Detection and Distribution of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in Campylobacter jejuni Isolates from Chicken Livers.

Campylobacter jejuni is the leading foodborne bacterial pathogen that causes human gastroenteritis worldwide linked to the consumption of undercooked broiler livers. Application of bacteriophages during poultry production has been used as an alternative approach to reduce contamination of poultry meat by Campylobacter. To make this approach effective, understanding the presence of the bacteriophage sequences in the CRISPR spacers in C. jejuni is critical as they may confer bacterial resistance to bacteriophage treatment. Therefore, in this study, we explored the distribution of the CRISPR arrays from 178 C. jejuni isolated from chicken livers between January and July 2018. Genomic DNA of C. jejuni isolates was extracted, and CRISPR type 1 sequences were amplified by PCR. Amplicons were purified and sequenced by the Sanger dideoxy sequencing method. Direct repeats (DRs) and spacers of CRISPR sequences were identified using the CRISPRFinder program. Further, spacer sequences were submitted to the CRISPRTarget to identify potential homology to bacteriophage types. Even though CRISPR-Cas is reportedly not an active system in Campylobacter, a total of 155 (87%) C. jejuni isolates were found to harbor CRISPR sequences; one type of DR was identified in all 155 isolates. The CRISPR loci lengths ranged from 97 to 431 nucleotides. The numbers of spacers ranged from one to six. A total of 371 spacer sequences were identified in the 155 isolates that could be grouped into 51 distinctive individual sequences. Further comparison of these 51 spacer sequences with those in databases showed that most spacer sequences were homologous to Campylobacter bacteriophage DA10. The results of our study provide important information relative to the development of an effective bacteriophage treatment to mitigate Campylobacter during poultry production.

Prevalence and Subtype Characterization of Campylobacter in Ceca of Commercial Broiler Chickens at Processing - A 452 Flock, Seven-year Survey.

Human Campylobacter infections have been associated with chicken and other poultry meat products. Environmental conditions such as temperature and season can affect Campylobacter recoverability from chicken meat products. In the presented study, we sought to investigate the relationship between ambient weather conditions and the isolation of Campylobacter from chicken flocks, as well as the subtype of these isolates. Campylobacter was isolated from the ceca of broilers collected in a commercial processing facility over 7 years, representing 452 flocks. Isolates were subjected to whole-genome sequencing and subtyping by multilocus sequence typing (MLST). Approximately 60% (269/452) of flocks sampled were positive for Campylobacter. There was no significant effect on the presence of detectable Campylobacter by month, season, temperature, or rainfall during grow-out or transportation. Sixty-eight different STs were detected; 45 C. jejuni and 23 C. coli. Diversity as measured by Shannon's diversity index was higher in the spring and fall than in mid-winter and summer. We concluded that in the warm temperate climate of the Southeastern U.S., seasonality does not affect the rate of Campylobacter isolation from broilers, but the diversity of isolates was higher in the milder spring and fall seasons.

Filter sterilized carcass rinsate for recovery of Salmonella species with various concentrations of cetylpyridinium chloride.

Controlling Salmonella in poultry processing continues to be important to processors and consumers. Cetylpyridinium chloride (CPC) has proven to be effective in vitro in controlling Salmonella. This study evaluated the recovery of Salmonella after overnight storage in 4°C filter-sterilized carcass rinsate containing CPC from 0.44 to 909 ppm (µg/mL). Ten Salmonella serotypes (18 strains), of which 6 serotypes are commonly isolated from poultry products, were grown in Bacto-Tryptic Soy Broth overnight at 37°C. Serial dilutions of a CPC/propylene glycol solution were prepared in 24-well tissue culture plates containing filter-sterilized carcass rinsate. Approximately 107 cfu/mL of each Salmonella serotype was added to the appropriate wells. Inoculated plates were stored overnight at 4°C. After storage, triplicate plates of brilliant green agar with sulfapyridine (BGS) were surface inoculated with 10 µL of the contents for each well, streaked for isolation, and incubated at 37°C for 24 h. Three replications were conducted. The presence of typical colonies on BGS plates was recorded as growth and verified through biochemical and serological testing. Of the serotypes chosen, Salmonella Kentucky, Dublin, and Enteritidis were the least resistant to CPC with a median minimum inhibitory concentration (MIC) of 14.22 µg/mL (range from 3.55 to 56.88 µg/mL); S. Typhimurium demonstrated a median MIC of 114.00 µg/mL (range from 28.44 to 114.00 µg/mL). Residual CPC potentially remaining attached to a carcass or in the weep after processing could potentially alter which Salmonella serotype is recovered from a carcass rinse due to different growth patterns during regulatory testing, with a potential for more virulent strains not to be recovered.

Exudate From Retail Chicken Liver Packaging Allows for Survival of Naturally Occurring Campylobacter, Coliforms, and Aerobic Microorganisms Under Drying Conditions.

Campylobacter spp. are a leading cause of human foodborne illness associated with chicken meat products in the United States. Chicken livers, including exudate from packaging, commonly carry Campylobacter and could be a source of illness if mishandled. Survivability of naturally occurring Campylobacter, total aerobic bacteria, and coliforms was determined under drying conditions in two consumer simulated environments: moist sponge and solid surface. Fresh chicken liver exudate was dispensed onto sponges and glass slides and allowed to dry under ambient conditions for 7 days. Bacterial concentration was measured at 0, 6, 24, 48, 72, and 168 h. Total aerobic population did not decrease by more than one log over 7 days and did not correlate to water activity or time in either simulation. Coliform concentrations increased in sponge simulations but decreased in solid surface simulations. Further, coliform concentrations were significantly higher in sponge simulations than in solid surface. Campylobacter was naturally present in exudate and survived at least to 6 h in every trial. Campylobacter was recoverable at 24 h in some sponge trials. However, Campylobacter concentration was strongly correlated to water activity. Fresh chicken liver exudate could present a risk of campylobacteriosis to consumers if mishandled even after drying.

Whole-genome sequencing of Listeria innocua recovered from retail milk and dairy products in Egypt.

The similarity of the Listeria innocua genome with Listeria monocytogenes and their presence in the same niche may facilitate gene transfer between them. A better understanding of the mechanisms responsible for bacterial virulence requires an in-depth knowledge of the genetic characteristics of these bacteria. In this context, draft whole genome sequences were completed on five L. innocua isolated from milk and dairy products in Egypt. The assembled sequences were screened for antimicrobial resistance and virulence genes, plasmid replicons and multilocus sequence types (MLST); phylogenetic analysis of the sequenced isolates was also performed. The sequencing results revealed the presence of only one antimicrobial resistance gene, fosX, in the L. innocua isolates. However, the five isolates carried 13 virulence genes involved in adhesion, invasion, surface protein anchoring, peptidoglycan degradation, intracellular survival, and heat stress; all five lacked the Listeria Pathogenicity Island 1 (LIPI-1) genes. MLST assigned these five isolates into the same sequence type (ST), ST-1085; however, single nucleotide polymorphism (SNP)-based phylogenetic analysis revealed 422-1,091 SNP differences between our isolates and global lineages of L. innocua. The five isolates possessed an ATP-dependent protease (clpL) gene, which mediates heat resistance, on a rep25 type plasmids. Blast analysis of clpL-carrying plasmid contigs showed approximately 99% sequence similarity to the corresponding parts of plasmids of L. monocytogenes strains 2015TE24968 and N1-011A previously isolated from Italy and the United States, respectively. Although this plasmid has been linked to L. monocytogenes that was responsible for a serious outbreak, this is the first report of L. innocua containing clpL-carrying plasmids. Various genetic mechanisms of virulence transfer among Listeria species and other genera could raise the possibility of the evolution of virulent strains of L. innocua. Such strains could challenge processing and preservation protocols and pose health risks from dairy products. Ongoing genomic research is necessary to identify these alarming genetic changes and develop preventive and control measures.

Despite Shared Geography, Campylobacter Isolated from Surface Water Are Genetically Distinct from Campylobacter Isolated from Chickens.

We tested the hypothesis that Campylobacter isolated from chicken ceca and river water in an overlapping geographic area would share genetic information. Isolates of C. jejuni from chicken ceca were collected from a commercial slaughter plant and isolates of C. jejuni were also collected from rivers and creeks in the same watershed. Isolates were subjected to whole-genome sequencing and the data were used for core genome multilocus sequence typing (cgMLST). Cluster analysis showed that there were four distinct subpopulations, two from chickens and two from water. Calculation of fixation statistic (Fst) showed that all four subpopulations were significantly distinct. Greater than 90% of the loci were differentiated by subpopulation. Only two genes showed clear differentiation of both chicken subpopulations from both water subpopulations. Sequence fragments of the CJIE4 bacteriophage family were found frequently in the main chicken subpopulation and the water outgroup subpopulation but were sparsely found in the main water population and not at all in the chicken outgroup. CRISPR spacers that targeted the phage sequences were common in the main water subpopulation, only once in the main chicken subpopulation, and not at all in the chicken or water outgroups. Restriction enzyme genes also showed a biased distribution. These data suggest that there is little transfer of C. jejuni genetic material between chickens and nearby river water. Campylobacter differentiation according to these two sources does not show clear evidence of evolutionary selection; the differentiation is probably due to geospatial isolation, genetic drift, and the action of CRISPRs and restriction enzymes. IMPORTANCE Campylobacter jejuni causes gastroenteritis in humans, and chickens and environmental water are leading sources of infection. We tested the hypothesis that Campylobacter isolated from chicken ceca and river water in an overlapping geographic area would share genetic information. Isolates of Campylobacter were collected from water and chicken sources in the same watershed and their genomes were sequenced and analyzed. Four distinct subpopulations were found. There was no evidence of sharing genetic material between the subpopulations. Phage profiles, CRISPR profiles and restriction systems differed by subpopulation.

Buffered Peptone Water Formulation Does Not Influence Growth of pESI-positive Salmonella enterica Serovar Infantis.

Salmonella enterica is a major cause of human foodborne illness and is often attributed to poultry food sources. S. enterica serovar Infantis, specifically those carrying the pESI plasmid, has become a frequently isolated serotype from poultry meat samples at processing and has caused numerous recent human infections. In 2016, the USDA-Food Safety and Inspection Service changed the official sampling method for raw poultry products from BPW to using neutralizing BPW (nBPW) as the rinsing agent in order to prevent residual antimicrobial effects from acidifying and oxidizing processing aids. This change was contemporaneous to the emergence of pESI-positive ser. Infantis as a prevalent serovar in poultry, prompting some to question if nBPW could be selecting for this prevalent serovar. We performed two experiments: a comparison of ser. Infantis growth in BPW versus nBPW, and a simulation of regulatory sampling methods. We found that when inoculated into both broths, ser. Infantis initially grows slightly slower in nBPW than in BPW but little difference was seen in abundance after 6 h of growth. Additionally, the use of nBPW to simulate poultry rinse sample and overnight cold shipping to a regulatory lab did not affect the survival or subsequent growth of ser. Infantis in BPW. We concluded that the change in USDA-FSIS methodology to include nBPW in sampling procedures has likely not affected the emergence of S. ser. Infantis as a prevalent serovar in chicken and turkey meat product samples.

Cecal Microbial Hydrogen Cycling Potential Is Linked to Feed Efficiency Phenotypes in Chickens.

In chickens, early life exposure to environmental microbes has long-lasting impacts on gastrointestinal (GI) microbiome development and host health and growth, via mechanisms that remain uncharacterized. In this study, we demonstrated that administrating a fecal microbiome transplant (FMT) from adults to day-of-hatch chicks results in significantly higher body mass of birds and decreased residual feed intake (RFI), implying enhanced feed efficiency, at 6 weeks of age. To assess the potential mechanisms through which FMT affects adult bird phenotype, we combined 16 S rRNA gene amplification, metagenomic, and comparative genomic approaches to survey the composition and predicted activities of the resident microbiome of various GI tract segments. Early life FMT exposure had a long-lasting significant effect on the microbial community composition and function of the ceca but not on other GI segments. Within the ceca of 6-week-old FMT birds, hydrogenotrophic microbial lineages and genes were most differentially enriched. The results suggest that thermodynamic regulation in the cecum, in this case via hydrogenotrophic methanogenic and sulfur-cycling lineages, potentially serving as hydrogen sinks, may enhance fermentative efficiency and dietary energy harvest capacity. Our study provides a specific mechanism of action through which early-life microbiome transplants modulate market-relevant phenotypes in poultry and, thereby, may represent a significant advance toward microbiome-focused sustainable agriculture.

Assessing Salmonella prevalence and complexity through processing using different culture methods.

Conventional Salmonella surveillance requires a week for isolation, confirmation, and subsequent serotyping. We previously showed that this could be reduced by 24 h by combining the pre-enrichment and enrichment steps into a single selective pre-enrichment step and was tested on directly after picking. The goal of this study was 2-fold: 1) to evaluate the use of selective pre-enrichment through each step of processing, including postintervention when the Salmonella load is reduced, and 2) to assess any changes in serovar populations in Salmonella positive samples. Duplicate carcass drip samples, each representative of 500 broiler carcasses, were collected by catching processing water drip under moving carcass shackle lines in each of three commercial broiler slaughter plants. Samples were collected post-pick, post-inside-outside bird wash (IOBW), and post-chill; duplicate wing rinses were performed pre- and post-antimicrobial parts dip. Each processing plant was sampled 6 times for a total of 180 samples collected. The number of Salmonella positives identified with selective pre-enrichment conditions (48/180) was similar to traditional selective enrichment culture conditions (52/180), showed good concordance in recovery rate between the 2 culture methods (Fisher's exact test, P = 0.72). We also found that the incidence of Salmonella reduced dramatically after antimicrobial intervention (post-pick 66.7% vs. post chill 8.3%). When serovar populations were evaluated in Salmonella positive samples using CRISPR-SeroSeq, we detected four different Salmonella serovars, Kentucky, Infantis, Schwarzengrund, and Typhimurium, and their incidence rose between post-pick and post-IOBW. The relative abundance of Infantis within individual samples increased between post-pick and post-IOBW while the relative abundance of the other 3 serovars decreased. These results suggest that a selective pre-enrichment step reduces the time required for Salmonella isolation without negatively affecting detection and serovar profiles in culture positive samples were not altered between culture conditions used.

Survey of Meat Collected from Commercial Broiler Processing Plants Suggests Low Levels of Semicarbazide Can Be Created during Immersion Chilling.

ABSTRACT: Semicarbazide (SEM) is routinely employed as an indicator for the use of nitrofurazone, a banned antimicrobial. The validity of SEM as a nitrofurazone marker has been scrutinized because of other possible sources of the compound. Nonetheless, a U.S. trade partner rejected skin-on chicken thighs because of SEM detection and suspected nitrofurazone use. Because nitrofurazone has been banned in U.S. broiler production since 2003, we hypothesized that incidental de novo SEM formation occurs during broiler processing. To assess this possibility, raw leg quarters were collected from 23 commercial broiler processing plants across the United States and shipped frozen to our laboratory, where liquid chromatography-mass spectrometry was used to quantitatively assess for SEM. Leg quarter samples were collected at four points along the processing line: hot rehang (transfer from the kill line to the evisceration line), prechill (before the chilling process), postchill (immediately following chilling), and at the point of pack. Thigh meat with skin attached was removed from 535 leg quarters and analyzed in triplicate for SEM concentrations. The concentrations ranged from 0 to 2.67 ppb, with 462 (86.4%) of 535 samples below the regulatory decision level of 0.5 ppb of SEM. The 73 samples over the 0.5-ppb limit came from 21 plants; 53 (72.6%) of positive samples were in meat collected after chilling (postchill or point of pack). The difference in both prevalence and concentration of SEM detected before and after chilling was highly significant (P < 0.0001). These data support our hypothesis that SEM detection in raw broiler meat is related to de novo creation of the chemical during processing.

Prevalence and Characterization of Quinolone Resistance in Campylobacter spp. Isolates in Chicken Livers from Retail Stores in Georgia, USA.

ABSTRACT: Campylobacter is a bacterial pathogen that causes human foodborne illnesses worldwide, and outbreaks have been associated with consumption of undercooked chicken livers. The objectives of this study were to compare two PCR assays of 250 Campylobacter isolates for identification to species, to assess antibiotic resistance of the isolates, and to analyze genetic diversity of the quinolone resistance determining regions (QRDRs) of the isolates. A double-blind design was used to identify the species of Campylobacter; 181 (72%) of the isolates were identified as Campylobacter jejuni, and 69 (28%) isolates were identified as Campylobacter coli by both PCR assays. A total of 93 (37.2%) isolates were resistant to at least one antibiotic. Among 88 C. jejuni isolates, 33 (18%) were resistant to nalidixic acid (NAL) and ciprofloxacin (CIP), 25 (14%) were resistant to tetracycline (TET), and 18 (10%) were resistant to NAL and TET. Two C. jejuni isolates were resistant to four of the tested antibiotics, and one isolate was resistant to five antibiotics. Two C. coli isolates were resistant to TET, and two were resistant to NAL, CIP, and TET. The amino acid sequences of the QRDRs for the isolates had eight point mutations and could be classified into 12 groups. Thirty-eight C. jejuni isolates resistant to NAL and CIP had a point mutation at residue 86 (substitution from threonine to isoleucine). However, six isolates without this substitution were resistant to NAL and/or CIP. Ten isolates with a point mutation at residue 86 were susceptible to NAL and CIP. This observation suggests that in addition to the substitution at residue 86 other mechanisms may confer resistance to quinolones. Further studies are needed to elucidate mechanisms for quinolone resistance in Campylobacter. The Campylobacter spp. isolated from chicken livers in this study were resistant to quinolones and other classes of antibiotics.

Horizontal Gene Transfer Is the Main Driver of Antimicrobial Resistance in Broiler Chicks Infected with Salmonella enterica Serovar Heidelberg.

The overuse and misuse of antibiotics in clinical settings and in food production have been linked to the increased prevalence and spread of antimicrobial resistance (AR). Consequently, public health and consumer concerns have resulted in a remarkable reduction in antibiotics used for food animal production. However, there are no data on the effectiveness of antibiotic removal in reducing AR shared through horizontal gene transfer (HGT). In this study, we used neonatal broiler chicks and Salmonella enterica serovar Heidelberg, a model food pathogen, to test if chicks raised antibiotic free harbor transferable AR. We challenged chicks with an antibiotic-susceptible S. Heidelberg strain using various routes of inoculation and determined if S. Heidelberg isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole-genome sequencing (WGS) to show that chicks grown without antibiotics harbored an antimicrobial resistant S. Heidelberg population at 14 days after challenge and chicks challenged orally acquired AR at a higher rate than chicks inoculated via the cloaca. Using 16S rRNA gene sequencing, we found that S. Heidelberg infection perturbed the microbiota of broiler chicks, and we used metagenomics and WGS to confirm that a commensal Escherichia coli population was the main reservoir of an IncI1 plasmid acquired by S. Heidelberg. The carriage of this IncI1 plasmid posed no fitness cost to S. Heidelberg but increased its fitness when exposed to acidic pH in vitro. These results suggest that HGT of plasmids carrying AR shaped the evolution of S. Heidelberg and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella enterica. IMPORTANCE The reported increase in antibiotic-resistant bacteria in humans has resulted in a major shift away from antibiotic use in food animal production. This shift has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, which in turn will allow the currently available antibiotic arsenal to be more effective. This change in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic-susceptible Salmonella enterica serovar Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggest that simply removing antibiotics from food animal production might not be sufficient to limit the spread of antimicrobial resistance.

Prevalence of Listeria Species and Listeria monocytogenes on Raw Produce Arriving at Frozen Food Manufacturing Facilities.

ABSTRACT: The ubiquity of Listeria monocytogenes in the environment affects the food industry and presents concerns for frozen food facilities. This study determined the prevalence and numbers of Listeria species and L. monocytogenes on raw produce arriving at frozen food facilities. Raw produce was collected using multilevel blinding protocols to ensure anonymity of participants and avoid traceback. Five raw vegetables were selected: corn, carrots, green beans, peas, and spinach. Raw products were collected after arrival at the facilities but before cleaning or other preprocessing steps that are typically performed inside the facility. The U.S. Food and Drug Administration's Bacteriological Analytical Manual method for detection of Listeria spp. and L. monocytogenes was followed, with PCR screening followed by selective plating methods. Listeria numbers were estimated from positive samples using the most-probable-number (MPN) methodology. A total of 290 samples were collected, with 96 and 17 samples positive for Listeria spp. (33.1%) and L. monocytogenes (5.9%), respectively. Enumeration data for the 96 Listeria spp. samples indicated 82 samples had greater than 100 MPN of Listeria spp. per g and 14 samples had less than 100 MPN Listeria spp. per g. The prevalence of Listeria spp. varied by commodity: spinach (66.7%), peas (50%), corn (32.2%), green beans (22.2%), and carrots (13%). L. monocytogenes prevalence was determined in corn (13.6%), peas (6.3%), and green beans (4.2%) arriving at processing facilities. Such data were previously unavailable to frozen vegetable processors and are valuable in implementing process control standards. The prevalence and pathogen concentration data from raw commodities found in this study can provide the industry with information to conduct more accurate quantitative risk assessments and a baseline to model and target appropriate pathogen reduction steps during processing.

Broiler chickens and early life programming: Microbiome transplant-induced cecal community dynamics and phenotypic effects.

The concept of successional trajectories describes how small differences in initial community composition can magnify through time and lead to significant differences in mature communities. For many animals, the types and sources of early-life exposures to microbes have been shown to have significant and long-lasting effects on the community structure and/or function of the microbiome. In modern commercial poultry production, chicks are reared as a single age cohort and do not directly encounter adult birds. This scenario is likely to initiate a trajectory of microbial community development that is significantly different than non-industrial settings where chicks are exposed to a much broader range of environmental and fecal inocula; however, the comparative effects of these two scenarios on microbiome development and function remain largely unknown. In this work, we performed serial transfers of cecal material through multiple generations of birds to first determine if serial transfers exploiting the ceca in vivo, rather than the external environment or artificial incubations, can produce a stable microbial community. Subsequently, we compared microbiome development between chicks receiving this passaged, i.e. host-selected, cecal material orally, versus an environmental inoculum, to test the hypothesis that the first exposure of newly hatched chicks to microbes determines early GI microbiome structure and may have longer-lasting effects on bird health and development. Cecal microbiome dynamics and bird weights were tracked for a two-week period, with half of the birds in each treatment group exposed to a pathogen challenge at 7 days of age. We report that: i) a relatively stable community was derived after a single passage of transplanted cecal material, ii) this cecal inoculum significantly but ephemerally altered community structure relative to the environmental inoculum and PBS controls, and iii) either microbiome transplant administered at day-of-hatch appeared to have some protective effects against pathogen challenge relative to uninoculated controls. Differentially abundant taxa identified across treatment types may inform future studies aimed at identifying strains associated with beneficial phenotypes.

An Open-Source Program (Haplo-ST) for Whole-Genome Sequence Typing Shows Extensive Diversity among Listeria monocytogenes Isolates in Outdoor Environments and Poultry Processing Plants.

A reliable and standardized classification of Listeria monocytogenes is important for accurate strain identification during outbreak investigations. Current whole-genome sequencing (WGS)-based approaches for strain characterization are either difficult to standardize, rendering them less suitable for data exchange, or are not freely available. Thus, we developed a portable and open-source tool, Haplo-ST, to improve standardization and provide maximum discriminatory potential to WGS data tied to a multilocus sequence typing (MLST) framework. Haplo-ST performs whole-genome MLST (wgMLST) for L. monocytogenes while allowing for data exchangeability worldwide. This tool takes in (i) raw WGS reads as input, (ii) cleans the raw data according to user-specified parameters, (iii) assembles genes across loci by mapping to genes from reference strains, and (iv) assigns allelic profiles to assembled genes and provides a wgMLST subtyping for each isolate. Data exchangeability relies on the tool assigning allelic profiles based on a centralized nomenclature defined by the widely used BIGSdb-Lm database. Tests of Haplo-ST's performance with simulated reads from L. monocytogenes reference strains demonstrated high sensitivity (97.5%), and coverage depths of ≥20× were found to be sufficient for wgMLST profiling. We then used Haplo-ST to characterize and differentiate between two groups of L. monocytogenes isolates derived from the natural environment and poultry processing plants. Phylogenetic reconstruction identified lineages within each group, and no lineage specificity was observed with isolate phenotypes (transient versus persistent) or origins. Genetic differentiation analyses between isolate groups identified 21 significantly differentiated loci, potentially enriched for adaptation and persistence of L. monocytogenes within poultry processing plants.IMPORTANCE We have developed an open-source tool (https://github.com/swarnalilouha/Haplo-ST) that provides allele-based subtyping of L. monocytogenes isolates at the whole-genome level. Along with allelic profiles, this tool also generates allele sequences and identifies paralogs, which is useful for phylogenetic tree reconstruction and deciphering relationships between closely related isolates. More broadly, Haplo-ST is flexible and can be adapted to characterize the genome of any haploid organism simply by installing an organism-specific gene database. Haplo-ST also allows for scalable subtyping of isolates; fewer reference genes can be used for low-resolution typing, whereas higher resolution can be achieved by increasing the number of genes used in the analysis. Our tool enabled clustering of L. monocytogenes isolates into lineages and detection of potential loci for adaptation and persistence in food processing environments. Findings from these analyses highlight the effectiveness of Haplo-ST in subtyping and evaluating relationships among isolates in studies of bacterial population genetics.

Draft Genome Sequences of Two Potentially Novel Bacillus Isolates from Backyard and Commercial Chicken Gastrointestinal Tracts.

Here, we present the draft genome sequences of two Bacillus strains, HF117_J1_D and USDA818B3_A, isolated in Pomona, California, from the gastrointestinal (GI) tract of backyard and commercial broiler chickens, respectively. The draft genomes of both strains appear to represent novel species.

Draft Genome Sequences of Two Paenibacillus Isolates from Goose and Chicken Gastrointestinal Material.

Here, we present the draft genome sequences of two Paenibacillus strains, An7 and USDA918EY, isolated from goose feces (Bend, OR, USA) and chicken ceca (Pomona, CA, USA), respectively. These data may assist with analyses of microorganisms associated with free-ranging and commercial avian species.

Population Analyses Reveal Preenrichment Method and Selective Enrichment Media Affect Salmonella Serovars Detected on Broiler Carcasses.

Poultry is a major Salmonella reservoir, but conventional culture-based methods typically identify the most abundant serovars while those less abundant remain undetected. Choice of enrichment procedure also introduces bias, and for broiler carcasses, a 1-min rinse before preenrichment is insufficient to release all Salmonella present. The inability to assess serovar diversity means that serovars more often associated with human illness may be masked by more abundant Salmonella. CRISPR-SeroSeq (serotyping by sequencing clustered regularly interspaced short palindromic repeats), an amplicon-based, next-generation sequencing tool, allows detection of multiple serovars and maps the relative serovar frequencies in a sample. To address the preceding limitations, CRISPR-SeroSeq was used on broiler carcasses collected prechilled at a commercial plant. Standard carcass rinse aliquot preenrichments and whole carcass preenrichments that were enriched in Rappaport-Vassiliadis (RV) and tetrathionate (TT) broths were compared. On average, five serovars were observed per carcass, including nine on one carcass. CRISPR-SeroSeq detected serovars comprising as little as 0.005% of the population. CRISPR-SeroSeq data matched (28 of 32) standard culture analysis for abundant serovars. Salmonella serovars Kentucky, Typhimurium, and Schwarzengrund were found on each carcass. Overall, serovar diversity was higher in whole carcass preenrichments that were enriched in RV (P < 0.05). Serovar Schwarzengrund was present at higher frequencies in whole carcass preenrichments compared with rinse aliquot preenrichments (t test, P < 0.05), suggesting it adheres more strongly to the carcass. Salmonella serovar Enteritidis was enriched eightfold more in TT than in RV, and serovars Schwarzengrund and Reading were preferentially enriched in RV. Comparison of preenriched and enriched samples suggests that selective enrichment in RV or TT was inhibitory to some serovars. This article addresses limitations of Salmonella surveillance protocols and provides information related to Salmonella population dynamics.

Dry and heat stress affects H2S production of Salmonella on selective plating media.

The pH of Salmonella pre-enrichment media can become acidic (pH 4.0-5.0) when feeds/ingredients are incubated for 24 h. Salmonella in feed that have been stressed by heat and desiccation exhibit different pH tolerances than non-stressed cultures. Acidic conditions can result in cell injury/death and affect biochemical pathways. In this study, eight serotypes of Salmonella were grown in sterile meat and bone meal that was subjected to desiccation and heat stress. Cultures of non-stressed and stressed isolates were subsequently exposed to acidic pH from 4.0 to 7.0 in 0.5 pH increments (3 replicates/pH increment) in citrate buffer. At 6 and 24 h, serial dilutions were plated in duplicate on XLT-4 (xylose lysine tergitol-4) agar. Four serotypes showed an impaired ability to decarboxylate lysine on XLT-4. This inability to decarboxylate lysine was dependent on isolate, stress status, and incubation time. When the isolates' ability to decarboxylate lysine was examined using biochemical tests, cultures were found to be able to decarboxylate lysine with the exception of S. Infantis. This suggests that XLT-4 contains a biochemical stressor(s) which affects the rate of decarboxylation by these Salmonella. These results suggest that acidic conditions may influence the detection and confirmation of Salmonella in feed.