...
Proceedings of The National Academy of Sciences, 2004
Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilizat... more Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilization. Y. pestis is a successful global pathogen, with active foci on all continents except Australia and Antarctica. Because the Y. pestis genome is highly monomorphic, previous attempts to characterize the population genetic structure within a single focus have been largely unsuccessful. Here we report that highly
Facile laboratory tools are needed to augment identification in contamination events to trace the... more 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.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2007
VNTRs are able to discriminate among closely related isolates of recently emerged clonal pathogen... more 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.
In order to design and validate a method to identify virulence genes of Salmonella typhimurium us... more In order to design and validate a method to identify virulence genes of Salmonella typhimurium using DNA microarray, a protocol was developed to label the isolated bacterial DNA directly and to use PCR amplification of limited numbers of genes to validate the hybridization signals. Therefore, a DNA microarray chip of 71 virulence genes of S. typhimurium was developed and evaluated using 10 isolates. Each gene was represented by 65bp oligonucleotide probes (oligoprobes) and immobilized on the surface of chemically modified slides. Whole DNA genomes were digested with Hinf1 and Sau3AI, labeled with a fluorescent tag of Cy3 and then hybridized. The presence of virulence genes in 10 strains of S. typhimurium was established by measuring a fluorescent signal above the background noise of the chip. PCR amplification of 10 genes (orgA, ORF319, ttrB, rmbA, misL, spi4F, spi4H, spi4N, rRNA, and purR) of S. typhimurium was used as a standard to verify the confidence level of the DNA microarray chip. In conclusion, using PCR amplification to increase the confidence level of the microarray hybridization data was successful.
ABSTRACT Over 100 individuals were sickened after ingesting an assortment of desserts linked to f... more ABSTRACT Over 100 individuals were sickened after ingesting an assortment of desserts linked to four staphylococcal food poisoning outbreaks leading investigators to products manufactured by an Illinois bakery. The investigative team identified substantial deviations from the current Good Manufacturing Practice Regulations, 21 CFR Part 110, during multiple visits to the bakery. A total of 299 environmental swabs and 16 raw ingredients were collected for bacteriological analysis. Staphylococcus aureus isolates recovered from these samples were evaluated using either serological or polymerase chain reaction methods for the identification of staphylococcal enterotoxins SEA‐SEE, SEG‐SEI, SE‐like SElJ‐SElU or their respective genes, and for Panton–Valentine leukocidin cytotoxin. A raw ingredient and 16% of the environmental samples revealed the presence of enterotoxigenic S. aureus capable of producing diverse combinations of toxins. Antimicrobial susceptibility testing found that all S. aureus isolates were resistant to one or more agent(s). Pulsed field gel electrophoresis characterization of the isolates identified six pattern types. Practical ApplicationsThis Staphylococcus aureus outbreak investigation revealed the prevalence and diversity of antimicrobial‐resistant strains of S. aureus carrying both classical staphylococcal enterotoxins (SEs) and genes for nonclassical SE and SE‐like enterotoxins found in the environment of an Illinois bakery. In this occurrence, nonclassical SE and SE‐like enterotoxin serotypes were coupled with classical SEs, and therefore detectable by commercially available kits. This outbreak investigation demonstrated the abundance of the nonclassical SE and SE‐like enterotoxin serotypes in the environment and the anticipated need for new screening method requirements.
Proceedings of the National Academy of Sciences of the United States of America, 2004
Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilizat... more Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilization. Y. pestis is a successful global pathogen, with active foci on all continents except Australia and Antarctica. Because the Y. pestis genome is highly monomorphic, previous attempts to characterize the population genetic structure within a single focus have been largely unsuccessful. Here we report that highly mutable marker loci allow determination of Y. pestis population genetic structure and tracking of transmission patterns at two spatial scales within a single focus. In addition, we found that in vitro mutation rates for these loci are similar to those observed in vivo, which allowed us to develop a mutation-rate-based model to examine transmission mechanisms. Our model suggests there are two primary components of plague ecology: a rapid expansion phase for population growth and dispersal followed by a slower persistence phase. This pattern seems consistent across local, regiona...
Virginia is the third largest producer of fresh-market tomatoes in the United States. Tomatoes gr... more 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.
The study objective was to characterize and analyse the distribution of enterotoxins and genes en... more The study objective was to characterize and analyse the distribution of enterotoxins and genes encoding enterotoxins in Staphylococcus aureus strains recovered from the 601 environment and ingredient samples obtained during multiple inspections of a bakery implicated in two separate staphylococcal food poisoning incidents. Staphylococcus aureus isolates were evaluated using serological assays for identification of classical staphylococcal enterotoxins (SEs) SEA-SEE and polymerase chain reaction for the detection of newly described SE and SE-like enterotoxin genes seg-seu. Pulsed field gel electrophoresis identified thirteen pattern types. During these investigations, a total of 585 environmental swabs and 16 raw ingredient samples were collected by investigators, 85 of which were confirmed to contain Staph. aureus; of those isolates, 95·3% (81/85) harboured enterotoxin genes and 4·7% (4/85) carried newly described SE and SE-like enterotoxin genes in the absence of classical enteroto...
Imported foods must meet the same U.S. Food and Drug Administration (FDA) standards as domestic f... more Imported foods must meet the same U.S. Food and Drug Administration (FDA) standards as domestic foods. The FDA determines whether an imported food is in compliance with the Federal Food, Drug, and Cosmetic Act. Pursuant to its regulatory activities, the FDA conducts compliance surveillance on imported foods offered for entry into the U.S. commerce. The National PulseNet Database is the molecular surveillance network for foodborne infections and is widely used to provide real-time subtyping support to epidemiologic investigations of foodborne diseases. FDA laboratories use pulsed-field gel electrophoresis to subtype foodborne pathogens recovered from imported foods and submit the molecular patterns to the National PulseNet Database at the Centers for Disease Control and Prevention. There were 60 isolates of Listeria monocytogenes in the FDA Field Accomplishment and Compliance Tracking System from 2001 to 2008 due to cheese imported from the following countries: Mexico (n=21 isolates)...
Proceedings of The National Academy of Sciences, 2004
Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilizat... more Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilization. Y. pestis is a successful global pathogen, with active foci on all continents except Australia and Antarctica. Because the Y. pestis genome is highly monomorphic, previous attempts to characterize the population genetic structure within a single focus have been largely unsuccessful. Here we report that highly
Facile laboratory tools are needed to augment identification in contamination events to trace the... more 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.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2007
VNTRs are able to discriminate among closely related isolates of recently emerged clonal pathogen... more 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.
In order to design and validate a method to identify virulence genes of Salmonella typhimurium us... more In order to design and validate a method to identify virulence genes of Salmonella typhimurium using DNA microarray, a protocol was developed to label the isolated bacterial DNA directly and to use PCR amplification of limited numbers of genes to validate the hybridization signals. Therefore, a DNA microarray chip of 71 virulence genes of S. typhimurium was developed and evaluated using 10 isolates. Each gene was represented by 65bp oligonucleotide probes (oligoprobes) and immobilized on the surface of chemically modified slides. Whole DNA genomes were digested with Hinf1 and Sau3AI, labeled with a fluorescent tag of Cy3 and then hybridized. The presence of virulence genes in 10 strains of S. typhimurium was established by measuring a fluorescent signal above the background noise of the chip. PCR amplification of 10 genes (orgA, ORF319, ttrB, rmbA, misL, spi4F, spi4H, spi4N, rRNA, and purR) of S. typhimurium was used as a standard to verify the confidence level of the DNA microarray chip. In conclusion, using PCR amplification to increase the confidence level of the microarray hybridization data was successful.
ABSTRACT Over 100 individuals were sickened after ingesting an assortment of desserts linked to f... more ABSTRACT Over 100 individuals were sickened after ingesting an assortment of desserts linked to four staphylococcal food poisoning outbreaks leading investigators to products manufactured by an Illinois bakery. The investigative team identified substantial deviations from the current Good Manufacturing Practice Regulations, 21 CFR Part 110, during multiple visits to the bakery. A total of 299 environmental swabs and 16 raw ingredients were collected for bacteriological analysis. Staphylococcus aureus isolates recovered from these samples were evaluated using either serological or polymerase chain reaction methods for the identification of staphylococcal enterotoxins SEA‐SEE, SEG‐SEI, SE‐like SElJ‐SElU or their respective genes, and for Panton–Valentine leukocidin cytotoxin. A raw ingredient and 16% of the environmental samples revealed the presence of enterotoxigenic S. aureus capable of producing diverse combinations of toxins. Antimicrobial susceptibility testing found that all S. aureus isolates were resistant to one or more agent(s). Pulsed field gel electrophoresis characterization of the isolates identified six pattern types. Practical ApplicationsThis Staphylococcus aureus outbreak investigation revealed the prevalence and diversity of antimicrobial‐resistant strains of S. aureus carrying both classical staphylococcal enterotoxins (SEs) and genes for nonclassical SE and SE‐like enterotoxins found in the environment of an Illinois bakery. In this occurrence, nonclassical SE and SE‐like enterotoxin serotypes were coupled with classical SEs, and therefore detectable by commercially available kits. This outbreak investigation demonstrated the abundance of the nonclassical SE and SE‐like enterotoxin serotypes in the environment and the anticipated need for new screening method requirements.
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Papers by Christine Keys