Comparative genomics of 28 Salmonella enterica isolates: evidence for CRISPR-mediated adaptive sublineage evolution.

Despite extensive surveillance, food-borne Salmonella enterica infections continue to be a significant burden on public health systems worldwide. As the S. enterica species comprises sublineages that differ greatly in antigenic representation, virulence, and antimicrobial resistance phenotypes, a better understanding of the species' evolution is critical for the prediction and prevention of future outbreaks. The roles that virulence and resistance phenotype acquisition, exchange, and loss play in the evolution of S. enterica sublineages, which to a certain extent are represented by serotypes, remains mostly uncharacterized. Here, we compare 17 newly sequenced and phenotypically characterized nontyphoidal S. enterica strains to 11 previously sequenced S. enterica genomes to carry out the most comprehensive comparative analysis of this species so far. These phenotypic and genotypic data comparisons in the phylogenetic species context suggest that the evolution of known S. enterica sublineages is mediated mostly by two mechanisms, (i) the loss of coding sequences with known metabolic functions, which leads to functional reduction, and (ii) the acquisition of horizontally transferred phage and plasmid DNA, which provides virulence and resistance functions and leads to increasing specialization. Matches between S. enterica clustered regularly interspaced short palindromic repeats (CRISPR), part of a defense mechanism against invading plasmid and phage DNA, and plasmid and prophage regions suggest that CRISPR-mediated immunity could control short-term phenotype changes and mediate long-term sublineage evolution. CRISPR analysis could therefore be critical in assessing the evolutionary potential of S. enterica sublineages and aid in the prediction and prevention of future S. enterica outbreaks.

Comparative genomics of the IncA/C multidrug resistance plasmid family.

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

Antimicrobial resistance-conferring plasmids with similarity to virulence plasmids from avian pathogenic Escherichia coli strains in Salmonella enterica serovar Kentucky isolates from poultry.

Salmonella enterica, a leading cause of food-borne gastroenteritis worldwide, may be found in any raw food of animal, vegetable, or fruit origin. Salmonella serovars differ in distribution, virulence, and host specificity. Salmonella enterica serovar Kentucky, though often found in the food supply, is less commonly isolated from ill humans. The multidrug-resistant isolate S. Kentucky CVM29188, isolated from a chicken breast sample in 2003, contains three plasmids (146,811 bp, 101,461 bp, and 46,121 bp), two of which carry resistance determinants (pCVM29188_146 [strAB and tetRA] and pCVM29188_101 [bla(CMY-2) and sugE]). Both resistance plasmids were transferable by conjugation, alone or in combination, to S. Kentucky, Salmonella enterica serovar Newport, and Escherichia coli recipients. pCVM29188_146 shares a highly conserved plasmid backbone of 106 kb (>90% nucleotide identity) with two virulence plasmids from avian pathogenic Escherichia coli strains (pAPEC-O1-ColBM and pAPEC-O2-ColV). Shared avian pathogenic E. coli (APEC) virulence factors include iutA iucABCD, sitABCD, etsABC, iss, and iroBCDEN. PCR analyses of recent (1997 to 2005) S. Kentucky isolates from food animal, retail meat, and human sources revealed that 172 (60%) contained similar APEC-like plasmid backbones. Notably, though rare in human- and cattle-derived isolates, this plasmid backbone was found at a high frequency (50 to 100%) among S. Kentucky isolates from chickens within the same time span. Ninety-four percent of the APEC-positive isolates showed resistance to tetracycline and streptomycin. Together, our findings of a resistance-conferring APEC virulence plasmid in a poultry-derived S. Kentucky isolate and of similar resistance/virulence plasmids in most recent S. Kentucky isolates from chickens and, to lesser degree, from humans and cattle highlight the need for additional research in order to examine the prevalence and spread of combined virulence and resistance plasmids in bacteria in agricultural, environmental, and clinical settings.

Altered utilization of N-acetyl-D-galactosamine by Escherichia coli O157:H7 from the 2006 spinach outbreak.

In silico analyses of previously sequenced strains of Escherichia coli O157:H7, EDL933 and Sakai, localized the gene cluster for the utilization of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam). This gene cluster encodes the Aga phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and other catabolic enzymes responsible for transport and catabolism of Aga. As the complete coding sequences for enzyme IIA (EIIA)(Aga/Gam), EIIB(Aga), EIIC(Aga), and EIID(Aga) of the Aga PTS are present, E. coli O157:H7 strains normally are able to utilize Aga as a sole carbon source. The Gam PTS complex, in contrast, lacks EIIC(Gam), and consequently, E. coli O157:H7 strains cannot utilize Gam. Phenotypic analyses of 120 independent isolates of E. coli O157:H7 from our culture collection revealed that the overwhelming majority (118/120) displayed the expected Aga+ Gam- phenotype. Yet, when 194 individual isolates, derived from a 2006 spinach-associated E. coli O157:H7 outbreak, were analyzed, all (194/194) displayed an Aga- Gam- phenotype. Comparison of aga/gam sequences from two spinach isolates with those of EDL933 and Sakai revealed a single nucleotide change (G:C-->A:T) in the agaF gene in the spinach-associated isolates. The base substitution in agaF, which encodes EIIA(Aga/Gam) of the PTS, changes a conserved glycine residue to serine (Gly91Ser). Pyrosequencing of this region showed that all spinach-associated E. coli O157:H7 isolates harbored this same G:C-->A:T substitution. Notably, when agaF+ was cloned into an expression vector and transformed into six spinach isolates, all (6/6) were able to grow on Aga, thus demonstrating that the Gly91Ser substitution underlies the Aga- phenotype in these isolates.

Exploring genotypic and phenotypic diversity of microbes using microarray approaches.

Application of genome-scale analysis like DNA microarray technology has revolutionized multiple scientific disciplines. Herein, a next generation of DNA microarrays, a DNA tiling approach that allows high throughput sampling of genomes with single-nucleotide precision, is described. As methods revealing a genomic scale examination of cellular phenotypes offer keen insights for genomic analyses, a high throughput system for whole cell phenotyping is similarly detailed. The merit of these technologies in discriminating pathogenic and commensal strains of microbes is emphasized using the microbe, Escherichia coli, as an example. Deployment of microarray strategies to assess closely-related microbial strains should help address diversity of organisms in their feral settings.

Chips and SNPs, bugs and thugs: a molecular sleuthing perspective.

Recent events both here and abroad have focused attention on the need for ensuring a safe and secure food supply. Although much has been written about the potential of particular select agents in bioterrorism, we must consider seriously the more mundane pathogens, especially those that have been implicated previously in foodborne outbreaks of human disease, as possible agents of bioterrorism. Given their evolutionary history, the enteric pathogens are more diverse than agents such as Bacillus anthracis, Francisella tularensis, or Yersinia pestis. This greater diversity, however, is a double-edged sword; although diversity affords the opportunity for unequivocal identification of an organism without the need for whole-genome sequencing, the same diversity can confound definitive forensic identification if boundaries are not well defined. Here, we discuss molecular approaches used for the identification of Salmonella enterica, Escherichia coli, and Shigella spp. and viral pathogens and discuss the utility of these approaches to the field of microbial molecular forensics.

Healthy plants: necessary for a balanced 'One Health' concept.

All life forms depend ultimately upon sunlight to create the energy 'currency' required for the functions of living. Green plants can make that conversion directly but the rest of us would perish without access to foods derived, directly or indirectly, from plants. We also require their fibre which we use for clothing, building and other purposes. However, plants, just as humans and animals, are attacked by pathogens that cause a myriad of symptoms that can lead to reduced yields, lower quality products and diminished nutritional value. Plant pathogens share many features with their human and animal counterparts. Some pathogens - whether of humans, animals, or plants - have nimble genomes or the ability to pirate genes from other organisms via mobile elements. Some have developed the ability to cross kingdoms in their host ranges. Many others share virulence factors, such as the type III secretion system (T3SS) or mechanisms for sensing population density, that work equally well in all kingdoms. Certain pathogens of hosts in all kingdoms rely upon insect vectors and use similar mechanisms to ensure dispersal (and sometimes survival) in this way. Plant-pathogen interactions have more direct consequence for humans when the microbes are human pathogens such as Escherichia coli 0157:H7 and Salmonella spp., which can contaminate fresh produce or when they produce metabolites, such as mycotoxins, which are harmful when consumed. Finally, national biosecurity concerns and the need for prevention, preparedness and forensic capabilities cross all kingdom barriers. Thus, our communities that focus on one of these kingdoms have much to learn from one another and a complete and balanced 'One Health' initiative must be tripartite, embracing the essential components of healthy plants, healthy animals and healthy people.

Optical mapping and 454 sequencing of Escherichia coli O157 : H7 isolates linked to the US 2006 spinach-associated outbreak.

Optical maps for five representative clinical, food-borne and bovine-derived isolates from the 2006 Escherichia coli O157 : H7 outbreak linked to fresh spinach in the United States showed a common set of 14 distinct chromosomal markers that define the outbreak strain. Partial 454 DNA sequencing was used to characterize the optically mapped chromosomal markers. The markers included insertions, deletions, substitutions and a simple single nucleotide polymorphism creating a BamHI site. The Shiga toxin gene profile of the spinach-associated outbreak isolates (stx1(-) stx2(+) stx2c(+)) correlated with prophage insertions different from those in the prototypical EDL933 and Sakai reference strains (stx1(+) stx2(+) stx2c(-)). The prophage occupying the yehV chromosomal position in the spinach-associated outbreak isolates was similar to the stx1(+) EDL933 cryptic prophage V, but it lacked the stx1 gene. In EDL933, the stx2 genes are within prophage BP933-W at the wrbA chromosomal locus; this locus was unoccupied in the spinach outbreak isolates. Instead, the stx2 genes were found within a chimeric BP933-W-like prophage with a different integrase, inserted at the argW locus in the outbreak isolates. An extra set of Shiga toxin genes, stx2c, was found in the outbreak isolates within a prophage integrated at the sbcB locus. The optical maps of two additional clinical isolates from the outbreak showed a single, different prophage variation in each, suggesting that changes occurred in the source strain during the course of this widespread, multi-state outbreak.

Optical maps distinguish individual strains of Escherichia coli O157 : H7.

Optical maps of 11 Escherichia coli O157 : H7 strains have been generated by the assembly of contiguous sets of restriction fragments across their entire 5.3 to 5.6 Mbp chromosomes. Each strain showed a distinct, highly individual configuration of 500-700 BamHI fragments, yielding a map resembling a DNA 'bar code'. The accuracy of optical mapping was assessed by comparing directly the in silico restriction maps of two wholly sequenced reference genomes of E. coli O157 : H7, i.e. EDL933 and the Sakai isolate (RIMD 0509952), with the optical maps of the same strains. The optical maps of nine other E. coli O157 : H7 strains were compared similarly, using the sequence-based maps of the Sakai and EDL933 strains as references. A total of 91 changes at 28 loci were positioned and sized; these included complex chromosomal inversions, insertions, deletions, substitutions, as well as a number of simple RFLPs. The optical maps defined unique genome landmarks in each of the strains and demonstrated the ability of optical mapping to distinguish and differentiate, at the individual level, strains of this important pathogen.

Interrogating genomic diversity of E. coli O157:H7 using DNA tiling arrays.

Here, we describe a novel microarray-based approach for investigating the genomic diversity of Escherichia coli O157:H7 in a semi-high throughput manner using a high density, oligonucleotide-based microarray. This microarray, designed to detect polymorphisms at each of 60,000 base-pair (bp) positions within an E. coli genome, is composed of overlapping 29-mer oligonucleotides specific for 60 equally spaced, 1000-bp loci of the E. coli O157:H7 strain EDL933 chromosome. By use of a novel 12-well microarray that permitted the simultaneous investigation of 12 strains, the genomes of 44 individual isolates of E. coli O157:H7 were interrogated. These analyses revealed more than 150 single nucleotide polymorphisms (SNPs) and several deletions and amplifications in the test strains. Pyrosequencing was used to confirm the usefulness of the novel SNPs by determining their allelic frequency among a collection of diverse isolates of E. coli O157:H7. The tiling DNA microarray system would be useful for the tracking and identification of individual strains of E. coli O157:H7 needed for forensic investigations.

Multiple antimicrobial resistance in plague: an emerging public health risk.

Antimicrobial resistance in Yersinia pestis is rare, yet constitutes a significant international public health and biodefense threat. In 1995, the first multidrug resistant (MDR) isolate of Y. pestis (strain IP275) was identified, and was shown to contain a self-transmissible plasmid (pIP1202) that conferred resistance to many of the antimicrobials recommended for plague treatment and prophylaxis. Comparative analysis of the DNA sequence of Y. pestis plasmid pIP1202 revealed a near identical IncA/C plasmid backbone that is shared by MDR plasmids isolated from Salmonella enterica serotype Newport SL254 and the fish pathogen Yersinia ruckeri YR71. The high degree of sequence identity and gene synteny between the plasmid backbones suggests recent acquisition of these plasmids from a common ancestor. In addition, the Y. pestis pIP1202-like plasmid backbone was detected in numerous MDR enterobacterial pathogens isolated from retail meat samples collected between 2002 and 2005 in the United States. Plasmid-positive strains were isolated from beef, chicken, turkey and pork, and were found in samples from the following states: California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York and Oregon. Our studies reveal that this common plasmid backbone is broadly disseminated among MDR zoonotic pathogens associated with agriculture. This reservoir of mobile resistance determinants has the potential to disseminate to Y. pestis and other human and zoonotic bacterial pathogens and therefore represents a significant public health concern.

Structure and distribution of the phosphoprotein phosphatase genes, prpA and prpB, among Shigella subgroups.

Phosphoprotein phosphatases encoded by the prpA and prpB genes function in signal transduction pathways for degradation of misfolded proteins in the extracytoplasmic compartments of Escherichia coli. In order to trace the evolution of prp genes and assess their roles in other enteric pathogens, the structure and distribution of these genes among closely related Shigella subgroups were studied. PCR amplification, probe hybridization studies and DNA sequencing were used to determine the prp genotypes of 58 strains from the four Shigella subgroups, Dysenteriae, Boydii, Sonnei and Flexneri. It was found that the prp alleles among Shigella subgroups were extremely susceptible to gene inactivation and that the mutations involved in prp allele inactivation were varied. They included IS insertions, gene replacement by an IS element, a small deletion within the gene or large deletion engulfing the entire gene region, and base substitutions that generated premature termination codons. As a result, of 58 strains studied, only eight (14 %) possessed intact prpA and prpB genes. Of the Shigella strains examined, 76 % (44/58) showed at least one of the prp alleles inactivated by one or more IS elements, including IS1, IS4, IS600 and IS629. Phylogenetic analysis revealed that IS elements have been independently acquired in multiple lineages of Shigella, suggesting that loss of functional alleles has been advantageous during Shigella strain evolution.

Molecular applications for identifying microbial pathogens in the post-9/11 era.

Rapid advances in molecular and optical technologies over the past 10 years have dramatically impacted the way biologic research is conducted today. Examples include microarrays, capillary sequencing, optical mapping and real-time sequencing (Pyrosequencing). These technologies are capable of rapidly delivering massive amounts of genetic information and are becoming routine mainstays of many laboratories. Fortunately, advances in scientific computing have provided the enormous computing power necessary to analyze these enormous data sets. The application of molecular technologies should prove useful to the burgeoning field of microbial forensics. In the post-9/11 era, when securing America's food supply is a major endeavor, the need for rapid identification of microbes that accidentally or intentionally find their way into foods is apparent. The principle that distinguishes a microbial forensic investigation from a molecular epidemiology study is that a biocrime has been committed. If proper attribution is to be attained, a link must be made between a particular microbe in the food and the perpetrator who placed it there. Therefore, the techniques used must be able to discriminate individual isolates of a particular microbe. A battery of techniques in development for distinguishing individual isolates of particular foodborne pathogens is discussed.

Limited boundaries for extensive horizontal gene transfer among Salmonella pathogens.

Recombination is thought to be rare within Salmonella, as evidenced by absence of gene transfer among SARC strains that represent the broad genetic diversity of the eight primary subspecies of this common facultative intracellular pathogen. We adopted a phylogenetic approach to assess recombination within the mutS gene of 70 SARB strains, a genetically homogeneous population of Salmonella enterica subspecies I strains, which have in common the ability to infect warm-blooded animals. We report here that SARB strains show evidence for widespread recombinational exchange in contrast to results obtained with strains exhibiting species-level genetic variation. Besides extensive allele shuffling, SARB strains showed notably larger recombinagenic patch sizes for mutS (at least approximately 1.1 kb) than previously reported for S. enterica SARC strains. Explaining these experimental dichotomies provides important insight for understanding microbial evolution, because they suggest likely ecologic and genetic barriers that limit extensive gene transfer in the feral setting.

Evolution of multi-gene segments in the mutS-rpoS intergenic region of Salmonella enterica serovar Typhimurium LT2.

The nucleotide sequence of the 12.6 kb region between the mutS and rpoS genes of Salmonella enterica serovar Typhimurium LT2 (S. typhimurium) was compared to other enteric bacterial mutS-rpoS intergenic regions. The mutS-rpoS region is composed of three distinct segments, designated HK, O and S, as defined by sequence similarities to contiguous ORFs in other bacteria. Inverted chromosomal orientations of each of these segments are found between the mutS and rpoS genes in related ENTEROBACTERIACEAE: The HK segment is distantly related to a cluster of seven ORFs found in Haemophilus influenzae and a cluster of five ORFs found between the mutS and rpoS genes in Escherichia coli K-12. The O segment is related to the mutS-rpoS intergenic region found in E. coli O157:H7 and Shigella dysenteriae type 1. The third segment, S, is common to diverse Salmonella species, but is absent from E. coli. Despite the extensive collinearity and conservation of the overall genetic maps of S. typhimurium and E. coli K-12, the insertions, deletions and inversions in the mutS-rpoS region provide evidence that this region of the chromosome is an active site for horizontal gene transfer and rearrangement.

Molecular analysis of mutS expression and mutation in natural isolates of pathogenic Escherichia coli.

Deficiencies in the MutS protein disrupt methyl-directed mismatch repair (MMR), generating a mutator phenotype typified by high mutation rates and promiscuous recombination. How such deficiencies might arise in the natural environment was determined by analysing pathogenic strains of Escherichia coli. Quantitative Western immunoblotting showed that the amount of MutS in a wild-type strain of the enterohaemorrhagic pathogen E. coli O157 : H7 decreased about 26-fold in stationary-phase cells as compared with the amount present during exponential-phase growth. The depletion of MutS in O157 : H7 is significantly greater than that observed for a laboratory-attenuated E. coli K-12 strain. In the case of stable mutators, mutS defects in strains identified among natural isolates were analysed, including two E. coli O157 : H7 strains, a diarrhoeagenic E. coli O55 : H7 strain, and a uropathogenic strain from the E. coli reference (ECOR) collection. No MutS could be detected in the four strains by Western immunoblot analyses. RNase T2 protection assays showed that the strains were either deficient in mutS transcripts or produced transcripts truncated at the 3' end. Nucleotide sequence analysis revealed extensive deletions in the mutS region of three strains, ranging from 7.5 to 17.3 kb relative to E. coli K-12 sequence, while the ECOR mutator contained a premature stop codon in addition to other nucleotide changes in the mutS coding sequence. These results provide insights into the status of the mutS gene and its product in pathogenic strains of E. coli.

The bacterial adaptive response gene, barA, encodes a novel conserved histidine kinase regulatory switch for adaptation and modulation of metabolism in Escherichia coli.

Histidine kinases are important prokaryotic determinants of cellular adaptation to environmental conditions, particularly stress. The highly conserved histidine kinase, BarA, encoded by the bacterial adaptive response gene, barA, is a member of the family of tripartite histidine kinases, and is involved in stress adaptation. BarA has been implicated to play a role during infection of epithelial cells. Homologues and orthologues of BarA have been found in pathogenic yeast, fungi, mould and in plants. The primary aim of this review is to assimilate evidence present in the current literature linking the role of BarA in stress response, and to support it with preliminary experimental evidence indicating that, it is indeed a global response regulator. In particular, the review focuses on the unusual domain structure of the BarA protein, its role in oxidative, weak acid, and osmotic stress responses and its role in biofilm formation. A preliminary genomic approach to identify downstream genes regulated by the BarA signaling pathway, using DNA microarray, is reported. The results demonstrate that BarA plays a global response regulatory role in cell division, carbon metabolism, iron metabolism and pili formation. The evolutionary significance of these types of histidine kinase sensors is reviewed in light of their roles in pathogenesis.