Steven L. Foley

Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production.

ABSTRACT Fresh and processed poultry have been frequently implicated in cases of human salmonellosis. Furthermore, increased consumption of meat and poultry has increased the potential for exposure to Salmonella enterica. While advances have been made in reducing the prevalence and frequency of Salmonella contamination in processed poultry, there is mounting pressure on commercial growers to prevent and/or eliminate these human pathogens in preharvest production facilities. Several factors contribute to Salmonella colonization in commercial poultry, including the serovar and the infectious dose. In the early 1900s, Salmonella enterica serovars Pullorum and Gallinarum caused widespread diseases in poultry, but vaccination and other voluntary programs helped eradicate pullorum disease and fowl typhoid from commercial flocks. However, the niche created by the eradication of these serovars was likely filled by S. Enteritidis, which proliferated in the bird populations. While this pathogen remains a significant problem in commercial egg and poultry production, its prevalence among poultry has been declining since the 1990s. Coinciding with the decrease of S. Enteritidis, S. Heidelberg and S. Kentucky have emerged as the predominant serovars in commercial broilers. In this review, we have highlighted bacterial genetic and host-related factors that may contribute to such shifts in Salmonella populations in commercial poultry and intervention strategies that could limit their colonization.

Salmonella Pathogenicity and Host Adaptation in Chicken-Associated Serovars

SUMMARY Enteric pathogens such as Salmonella enterica cause significant morbidity and mortality. S. enterica serovars are a diverse group of pathogens that have evolved to survive in a wide range of environments and across multiple hosts. S. enterica serovars such as S. Typhi, S. Dublin, and S. Gallinarum have a restricted host range, in which they are typically associated with one or a few host species, while S. Enteritidis and S. Typhimurium have broad host ranges. This review examines how S. enterica has evolved through adaptation to different host environments, especially as related to the chicken host, and continues to be an important human pathogen. Several factors impact host range, and these include the acquisition of genes via horizontal gene transfer with plasmids, transposons, and phages, which can potentially expand host range, and the loss of genes or their function, which would reduce the range of hosts that the organism can infect. S. Gallinarum, with a limited host range, has a large number of pseudogenes in its genome compared to broader-host-range serovars. S. enterica serovars such as S. Kentucky and S. Heidelberg also often have plasmids that may help them colonize poultry more efficiently. The ability to colonize different hosts also involves interactions with the hosts immune system and commensal organisms that are present. Thus, the factors that impact the ability of Salmonella to colonize a particular host species, such as chickens, are complex and multifactorial, involving the host, the pathogen, and extrinsic pressures. It is the interplay of these factors which leads to the differences in host ranges that we observe today.

Molecular Characterization of Resistance to Extended-Spectrum Cephalosporins in Clinical Escherichia coli Isolates from Companion Animals in the United States

ABSTRACT Resistance to extended-spectrum cephalosporins (ESC) among members of the family Enterobacteriaceae occurs worldwide; however, little is known about ESC resistance in Escherichia coli strains from companion animals. Clinical isolates of E. coli were collected from veterinary diagnostic laboratories throughout the United States from 2008 to 2009. E. coli isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime (MIC ≥ 16 μg/ml) and extended-spectrum-β-lactamase (ESBL) phenotypes were analyzed. PCR and sequencing were used to detect mutations in ESBL-encoding genes and the regulatory region of the chromosomal gene ampC. Conjugation experiments and plasmid identification were conducted to examine the transferability of resistance to ESCs. All isolates carried the blaCTX-M-1-group β-lactamase genes in addition to one or more of the following β-lactamase genes: blaTEM, blaSHV-3, blaCMY-2, blaCTX-M-14-like, and blaOXA-1. Different blaTEM sequence variants were detected in some isolates (n = 40). Three isolates harbored a blaTEM-181 gene with a novel mutation resulting in an Ala184Val substitution. Approximately 78% of the isolates had mutations in promoter/attenuator regions of the chromosomal gene ampC, one of which was a novel insertion of adenine between bases −28 and −29. Plasmids ranging in size from 11 to 233 kbp were detected in the isolates, with a common plasmid size of 93 kbp identified in 60% of isolates. Plasmid-mediated transfer of β-lactamase genes increased the MICs (≥16-fold) of ESCs for transconjugants. Replicon typing among isolates revealed the predominance of IncI and IncFIA plasmids, followed by IncFIB plasmids. This study shows the emergence of conjugative plasmid-borne ESBLs among E. coli strains from companion animals in the United States, which may compromise the effective therapeutic use of ESCs in veterinary medicine.

Horizontal Gene Transfer of a ColV Plasmid Has Resulted in a Dominant Avian Clonal Type of Salmonella enterica Serovar Kentucky

Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%), Typhimurium (15.0%) and Heidelberg (1.7%). We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard.

Meta-Analysis of Pulsed-Field Gel Electrophoresis Fingerprints Based on a Constructed Salmonella Database

A database was constructed consisting of 45,923 Salmonella pulsed-field gel electrophoresis (PFGE) patterns. The patterns, randomly selected from all submissions to CDC PulseNet during 2005 to 2010, included the 20 most frequent serotypes and 12 less frequent serotypes. Meta-analysis was applied to all of the PFGE patterns in the database. In the range of 20 to 1100 kb, serotype Enteritidis averaged the fewest bands at 12 bands and Paratyphi A the most with 19, with most serotypes in the 13−15 range among the 32 serptypes. The 10 most frequent bands for each of the 32 serotypes were sorted and distinguished, and the results were in concordance with those from distance matrix and two-way hierarchical cluster analyses of the patterns in the database. The hierarchical cluster analysis divided the 32 serotypes into three major groups according to dissimilarity measures, and revealed for the first time the similarities among the PFGE patterns of serotype Saintpaul to serotypes Typhimurium, Typhimurium var. 5-, and I 4,[5],12:i:-; of serotype Hadar to serotype Infantis; and of serotype Muenchen to serotype Newport. The results of the meta-analysis indicated that the pattern similarities/dissimilarities determined the serotype discrimination of PFGE method, and that the possible PFGE markers may have utility for serotype identification. The presence of distinct, serotype specific patterns may provide useful information to aid in the distribution of serotypes in the population and potentially reduce the need for laborious analyses, such as traditional serotyping.

Multilocus sequence typing of Salmonella strains by high-throughput sequencing of selectively amplified target genes.

Rapid development of next generation sequencing (NGS) technologies in recent years has made whole genome sequencing of bacterial genomes widely accessible. However, it is often unnecessary or not feasible to sequence the whole genome for most applications of genetic analyses in bacteria. Selectively capturing defined genomic regions followed by NGS analysis could be a promising approach for high-resolution molecular typing of a large set of strains. In this study, we describe a novel and straightforward PCR-based target-capturing method, hairpin-primed multiplex amplification (HPMA), which allows for simultaneous amplification of numerous target genes. To test the feasibility of NGS-based strain typing using HPMA, 20 target gene sequences were simultaneously amplified with barcode tagging in each of 41 Salmonella strains. The amplicons were then pooled and analyzed by 454 pyrosequencing. Analysis of the sequence data, as an extension of multilocus sequence typing (MLST), demonstrated the utility and potential of this novel typing method, MLST-seq, as a high-resolution strain typing method. With the rapidly increasing sequencing capacity of NGS, MLST-seq or its variations using different target enrichment methods can be expected to become a high-resolution typing method in the near future for high-throughput analysis of a large collection of bacterial strains.

Data mining tools for Salmonella characterization: application to gel-based fingerprinting analysis

BackgroundPulsed field gel electrophoresis (PFGE) is currently the most widely and routinely used method by the Centers for Disease Control and Prevention (CDC) and state health labs in the United States for Salmonella surveillance and outbreak tracking. Major drawbacks of commercially available PFGE analysis programs have been their difficulty in dealing with large datasets and the limited availability of analysis tools. There exists a need to develop new analytical tools for PFGE data mining in order to make full use of valuable data in large surveillance databases.ResultsIn this study, a software package was developed consisting of five types of bioinformatics approaches exploring and implementing for the analysis and visualization of PFGE fingerprinting. The approaches include PFGE band standardization, Salmonella serotype prediction, hierarchical cluster analysis, distance matrix analysis and two-way hierarchical cluster analysis. PFGE band standardization makes it possible for cross-group large dataset analysis. The Salmonella serotype prediction approach allows users to predict serotypes of Salmonella isolates based on their PFGE patterns. The hierarchical cluster analysis approach could be used to clarify subtypes and phylogenetic relationships among groups of PFGE patterns. The distance matrix and two-way hierarchical cluster analysis tools allow users to directly visualize the similarities/dissimilarities of any two individual patterns and the inter- and intra-serotype relationships of two or more serotypes, and provide a summary of the overall relationships between user-selected serotypes as well as the distinguishable band markers of these serotypes. The functionalities of these tools were illustrated on PFGE fingerprinting data from PulseNet of CDC.ConclusionsThe bioinformatics approaches included in the software package developed in this study were integrated with the PFGE database to enhance the data mining of PFGE fingerprints. Fast and accurate prediction makes it possible to elucidate Salmonella serotype information before conventional serological methods are pursued. The development of bioinformatics tools to distinguish the PFGE markers and serotype specific patterns will enhance PFGE data retrieval, interpretation and serotype identification and will likely accelerate source tracking to identify the Salmonella isolates implicated in foodborne diseases.

Massively parallel sequencing of enriched target amplicons for high-resolution genotyping of Salmonella serovars.

With next generation sequencing (NGS) technology, it is now possible to carry out in-depth, large-scale sequencing projects, such as whole genome sequencing, in a fast and inexpensive manner. However, often it is more practical and convenient to sequence and analyze multiple, smaller regions of the bacterial genome to gain valuable information about an organism. One such application is genotyping of bacterial strains by multilocus sequence typing (MLST) that involves PCR and sequencing analysis of typically 7 housekeeping genes. Recently, we described a novel MLST method, called MLST-seq that combines a PCR-based target enrichment method and NGS technology to simultaneously analyze numerous target gene sequences, thereby improving the resolution and high-throughput capacity of current MLST approaches. However, the performance of the MLST-seq method was hampered from a substantial bias in target enrichment step. In this study, we used an improved target enrichment method using hairpin selectors to amplify 21 target genes simultaneously from each of 41 Salmonella strains. The resulting amplicons tagged with strain-specific barcodes were pooled and sequenced en masse by 454 pyrosequencing. Analysis of sequence data from 38 Salmonella strains using combinations of 3, 7 and 14 target genes resulted in 23, 32 and 37 distinct allelic profiles, respectively. These results demonstrated that MLST-seq with an increased number of target genes is an efficient way to improve discrimination among closely-related strains of Salmonella. With the rapidly increasing sequencing capacity of NGS technologies combined with further improvements in target capturing methods, MLST-seq could become a promising approach to perform high-resolution strain typing of a large collection of Salmonella, and likely other genera in a labor- and cost-efficient manner in the future.

Salmonella Heidelberg in Layer Hens and Egg Production: Incidence and Potential Issues

Abstract Food-borne Salmonella continues to be a public health issue both from an illness standpoint as well as economically. Poultry and eggs represent a primary source of food-borne Salmonella with several serovars usually being the primary isolates specifically identified as originating from these food product sources. Salmonella Heidelberg has emerged as a serovar of increasing prominence, particularly in some of the more recent egg-associated outbreaks. In this chapter the incidence of S. Heidelberg in poultry and eggs as well as characteristics and ability to cause disease as a food-borne pathogen are discussed. Particular issues such as virulence capabilities and antimicrobial resistance are also described. Finally, the adaptability of S. Heidelberg and implications are summarized.Food-borne Salmonella continues to be a public health issue both from an illness standpoint as well as economically. Poultry and eggs represent a primary source of food-borne Salmonella with several serovars usually being the primary isolates specifically identified as originating from these food product sources. Salmonella Heidelberg has emerged as a serovar of increasing prominence, particularly in some of the more recent egg-associated outbreaks. In this chapter the incidence of S. Heidelberg in poultry and eggs as well as characteristics and ability to cause disease as a food-borne pathogen are discussed. Particular issues such as virulence capabilities and antimicrobial resistance are also described. Finally, the adaptability of S. Heidelberg and implications are summarized.

Structure and Inhibitor Specificity of L,D-Transpeptidase (LdtMt2) from Mycobacterium tuberculosis and Antibiotic Resistance: Calcium Binding Promotes Dimer Formation

The final step of peptidoglycan (PG) synthesis in all bacteria is the formation of cross-linkage between PG-stems. The cross-linking between amino acids in different PG chains gives the peptidoglycan cell wall a 3-dimensional structure and adds strength and rigidity to it. There are two distinct types of cross-linkages in bacterial cell walls. D,D-transpeptidase (D,D-TPs) generate the classical 4➔3 cross-linkages and the L,D-transpeptidase (L,D-TPs) generate the 3➔3 non-classical peptide cross-linkages. The present study is aimed at understanding the nature of drug resistance associated with L,D-TP and gaining insights for designing novel antibiotics against multi-drug resistant bacteria. Penicillin and cephalosporin classes of β-lactams cannot inhibit L,D-TP function; however, carbapenems inactivate its function. We analyzed the structure of L,D-TP of Mycobacterium tuberculosis in the apo form and in complex with meropenem and imipenem. The periplasmic region of L,D-TP folds into three domains. The catalytic residues are situated in the C-terminal domain. The acylation reaction occurs between carbapenem antibiotics and the catalytic Cys-354 forming a covalent complex. This adduct formation mimics the acylation of L,D-TP with the donor PG-stem. A novel aspect of this study is that in the crystal structures of the apo and the carbapenem complexes, the N-terminal domain has a muropeptide unit non-covalently bound to it. Another interesting observation is that the calcium complex crystallized as a dimer through head and tail interactions between the monomers.