Rajesh Nayak

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.

Molecular typing methodologies for microbial source tracking and epidemiological investigations of Gram-negative bacterial foodborne pathogens ☆

Gram-negative bacterial foodborne pathogens are a worldwide cause of morbidity and mortality. The ability to carry out epidemiological investigations to determine the primary sources of bacterial contamination is important to improve public health. Multiple methods are available for bacterial source tracking and to determine the distribution of pathogens isolated from sick patients. The molecular based typing methods available fall into three general categories: those based on restriction analysis of the bacterial DNA; those based on polymerase chain reaction (PCR) amplification of particular genetic targets; and those based on the identification of DNA sequence polymorphisms. The techniques that are examined in this review include: plasmid analysis, restriction fragment length polymorphism methods, pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, PCR-based genotyping, variable number of tandem repeat analysis, multilocus sequence typing, and single nucleotide polymorphism analysis. These methods are described along with a discussion of the strengths and weaknesses of the techniques for genotyping the major Gram-negative foodborne pathogens--Campylobacter spp., Salmonella enterica, Shigella spp., Escherichia coli, and Yersinia enterocolitica.

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.

Emergence of a New Delhi Metallo-β-Lactamase (NDM-1)-Encoding Gene in Clinical Escherichia coli Isolates Recovered from Companion Animals in the United States

The rapid spread of extended-spectrum β-lactamases (ESBLs) and quinolone resistance in Escherichia coli in companion animals (1-2) has increased concern among veterinarians9 to find an alternative therapy to treat clinical infections caused by these resistant organisms.…

Longitudinal study of distributions of similar antimicrobial-resistant Salmonella serovars in pigs and their environment in two distinct swine production systems.

ABSTRACT The aim of this longitudinal study was to determine and compare the prevalences and genotypic profiles of antimicrobial-resistant (AR) Salmonella isolates from pigs reared in antimicrobial-free (ABF) and conventional production systems at farm, at slaughter, and in their environment. We collected 2,889 pig fecal and 2,122 environmental (feed, water, soil, lagoon, truck, and floor swabs) samples from 10 conventional and eight ABF longitudinal cohorts at different stages of production (farrowing, nursery, finishing) and slaughter (postevisceration, postchill, and mesenteric lymph nodes [MLN]). In addition, we collected 1,363 carcass swabs and 205 lairage and truck samples at slaughter. A total of 1,090 Salmonella isolates were recovered from the samples; these were isolated with a significantly higher prevalence in conventionally reared pigs (4.0%; n = 66) and their environment (11.7%; n = 156) than in ABF pigs (0.2%; n = 2) and their environment (0.6%; n = 5) (P < 0.001). Salmonella was isolated from all stages at slaughter, including the postchill step, in the two production systems. Salmonella prevalence was significantly higher in MLN extracted from conventional carcasses than those extracted from ABF carcasses (P < 0.001). We identified a total of 24 different serotypes, with Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Anatum, Salmonella enterica serovar Infantis, and Salmonella enterica serovar Derby being predominant. The highest frequencies of antimicrobial resistance (AR) were exhibited to tetracycline (71%), sulfisoxazole (42%), and streptomycin (17%). Multidrug resistance (resistance to ≥3 antimicrobials; MDR) was detected in 27% (n = 254) of the Salmonella isolates from the conventional system. Our study reports a low prevalence of Salmonella in both production systems in pigs on farms, while a higher prevalence was detected among the carcasses at slaughter. The dynamics of Salmonella prevalence in pigs and carcasses were reciprocated in the farm and slaughter environment, clearly indicating an exchange of this pathogen between the pigs and their surroundings. Furthermore, the phenotypic and genotypic fingerprint profile results underscore the potential role played by environmental factors in dissemination of AR Salmonella to pigs.

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.