Poultry Processing Interventions Reduce Salmonella Serovar Complexity on Postchill Young Chicken Carcasses as Determined by Deep Serotyping.

Nearly 20% of salmonellosis cases are attributed to broilers, with renewed efforts to reduce Salmonella during broiler production and processing. A limitation to Salmonella culture is that often a single colony is picked for characterization, favoring isolation of the most abundant serovar found in a sample, while low abundance serovars can remain undetected. We used a deep serotyping approach, CRISPR-SeroSeq (serotyping by sequencing the clustered regularly interspaced palindromic repeats), to assess Salmonella serovar complexity during broiler processing and to determine the impact of antimicrobial interventions upon serovar population dynamics. Paired hot rehang and postchill young chicken carcasses were collected from establishments across the United States from August to November 2022. CRISPR-SeroSeq was performed on Salmonella culture-positive hot rehang (n = 153) and postchill (n = 38) samples, including 31 paired hot rehang and postchill samples. Multiple serovars were detected in 48.4% (74/153) and 7.9% (3/38) of hot rehang and postchill samples, respectively. On average, hot rehang carcasses contained 1.6 serovars, compared to 1.1 serovars at postchill (Mann Whitney U, p = 0.00018). Nineteen serovars were identified with serovar Kentucky the most common at hot rehang (72.5%; 111/153) and postchill (73.7%; 28/38). Serovar Infantis prevalence was higher at hot rehang (39.9%; 61/153) than in postchill (7.9%; 3/38). At hot rehang, serovar Enteritidis was outnumbered by other serovars 81.3% (13/16) of the time but was always the single or most abundant serovar detected when it was present at postchill (n = 5). We observed 98.4% (188/191) concordance between traditional isolation with serotyping and CRISPR-SeroSeq. Deep serotyping was able to explain serovar discrepancies between paired hot rehang and postchill samples when only traditional isolation and serotyping methods were used. These data demonstrate that processing interventions are effective in reducing Salmonella serovar complexity.

Genomic Diversity, Antimicrobial Resistance, and Virulence Gene Profiles of Salmonella Serovar Kentucky Isolated from Humans, Food, and Animal Ceca Content Sources in the United States.

Salmonella serovar Kentucky is frequently isolated from chickens and dairy cattle, but recovery from humans is comparatively low based on the U.S. National Antimicrobial Resistance Monitoring System (NARMS) reports. We aimed to better describe the genetic diversity, antimicrobial resistance, and virulence determinants of Salmonella Kentucky isolates from humans, food animal ceca, retail meat and poultry products, imported foods and food products, and other samples. We analyzed the genomes of 774 Salmonella Kentucky isolates and found that 63% (54/86) of human isolates were sequence type (ST)198, 33% (29/86) were ST152, and 3.5% (3/86) were ST314. Ninety-one percent (570/629) of cecal isolates and retail meat and poultry isolates were ST152 or ST152-like (one allele difference), and 9.2% (58/629) were ST198. Isolates from imported food were mostly ST198 (60%, 22/37) and ST314 (29.7%, 11/37). ST198 isolates clustered into two main lineages. Clade ST198.2 comprised almost entirely isolates from humans and imported foods, all containing triple mutations in the quinolone resistance-determining region (QRDR) that confer resistance to fluoroquinolones. Clade ST198.1 contained isolates from humans, ceca, retail meat and poultry products, and imported foods that largely lacked QRDR mutations. ST152 isolates from cattle had a lineage (Clade 2) distinct from ST152 isolates from chicken (Clade 4), and half of ST152 human isolates clustered within two other clades (Clades 1 and 3), largely distinct from Clades 2 and 4. Although clinical illness associated with Salmonella Kentucky is low, ST198 appears to account for most human infections in the Unites States but is uncommon among ceca of domestic food animals and retail meat and poultry products. These findings, combined with human exposure data, suggest that fluoroquinolone-resistant ST198 infections may be linked to the consumption of food products that are imported or consumed while traveling. We also found unique differences in the composition of virulence genes and antimicrobial resistance genes among the clades, which may provide clues to the host specificity and pathogenicity of Salmonella Kentucky lineages.

Use of Whole Genome Sequencing by the Federal Interagency Collaboration for Genomics for Food and Feed Safety in the United States.

ABSTRACT: This multiagency report developed by the Interagency Collaboration for Genomics for Food and Feed Safety provides an overview of the use of and transition to whole genome sequencing (WGS) technology for detection and characterization of pathogens transmitted commonly by food and for identification of their sources. We describe foodborne pathogen analysis, investigation, and harmonization efforts among the following federal agencies: National Institutes of Health; Department of Health and Human Services, Centers for Disease Control and Prevention (CDC) and U.S. Food and Drug Administration (FDA); and the U.S. Department of Agriculture, Food Safety and Inspection Service, Agricultural Research Service, and Animal and Plant Health Inspection Service. We describe single nucleotide polymorphism, core-genome, and whole genome multilocus sequence typing data analysis methods as used in the PulseNet (CDC) and GenomeTrakr (FDA) networks, underscoring the complementary nature of the results for linking genetically related foodborne pathogens during outbreak investigations while allowing flexibility to meet the specific needs of Interagency Collaboration partners. We highlight how we apply WGS to pathogen characterization (virulence and antimicrobial resistance profiles) and source attribution efforts and increase transparency by making the sequences and other data publicly available through the National Center for Biotechnology Information. We also highlight the impact of current trends in the use of culture-independent diagnostic tests for human diagnostic testing on analytical approaches related to food safety and what is next for the use of WGS in the area of food safety.

Long-Read Sequencing Reveals Evolution and Acquisition of Antimicrobial Resistance and Virulence Genes in Salmonella enterica.

Salmonella enterica is a significant and phylogenetically diverse zoonotic pathogen. To understand its genomic heterogeneity and antimicrobial resistance, we performed long-read sequencing on Salmonella isolated from retail meats and food animals. A collection of 134 multidrug-resistant isolates belonging to 33 serotypes were subjected to PacBio sequencing. One major locus of diversity among these isolates was the presence and orientation of Salmonella pathogenic islands (SPI), which varied across different serotypes but were largely conserved within individual serotypes. We also identified insertion of an IncQ resistance plasmid into the chromosome of fourteen strains of serotype I 4,[5],12:i:- and the Salmonella genomic island 1 (SGI-1) in five serotypes. The presence of various SPIs, SGI-1 and integrated plasmids contributed significantly to the genomic variability and resulted in chromosomal resistance in 55.2% (74/134) of the study isolates. A total of 93.3% (125/134) of isolates carried at least one plasmid, with isolates carrying up to seven plasmids. We closed 233 plasmid sequences of thirteen replicon types, along with twelve hybrid plasmids. Some associations between Salmonella isolate source, serotype, and plasmid type were seen. For instance, IncX plasmids were more common in serotype Kentucky from retail chicken. Plasmids IncC and IncHI had on average more than five antimicrobial resistance genes, whereas in IncX, it was less than one per plasmid. Overall, 60% of multidrug resistance (MDR) strains that carried >3 AMR genes also carried >3 heavy metal resistance genes, raising the possibility of co-selection of antimicrobial resistance in the presence of heavy metals. We also found nine isolates representing four serotypes that carried virulence plasmids with the spv operon. Together, these data demonstrate the power of long-read sequencing to reveal genomic arrangements and integrated plasmids with a high level of resolution for tracking and comparing resistant strains from different sources. Additionally, the findings from this study will help expand the reference set of closed Salmonella genomes that can be used to improve genome assembly from short-read data commonly used in One Health antimicrobial resistance surveillance.

Core Genome MLST for Source Attribution of Campylobacter coli.

Campylobacter species are among the leading foodborne bacterial agents of human diarrheal illness. The majority of campylobacteriosis has been attributed to Campylobacter jejuni (85% or more), followed by Campylobacter coli (5-10%). The distribution of C. jejuni and C. coli varies by host organism, indicating that the contribution to human infection may differ between isolation sources. To address the relative contribution of each source to C. coli infections in humans, core genome multilocus sequence type with a 200-allele difference scheme (cgMLST200) was used to determine cgMLST type for 3,432 C. coli isolated from food animals (n = 2,613), retail poultry meats (n = 389), human clinical settings (n = 285), and environmental sources (n = 145). Source attribution was determined by analyzing the core genome with a minimal multilocus distance methodology (MMD). Using MMD, a higher proportion of the clinical C. coli population was attributed to poultry (49.6%) and environmental (20.9%) sources than from cattle (9.8%) and swine (3.2%). Within the population of C. coli clinical isolates, 70% of the isolates that were attributed to non-cecal retail poultry, dairy cattle, beef cattle and environmental waters came from two cgMLST200 groups from each source. The most common antibiotic resistance genes among all C. coli were tetO (65.6%), bla OXA - 193 (54.2%), aph(3')-IIIa (23.5%), and aadE-Cc (20.1%). Of the antibiotic resistance determinants, only one gene was isolated from a single source: bla OXA - 61 was only isolated from retail poultry. Within cgMLST200 groups, 17/17 cgMLST200-435 and 89/92 cgMLST200-707 isolates encoded for aph(3')-VIIa and 16/16 cgMLST200-319 harbored aph(2')-If genes. Distribution of bla OXA alleles showed 49/50 cgMLST200-5 isolates contained bla OXA - 498 while bla OXA - 460 was present in 37/38 cgMLST200-650 isolates. The cgMLST200-514 group revealed both ant(6)-Ia and sat4 resistance genes in 23/23 and 22/23 isolates, respectively. Also, cgMLST200-266 and cgMLST200-84 had GyrAT86I mutation with 16/16 (100%) and 14/15 (93.3%), respectively. These findings illustrate how cgMLST and MMD methods can be used to evaluate the relative contribution of known sources of C. coli to the human burden of campylobacteriosis and how cgMLST typing can be used as an indicator of antimicrobial resistance in C. coli.

AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence.

Antimicrobial resistance (AMR) is a significant public health threat. With the rise of affordable whole genome sequencing, in silico approaches to assessing AMR gene content can be used to detect known resistance mechanisms and potentially identify novel mechanisms. To enable accurate assessment of AMR gene content, as part of a multi-agency collaboration, NCBI developed a comprehensive AMR gene database, the Bacterial Antimicrobial Resistance Reference Gene Database and the AMR gene detection tool AMRFinder. Here, we describe the expansion of the Reference Gene Database, now called the Reference Gene Catalog, to include putative acid, biocide, metal, stress resistance genes, in addition to virulence genes and species-specific point mutations. Genes and point mutations are classified by broad functions, as well as more detailed functions. As we have expanded both the functional repertoire of identified genes and functionality, NCBI released a new version of AMRFinder, known as AMRFinderPlus. This new tool allows users the option to utilize only the core set of AMR elements, or include stress response and virulence genes, too. AMRFinderPlus can detect acquired genes and point mutations in both protein and nucleotide sequence. In addition, the evidence used to identify the gene has been expanded to include whether nucleotide or protein sequence was used, its location in the contig, and presence of an internal stop codon. These database improvements and functional expansions will enable increased precision in identifying AMR genes, linking AMR genotypes and phenotypes, and determining possible relationships between AMR, virulence, and stress response.

Carriage and Gene Content Variability of the pESI-Like Plasmid Associated with Salmonella Infantis Recently Established in United States Poultry Production.

Salmonella Infantis carrying extended spectrum ß-lactamase blaCTX-M-65 on a pESI-like megaplasmid has recently emerged in United States poultry. In order to determine the carriage rate and gene content variability of this plasmid in U.S. Salmonella Infantis, whole genome sequences of Salmonella isolates from humans and animals in the U.S. and internationally containing the pESI-like plasmid were analyzed. The U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) identified 654 product sampling isolates containing pESI-like plasmids through hazard analysis and critical control point (HACCP) verification testing in 2017 and 2018. The Centers for Disease Control and Prevention identified 55 isolates with pESI-like plasmids in 2016-2018 through the National Antimicrobial Resistance Monitoring System. Approximately 49% of pESI-like plasmids from FSIS verification isolates and 71% from CDC NARMS contained blaCTX-M-65. Pan-plasmid genome analysis was also performed. All plasmids contained traN and more than 95% contained 172 other conserved genes; 61% contained blaCTX-M-65. In a hierarchical clustering analysis, some plasmids from U.S. animal sources clustered together and some plasmids from South America clustered together, possibly indicating multiple plasmid lineages. However, most plasmids contained similar genes regardless of origin. Carriage of the pESI-like plasmid in U.S. appears to be limited to Salmonella Infantis and carriage rates increased from 2017 to 2018.

Rapid, multiplexed, whole genome and plasmid sequencing of foodborne pathogens using long-read nanopore technology.

U.S. public health agencies have employed next-generation sequencing (NGS) as a tool to quickly identify foodborne pathogens during outbreaks. Although established short-read NGS technologies are known to provide highly accurate data, long-read sequencing is still needed to resolve highly-repetitive genomic regions and genomic arrangement, and to close the sequences of bacterial chromosomes and plasmids. Here, we report the use of long-read nanopore sequencing to simultaneously sequence the entire chromosome and plasmid of Salmonella enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. We developed a rapid and random sequencing approach coupled with de novo genome assembly within a customized data analysis workflow that uses publicly-available tools. In sequencing runs as short as four hours, using the MinION instrument, we obtained full-length genomes with an average identity of 99.87% for Salmonella Bareilly and 99.89% for E. coli in comparison to the respective MiSeq references. These nanopore-only assemblies provided readily available information on serotype, virulence factors, and antimicrobial resistance genes. We also demonstrate the potential of nanopore sequencing assemblies for rapid preliminary phylogenetic inference. Nanopore sequencing provides additional advantages as very low capital investment and footprint, and shorter (10 hours library preparation and sequencing) turnaround time compared to other NGS technologies.

Genomic Analysis of Emerging Florfenicol-Resistant Campylobacter coli Isolated from the Cecal Contents of Cattle in the United States.

Genomic analyses were performed on florfenicol-resistant (FFNr) Campylobacter coli isolates recovered from cattle, and the cfr(C) gene-associated multidrug resistance (MDR) plasmid was characterized. Sixteen FFNrC. coli isolates recovered between 2013 and 2018 from beef cattle were sequenced using MiSeq. Genomes and plasmids were found to be closed for three of the isolates using the PacBio system. Single nucleotide polymorphisms (SNPs) across the genome and the structures of MDR plasmids were investigated. Conjugation experiments were performed to determine the transferability of cfr(C)-associated MDR plasmids. The spectrum of resistance encoded by the cfr(C) gene was further investigated by agar dilution antimicrobial susceptibility testing. All 16 FFNr isolates were MDR and exhibited coresistance to ciprofloxacin, nalidixic acid, clindamycin, and tetracycline. All isolates shared the same resistance genotype, carrying aph (3')-III, hph, ΔaadE (truncated), blaOXA-61, cfr(C), and tet(O) genes plus a mutation of GyrA (T86I). The cfr(C), aph (3')-III, hph, ΔaadE, and tet(O) genes were colocated on transferable MDR plasmids ranging in size from 48 to 50 kb. These plasmids showed high sequence homology with the pTet plasmid and carried several Campylobacter virulence genes, including virB2, virB4, virB5, VirB6, virB7, virB8, virb9, virB10, virB11, and virD4 The cfr(C) gene conferred resistance to florfenicol (8 to 32 µg/ml), clindamycin (512 to 1,024 µg/ml), linezolid (128 to 512 µg/ml), and tiamulin (1,024 µg/ml). Phylogenetic analysis showed SNP differences ranging from 11 to 2,248 SNPs among the 16 isolates. The results showed that the cfr(C) gene located in the conjugative pTet MDR/virulence plasmid is present in diverse strains, where it confers high levels of resistance to several antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. This report highlights the power of genomic antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies.IMPORTANCECampylobacter is a leading cause of foodborne diarrheal illness worldwide, with more than one million cases each year in the United States alone. The global emergence of antimicrobial resistance in this pathogen has become a growing public health concern. Florfenicol-resistant (FFNr) Campylobacter has been very rare in the United States. In this study, we employed whole-genome sequencing to characterize 16 multidrug-resistant Campylobacter coli isolates recovered from cattle in the United States. A gene [cfr(C)] was found to be responsible for resistance not only to florfenicol but also to several other antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. The results showed that cfr(C) is located in a conjugative pTet MDR/virulence plasmid. This report highlights the power of antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies.

Antimicrobial Resistance Genes, Cassettes, and Plasmids Present in Salmonella enterica Associated With United States Food Animals.

The ability of antimicrobial resistance (AR) to transfer, on mobile genetic elements (MGEs) between bacteria, can cause the rapid establishment of multidrug resistance (MDR) in bacteria from animals, thus creating a foodborne risk to human health. To investigate MDR and its association with plasmids in Salmonella enterica, whole genome sequence (WGS) analysis was performed on 193 S. enterica isolated from sources associated with United States food animals between 1998 and 2011; 119 were resistant to at least one antibiotic tested. Isolates represented 86 serotypes and variants, as well as diverse phenotypic resistance profiles. A total of 923 AR genes and 212 plasmids were identified among the 193 strains. Every isolate contained at least one AR gene. At least one plasmid was detected in 157 isolates. Genes were identified for resistance to aminoglycosides (n = 472), ß-lactams (n = 84), tetracyclines (n = 171), sulfonamides (n = 91), phenicols (n = 42), trimethoprim (n = 8), macrolides (n = 5), fosfomycin (n = 48), and rifampicin (n = 2). Plasmid replicon types detected in the isolates were A/C (n = 32), ColE (n = 76), F (n = 43), HI1 (n = 4), HI2 (n = 20), I1 (n = 62), N (n = 4), Q (n = 7), and X (n = 35). Phenotypic resistance correlated with the AR genes identified in 95.4% of cases. Most AR genes were located on plasmids, with many plasmids harboring multiple AR genes. Six antibiotic resistance cassette structures (ARCs) and one pseudo-cassette were identified. ARCs contained between one and five resistance genes (ARC1: sul2, strAB, tetAR; ARC2: aac3-iid; ARC3: aph, sph; ARC4: cmy-2; ARC5: floR; ARC6: tetB; pseudo-ARC: aadA, aac3-VIa, sul1). These ARCs were present in multiple isolates and on plasmids of multiple replicon types. To determine the current distribution and frequency of these ARCs, the public NCBI database was analyzed, including WGS data on isolates collected by the USDA Food Safety and Inspection Service (FSIS) from 2014 to 2018. ARC1, ARC4, and ARC5 were significantly associated with cattle isolates, while ARC6 was significantly associated with chicken isolates. This study revealed that a diverse group of plasmids, carrying AR genes, are responsible for the phenotypic resistance seen in Salmonella isolated from United States food animals. It was also determined that many plasmids carry similar ARCs.

Proposed Epidemiological Cutoff Values for Ceftriaxone, Cefepime, and Colistin in Salmonella.

We tested a diverse set of 500 isolates of nontyphoidal Salmonella enterica subsp. enterica from various animal, food, and human clinical sources for susceptibility to antimicrobials currently lacking epidemiological cutoff values (ECOFFs) set by the European Committee on Antimicrobial Susceptibility Testing. A consortium of five different laboratories each tested 100 isolates, using broth microdilution panels containing twofold dilutions of ceftriaxone, cefepime, and colistin to determine the minimum inhibitory concentrations of each drug when tested against the Salmonella isolates. Based on the resulting data, new ECOFFs of 0.25 µg/mL for ceftriaxone, 0.12 µg/mL for cefepime, and 2 µg/mL for colistin have been proposed. These thresholds will aid in the identification of Salmonella that have phenotypically detectable resistance mechanisms to these important antimicrobials.

Benchmark datasets for phylogenomic pipeline validation, applications for foodborne pathogen surveillance.

BACKGROUND: As next generation sequence technology has advanced, there have been parallel advances in genome-scale analysis programs for determining evolutionary relationships as proxies for epidemiological relationship in public health. Most new programs skip traditional steps of ortholog determination and multi-gene alignment, instead identifying variants across a set of genomes, then summarizing results in a matrix of single-nucleotide polymorphisms or alleles for standard phylogenetic analysis. However, public health authorities need to document the performance of these methods with appropriate and comprehensive datasets so they can be validated for specific purposes, e.g., outbreak surveillance. Here we propose a set of benchmark datasets to be used for comparison and validation of phylogenomic pipelines. METHODS: We identified four well-documented foodborne pathogen events in which the epidemiology was concordant with routine phylogenomic analyses (reference-based SNP and wgMLST approaches). These are ideal benchmark datasets, as the trees, WGS data, and epidemiological data for each are all in agreement. We have placed these sequence data, sample metadata, and "known" phylogenetic trees in publicly-accessible databases and developed a standard descriptive spreadsheet format describing each dataset. To facilitate easy downloading of these benchmarks, we developed an automated script that uses the standard descriptive spreadsheet format. RESULTS: Our "outbreak" benchmark datasets represent the four major foodborne bacterial pathogens (Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter jejuni) and one simulated dataset where the "known tree" can be accurately called the "true tree". The downloading script and associated table files are available on GitHub: https://github.com/WGS-standards-and-analysis/datasets. DISCUSSION: These five benchmark datasets will help standardize comparison of current and future phylogenomic pipelines, and facilitate important cross-institutional collaborations. Our work is part of a global effort to provide collaborative infrastructure for sequence data and analytic tools-we welcome additional benchmark datasets in our recommended format, and, if relevant, we will add these on our GitHub site. Together, these datasets, dataset format, and the underlying GitHub infrastructure present a recommended path for worldwide standardization of phylogenomic pipelines.

Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl-L-alanine amidase as a potential antimicrobial to control the bacterium.

Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal, and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to identify putative prophage lysins or autolysins by BLAST analyses encoded by the genomes of C. perfringens isolates. A predicted N-acetylmuramoyl-L-alanine amidase or MurNAc-LAA (also known as peptidoglycan aminohydrolase, NAMLA amidase, NAMLAA, amidase 3, and peptidoglycan amidase; EC 3.5.1.28) was identified that would hydrolyze the amide bond between N-acetylmuramoyl and L-amino acids in certain cell wall glycopeptides. The gene encoding this protein was subsequently cloned from genomic DNA of a C. perfringens isolate by polymerase chain reaction, and the gene product (PlyCpAmi) was expressed to determine if it could be utilized as an antimicrobial to control the bacterium. By spot assay, lytic zones were observed for the purified amidase and the E. coli expression host cellular lysate containing the amidase gene. Turbidity reduction and plate counts of C. perfringens cultures were significantly reduced by the expressed protein and observed morphologies for cells treated with the amidase appeared vacuolated, non-intact, and injured compared to the untreated cells. Among a variety of C. perfringens strains, there was little gene sequence heterogeneity that varied from 1 to 21 nucleotide differences. The results further demonstrate that it is possible to discover lytic proteins encoded in the genomes of bacteria that could be utilized to control bacterial pathogens.

The poultry-associated microbiome: network analysis and farm-to-fork characterizations.

Microbial communities associated with agricultural animals are important for animal health, food safety, and public health. Here we combine high-throughput sequencing (HTS), quantitative-PCR assays, and network analysis to profile the poultry-associated microbiome and important pathogens at various stages of commercial poultry production from the farm to the consumer. Analysis of longitudinal data following two flocks from the farm through processing showed a core microbiome containing multiple sequence types most closely related to genera known to be pathogenic for animals and/or humans, including Campylobacter, Clostridium, and Shigella. After the final stage of commercial poultry processing, taxonomic richness was ca. 2-4 times lower than the richness of fecal samples from the same flocks and Campylobacter abundance was significantly reduced. Interestingly, however, carcasses sampled at 48 hr after processing harboured the greatest proportion of unique taxa (those not encountered in other samples), significantly more than expected by chance. Among these were anaerobes such as Prevotella, Veillonella, Leptrotrichia, and multiple Campylobacter sequence types. Retail products were dominated by Pseudomonas, but also contained 27 other genera, most of which were potentially metabolically active and encountered in on-farm samples. Network analysis was focused on the foodborne pathogen Campylobacter and revealed a majority of sequence types with no significant interactions with other taxa, perhaps explaining the limited efficacy of previous attempts at competitive exclusion of Campylobacter. These data represent the first use of HTS to characterize the poultry microbiome across a series of farm-to-fork samples and demonstrate the utility of HTS in monitoring the food supply chain and identifying sources of potential zoonoses and interactions among taxa in complex communities.

Influence of primer sequences and DNA extraction method on detection of non-O157 Shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, particularly those caused by the "big six" or "top six" non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx(1), stx(2), and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx(1d), stx(2e), and stx(2g), are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P < 0.0001) and eae assay (P < 0.0001), thereby increasing the ability to detect the targets. The use of different stx primers and probes increased the subtypes detected to include stx(1d), stx(2e), and stx(2g), and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.

Isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 from ground beef using modified rainbow agar and post-immunomagnetic separation acid treatment.

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coli (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121, and O145 from ground beef enrichments. A post-immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (1 to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111, O121, and O145 from raw ground beef.

Chicken intestine microbiota following the administration of lupulone, a hop-based antimicrobial.

The use of antibiotic growth promotants in poultry rearing is a public health concern due to antibiotic resistance in bacteria and the harborage of resistance genes. Lupulone, a hop ß-acid from Humulus lupulus, has been considered as a potential feed additive growth promotant. Here, the effect of lupulone was evaluated for its effect on the microbiota of the chicken intestine. The intestinal microbiota of broilers was quantified after the addition of 125 mg L(-1) lupulone to water and challenge with Clostridium perfringens. Microbial DNA was extracted from the broiler midgut and cecal sections and bacterial groups were quantified using real-time PCR. The predominant cecal bacterial groups were Clostridium leptum subgroup 16S rRNA gene Cluster IV, Clostridium coccoides subgroup 16S rRNA gene Clusters XIVa and XIVb and Bacteroides, whereas Lactobacillus, the Enterobacteriaceae family and Enterococcus dominated the midgut. Lupulone at 125 mg L(-1) significantly decreased the C. perfringens subgroup 16S rRNA gene Cluster I, which contains several pathogenic species, in both the midgut and the cecum and Lactobacillus in the midgut. No significant changes were noted in the overall microbiota for the cecum or the midgut. Lupulone warrants further evaluation as a botanical agent to mitigate C. perfringens overgrowth in antibiotic-free reared poultry.

Pulsed-field gel electrophoresis subtypes of shiga toxin-producing Escherichia coli O157 isolated from ground beef and humans, United States, 2001-2006.

Pulsed-field gel electrophoresis XbaI patterns of Shiga toxin-producing Escherichia coli O157 (STEC O157) isolates (n = 156) found in ground beef sampled from U.S. processing plants and retail stores during 2001 to 2006 were summarized and compared with XbaI patterns from human STEC O157 isolates (n = 14,591) in the national PulseNet E. coli database. Four ground beef samples contained more than one pulsed-field gel electrophoresis subtype of STEC O157. Of the 117 unique patterns found in ground beef, 100 (85%) appeared only once, and 17 (15%) were found in more than one isolate. The six patterns that appeared most frequently in human isolates were also found among the eight most common ground beef patterns. The yearly proportion of human isolates with the two most common patterns changed inversely, such that these patterns traded dominance over the study period. Human isolates with patterns that were first detected in both ground beef and humans contemporaneously were clustered in a 6-month window around the time of the respective ground beef sample. Of the 156 ground beef isolates, 82 (53%) were indistinguishable from at least one human isolate in this 6-month window. The yearly proportions of human STEC O157 isolates that were indistinguishable from ground beef isolates decreased significantly from 2002 to 2003 (12.3-0.8%), and then increased significantly from 2003 to 2006 (overall 0.8-12.6%). This increase in the numbers of human isolates that matched a ground beef isolate occurred during a period of relatively consistent rates of ground beef contamination with STEC O157. Pattern similarity of STEC O157 isolates derived from ground beef and clinical cases may serve as a good predictor of human incidence trends.