Targeted Amplicon Sequencing for Single-Nucleotide-Polymorphism Genotyping of Attaching and Effacing Escherichia coli O26:H11 Cattle Strains via a High-Throughput Library Preparation Technique.

Enterohemorrhagic Escherichia coli (EHEC) O26:H11, a serotype within Shiga toxin-producing E. coli (STEC) that causes severe human disease, has been considered to have evolved from attaching and effacing E. coli (AEEC) O26:H11 through the acquisition of a Shiga toxin-encoding gene. Targeted amplicon sequencing using next-generation sequencing technology of 48 phylogenetically informative single-nucleotide polymorphisms (SNPs) and three SNPs differentiating Shiga toxin-positive (stx-positive) strains from Shiga toxin-negative (stx-negative) strains were used to infer the phylogenetic relationships of 178 E. coli O26:H11 strains (6 stx-positive strains and 172 stx-negative AEEC strains) from cattle feces to 7 publically available genomes of human clinical strains. The AEEC cattle strains displayed synonymous SNP genotypes with stx2-positive sequence type 29 (ST29) human O26:H11 strains, while stx1 ST21 human and cattle strains clustered separately, demonstrating the close phylogenetic relatedness of these Shiga toxin-negative AEEC cattle strains and human clinical strains. With the exception of seven stx-negative strains, five of which contained espK, three stx-related SNPs differentiated the STEC strains from non-STEC strains, supporting the hypothesis that these AEEC cattle strains could serve as a potential reservoir for new or existing pathogenic human strains. Our results support the idea that targeted amplicon sequencing for SNP genotyping expedites strain identification and genetic characterization of E. coli O26:H11, which is important for food safety and public health.

Prevalence and distribution of Listeria monocytogenes inlA alleles prone to phase variation and inlA alleles with premature stop codon mutations among human, food, animal, and environmental isolates.

In Listeria monocytogenes, 18 mutations leading to premature stop codons (PMSCs) in the virulence gene inlA have been identified to date. While most of these mutations represent nucleotide substitutions, a frameshift deletion in a 5' seven-adenine homopolymeric tract (HT) in inlA has also been reported. This HT may play a role in phase variation and was first identified among L. monocytogenes lineage II ribotype DUP-1039C isolates. In order to better understand the distribution of different inlA mutations in this ribotype, a newly developed multiplex real-time PCR assay was used to screen 368 DUP-1039C isolates from human, animal, and food-associated sources for three known 5' inlA HT alleles: (i) wild-type (WT) (A7), (ii) frameshift (FS) (A6), and (iii) guanine interruption (A2GA4) alleles. Additionally, 228 DUP-1039C isolates were screened for all inlA PMSCs; data on the presence of all inlA PMSCs for the other 140 isolates were obtained from previous studies. The statistical analysis based on 191 epidemiologically unrelated strains showed that strains with inlA PMSC mutations (n = 41) were overrepresented among food-associated isolates, while strains encoding full-length InlA (n = 150) were overrepresented among isolates from farm animals and their environments. Furthermore, the A6 allele was overrepresented and the A7 allele was underrepresented among food isolates, while the A6 allele was underrepresented among farm and animal isolates. Our results indicate that genetic variation in inlA contributes to niche adaptation within the lineage II subtype DUP-1039C.

Genetic Diversity and Pathogenic Potential of Attaching and Effacing Escherichia coli O26:H11 Strains Recovered from Bovine Feces in the United States.

Escherichia coli O26 has been identified as the most common non-O157 Shiga toxin-producing E. coli (STEC) serogroup to cause human illnesses in the United States and has been implicated in outbreaks around the world. E. coli has high genomic plasticity, which facilitates the loss or acquisition of virulence genes. Attaching and effacing E. coli (AEEC) O26 strains have frequently been isolated from bovine feces, and there is a need to better characterize the relatedness of these strains to defined molecular pathotypes and to describe the extent of their genetic diversity. High-throughput real-time PCR was used to screen 178 E. coli O26 isolates from a single U.S. cattle feedlot, collected from May to July 2011, for the presence or absence of 25 O26 serogroup-specific and virulence-associated markers. The selected markers were capable of distinguishing these strains into molecularly defined groups (yielding 18 unique marker combinations). Analysis of the clustered regularly interspaced short palindromic repeat 1 (CRISPR1) and CRISPR2a loci further discriminated isolates into 24 CRISPR types. The combination of molecular markers and CRISPR typing provided 20.8% diversity. The recent CRISPR PCR target SP_O26-E, which was previously identified only in stx2-positive O26:H11 human clinical strains, was identified in 96.4% (161/167 [95% confidence interval, 99.2 to 93.6%]) of the stx-negative AEEC O26:H11 bovine fecal strains. This supports that these stx-negative strains may have previously contained a prophage carrying stx or could acquire this prophage, thus possibly giving them the potential to become pathogenic to humans. These results show that investigation of specific genetic markers may further elucidate our understanding of the genetic diversity of AEEC O26 strains in bovine feces.

Listeria floridensis sp. nov., Listeria aquatica sp. nov., Listeria cornellensis sp. nov., Listeria riparia sp. nov. and Listeria grandensis sp. nov., from agricultural and natural environments.

Sampling of agricultural and natural environments in two US states (Colorado and Florida) yielded 18 Listeria-like isolates that could not be assigned to previously described species using traditional methods. Using whole-genome sequencing and traditional phenotypic methods, we identified five novel species, each with a genome-wide average BLAST nucleotide identity (ANIb) of less than 85% to currently described species. Phylogenetic analysis based on 16S rRNA gene sequences and amino acid sequences of 31 conserved loci showed the existence of four well-supported clades within the genus Listeria; (i) a clade representing Listeria monocytogenes, L. marthii, L. innocua, L. welshimeri, L. seeligeri and L. ivanovii, which we refer to as Listeria sensu stricto, (ii) a clade consisting of Listeria fleischmannii and two newly described species, Listeria aquatica sp. nov. (type strain FSL S10-1188(T) = DSM 26686(T) = LMG 28120(T) = BEI NR-42633(T)) and Listeria floridensis sp. nov. (type strain FSL S10-1187(T) = DSM 26687(T) = LMG 28121(T) = BEI NR-42632(T)), (iii) a clade consisting of Listeria rocourtiae, L. weihenstephanensis and three novel species, Listeria cornellensis sp. nov. (type strain TTU A1-0210(T) = FSL F6-0969(T) = DSM 26689(T) = LMG 28123(T) = BEI NR-42630(T)), Listeria grandensis sp. nov. (type strain TTU A1-0212(T) = FSL F6-0971(T) = DSM 26688(T) = LMG 28122(T) = BEI NR-42631(T)) and Listeria riparia sp. nov. (type strain FSL S10-1204(T) = DSM 26685(T) = LMG 28119(T) = BEI NR- 42634(T)) and (iv) a clade containing Listeria grayi. Genomic and phenotypic data suggest that the novel species are non-pathogenic.

Landscape and meteorological factors affecting prevalence of three food-borne pathogens in fruit and vegetable farms.

Produce-related outbreaks have been traced back to the preharvest environment. A longitudinal study was conducted on five farms in New York State to characterize the prevalence, persistence, and diversity of food-borne pathogens in fresh produce fields and to determine landscape and meteorological factors that predict their presence. Produce fields were sampled four times per year for 2 years. A total of 588 samples were analyzed for Listeria monocytogenes, Salmonella, and Shiga toxin-producing Escherichia coli (STEC). The prevalence measures of L. monocytogenes, Salmonella, and STEC were 15.0, 4.6, and 2.7%, respectively. L. monocytogenes and Salmonella were detected more frequently in water samples, while STEC was detected with equal frequency across all sample types (soil, water, feces, and drag swabs). L. monocytogenes sigB gene allelic types 57, 58, and 61 and Salmonella enterica serovar Cerro were repeatedly isolated from water samples. Soil available water storage (AWS), temperature, and proximity to three land cover classes (water, roads and urban development, and pasture/hay grass) influenced the likelihood of detecting L. monocytogenes. Drainage class, AWS, and precipitation were identified as important factors in Salmonella detection. This information was used in a geographic information system framework to hypothesize locations of environmental reservoirs where the prevalence of food-borne pathogens may be elevated. The map indicated that not all croplands are equally likely to contain environmental reservoirs of L. monocytogenes. These findings advance recommendations to minimize the risk of preharvest contamination by enhancing models of the environmental constraints on the survival and persistence of food-borne pathogens in fields.

Substantial within-animal diversity of Salmonella isolates from lymph nodes, feces, and hides of cattle at slaughter.

Lymph nodes (mandibular, mesenteric, mediastinal, and subiliac; n = 68) and fecal (n = 68) and hide (n = 35) samples were collected from beef carcasses harvested in an abattoir in Mexico. Samples were analyzed for Salmonella, and presumptive colonies were subjected to latex agglutination. Of the isolates recovered, a subset of 91 was characterized by serotyping, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility phenotyping. Salmonella was isolated from 100% (hide), 94.1% (feces), 91.2% (mesenteric), 76.5% (subiliac), 55.9% (mandibular), and 7.4% (mediastinal) of samples. From the 87 typeable isolates, eight Salmonella enterica serotypes, including Kentucky (32.2%), Anatum (29.9%), Reading (17.2%), Meleagridis (12.6%), Cerro (4.6%), Muenster (1.1%), Give (1.1%), and Mbandaka (1.1%), were identified. S. Meleagridis was more likely (P = 0.03) to be recovered from lymph nodes than from feces or hides, whereas S. Kentucky was more likely (P = 0.02) to be recovered from feces and hides than from lymph nodes. The majority (59.3%) of the Salmonella isolates were pansusceptible; however, multidrug resistance was observed in 13.2% of isolates. Typing by PFGE revealed that Salmonella strains generally clustered by serotype, but some serotypes (Anatum, Kentucky, Meleagridis, and Reading) were comprised of multiple PFGE subtypes. Indistinguishable PFGE subtypes and, therefore, serotypes were isolated from multiple sample types, and multiple PFGE subtypes were commonly observed within an animal. Given the overrepresentation of some serotypes within lymph nodes, we hypothesize that certain Salmonella strains may be better at entering the bovine host than other Salmonella strains or that some may be better adapted for survival within lymph nodes. Our data provide insight into the ecology of Salmonella within cohorts of cattle and offer direction for intervention opportunities.

Genome sequencing identifies Listeria fleischmannii subsp. coloradonensis subsp. nov., isolated from a ranch.

Twenty Listeria-like isolates were obtained from environmental samples collected on a cattle ranch in northern Colorado; all of these isolates were found to share an identical partial sigB sequence, suggesting close relatedness. The isolates were similar to members of the genus Listeria in that they were Gram-stain-positive, short rods, oxidase-negative and catalase-positive; the isolates were similar to Listeria fleischmannii because they were non-motile at 25 °C. 16S rRNA gene sequencing for representative isolates and whole genome sequencing for one isolate was performed. The genome of the type strain of Listeria fleischmannii (strain LU2006-1(T)) was also sequenced. The draft genomes were very similar in size and the average MUMmer nucleotide identity across 91% of the genomes was 95.16%. Genome sequence data were used to design primers for a six-gene multi-locus sequence analysis (MLSA) scheme. Phylogenies based on (i) the near-complete 16S rRNA gene, (ii) 31 core genes and (iii) six housekeeping genes illustrated the close relationship of these Listeria-like isolates to Listeria fleischmannii LU2006-1(T). Sufficient genetic divergence of the Listeria-like isolates from the type strain of Listeria fleischmannii and differing phenotypic characteristics warrant these isolates to be classified as members of a distinct infraspecific taxon, for which the name Listeria fleischmannii subsp. coloradonensis subsp. nov. is proposed. The type strain is TTU M1-001(T) ( =BAA-2414(T) =DSM 25391(T)). The isolates of Listeria fleischmannii subsp. coloradonensis subsp. nov. differ from the nominate subspecies by the inability to utilize melezitose, turanose and sucrose, and the ability to utilize inositol. The results also demonstrate the utility of whole genome sequencing to facilitate identification of novel taxa within a well-described genus. The genomes of both subspecies of Listeria fleischmannii contained putative enhancin genes; the Listeria fleischmannii subsp. coloradonensis subsp. nov. genome also encoded a putative mosquitocidal toxin. The presence of these genes suggests possible adaptation to an insect host, and further studies are needed to probe niche adaptation of Listeria fleischmannii.

Prevalence of Foodborne Pathogens in Pacific Northwest Beef Feedlot Cattle Fed Two Different Direct-Fed Microbials.

In recent years, there has been an increased interest in beef cattle shedding of foodborne pathogens due to the potential to contaminate surrounding food crops; however, the number of studies published on this topic has declined as the majority of research has emphasized on postharvest mitigation efforts. A field study was conducted to determine the prevalence of pathogens and indicator bacteria in beef cattle fed two different direct-fed microbials (DFMs). Fecal samples from a total of 3,708 crossbred yearling cattle randomly assigned to 16 pens and two treatment groups at a commercial cattle feedlot were taken. During the study period, diets were supplemented with two different DFMs i.) Lactobacillus acidophilus (NP51) and Propionibacterium freudenreichii (NP24) (9 log10CFU/head/day), and ii.) Lactobacillus salivarius (L28) (6 log10CFU/head/day). Fecal samples from pen floors were collected on days 0, 21, 42, 63, 103, and analyzed for the presence of Salmonella and E. coli O157:H7 and concentration of E. coli O157:H7, Enterobacteriaceae, and C. perfringens. Fecal samples collected from cattle fed L28 had significantly lower concentration of C. perfringens (p < 0.05) and had a similar prevalence with no significant differences in E. coli O157:H7 as those fed NP51/NP24 through the study until day 103. On day 103, the prevalence in cattle fed L28 was 40% with a concentration of 0.95 log10MPN/g while those fed NP51/NP24 were 65% with a concentration of 1.2 log10MPN/g. Cattle supplemented with NP51/NP24 achieved a significant log reduction of EB by 2.4 log10CFU/g over the course of the 103-day supplementation period compared to L28. Salmonella prevalence was also measured, but not detected in any samples at significant amounts to draw conclusions. It is evident that E. coli O157:H7 and other foodborne pathogens are still prevalent in cattle operations and that preharvest mitigation strategies should be considered to reduce the risk to beef products.

Effects of a novel direct-fed microbial on growth performance, carcass characteristics, nutrient digestibility, and ruminal morphology of beef feedlot steers.

The effects of a novel direct-fed microbial (DFM) on feedlot performance, carcass characteristics, digestibility, ruminal morphology, and volatile fatty acid (VFA) profile of finishing steers were evaluated. Single-source Angus-crossbred yearling steers (n = 144; initial body weight (BW) = 371 ±â€…19 kg) were used in a randomized complete block design. Steers were blocked by initial BW and randomly assigned to treatments (12 pens/treatment; 4 steers/pen). Treatments included (A) CONTROL (no DFM, tylosin, or monensin, (B) MONTY (monensin sodium [330 mg/animal-daily] and tylosin phosphate [90 mg/animal-daily]), and (C) MONPRO (monensin sodium [same as previous] and Lactobacillus salivarius L28 [1 × 106 CFU/animal-daily]). Treatments were included in a steam-flaked corn-based finisher diet offered once daily using a clean-bunk management for ~149 d. The digestibility assessment was performed from days 70 to 74. Ruminal fluid and rumen tissue samples were collected at the slaughter for VFA profile and papillae morphology analyses, respectively. Data were analyzed using the GLIMMIX procedure of SAS with pen serving as the experimental unit, treatment as fixed effect, and BW block as random effect. Steers offered MONPRO had on average 5.3% less (P < 0.01) dry matter intake (9.56 kg/d) compared with either CONTROL (10.16 kg/d) or MONTY (9.96 kg/d). The carcass-adjusted final BW (613 kg; P = 0.23), overall average daily gain (1.64 kg/d; P = 0.23), and gain-efficiency (0.165; P = 0.61) were not affected by treatments. Steers offered CONTROL had greater (P < 0.01) marbling score and tended (P = 0.06) to have less carcasses grading Select and tended (P = 0.10) to have more carcasses grading Upper-Choice, while other carcass characteristics and liver-abscesses were not affected (P ≥ 0.23) by treatments. The digestibility of nutrients (P ≥ 0.13) and the ruminal VFA profile (P ≥ 0.12) were not affected by treatments. Steers offered MONPRO tended (P = 0.09) to have 16% greater average papillae number compared to other treatments. Yearlings offered finishing diets containing L. salivarius L28 plus monensin did not affect growth performance, digestibility, or ruminal VFA, but reduced feed intake. Carcass quality was negatively affected by treatments, while animals consuming L. salivarius L28 and monensin tended to improve ruminal morphology. Current findings in ruminal morphology and feed intake may warrant further assessment of diets containing L. salivarius L28 on beef cattle food safety aspects.

Antimicrobial resistance is a growing concern to public health and medically important antibiotics have been listed in the Veterinary Feed Directive. Nutritional technologies, such as direct-fed microbials, are being increasingly studied for the development of an effective use on beef cattle production systems. The newly isolated strain of Lactobacillus salivarius L28 has demonstrated pathogenic inhibition of Escherichia coli, Salmonella, and Listeria monocytogenes on in vitro assessments. The potential benefits have warranted the exploration of L. salivarius L28 in a feedlot setting. Single-source Angus-crossbred yearling steers were offered steam-flaked corn-based finishing diets containing no feed additive, or either a combination of tylosin plus monensin or L. salivarius L28 plus monensin. Steers offered L. salivarius L28 plus monensin consumed 5.3% less feed compared with other treatments, while other growth performance variables and the digestibility of nutrients were not affected. Carcasses from cattle supplemented with monensin had slightly lower carcass quality grades than those not supplemented with monensin. Lactobacillus salivarius L28 plus monensin tended to improve steers ruminal morphology. Current findings may warrant further food safety assessments when cattle are offered diets containing L. salivarius L28.

Reduction of Pathogens in Feces and Lymph Nodes Collected from Beef Cattle Fed Lactobacillus salivarius (L28), Lactobacillus acidophilus (NP51) and Propionibacterium freudenreichii (NP28), Commercially Available Direct-Fed Microbials.

The purpose of the study was to evaluate the prevalence and concentration of foodborne pathogens in the feces and peripheral lymph nodes (PLNs) of beef cattle when supplemented with direct-fed microbials (DFMs) in feedlots. Fecal samples were collected from the pen floors over a 5-month period at three different feedlots in a similar geographical location in Nebraska, where each feed yard represented a treatment group: (i.) control: no supplement, (ii.) Bovamine Defend: supplemented with NP51 and NP24 at a target dose of 9 log10CFU/g/head/day, and (iii.) Probicon: supplemented with L28 at a target dose of 6 log10CFU/g/head/day. Each fecal sample was tested for the prevalence of E. coli O157:H7 and Salmonella, and concentration of E. coli O157:H7, Enterobacteriaceae and Clostridium perfringens. Cattle were harvested and PLNs were collected on the harvest floor. Real-time Salmonella PCR assays were performed for each PLN sample to determine Salmonella presence. The cattle supplemented with both DFMs had reduced foodborne pathogens in fecal samples, but feces collected from the pens housing the cattle supplemented with Probicon consistently had significantly less E. coli O157:H7 and Salmonella prevalence as well as a lower C. perfringens concentration. While DFMs do not eliminate foodborne pathogens in fecal shedding and PLNs, the use of DFMs as a pre-harvest intervention allows for an effective way to target multiple pathogens reducing the public health risks and environmental dissemination from cattle.

Variation in Listeria monocytogenes dose responses in relation to subtypes encoding a full-length or truncated internalin A.

Internalin A (InlA; encoded by inlA) facilitates the crossing of the intestinal barrier by Listeria monocytogenes. Mutations leading to a premature stop codon (PMSC) in inlA and thus attenuated mammalian virulence have been reported. We recently characterized 502 L. monocytogenes food isolates from a retail survey and 507 human clinical isolates from multiple U.S. states with respect to the presence/absence of inlA mutations. The objective of this study was to investigate the hypothesis that dose responses for human listeriosis vary between L. monocytogenes strains with and those without a PMSC in inlA. Subtype-specific prevalence and concentration distributions in food, along with epidemiologic and consumption data, were input into established dose-response models to generate an r value (probability of a cell causing illness). Under the conservative assumption that L. monocytogenes levels at retail represent levels consumed, mean log(10) r values were -8.1 and -10.7 for L. monocytogenes subtypes with genes encoding a full-length and a truncated InlA, respectively. L. monocytogenes carrying a 5' frameshift mutation in a homopolymeric tract showed a mean log(10) r value of -12.1. Confidence intervals for the r values and their differences varied depending on subtypes. When the increase in concentration of L. monocytogenes subtypes between retail and consumption was considered, mean log(10) r values were reduced to -10.4, -13.8, and -12.8 for the subtypes with genes encoding a full-length InlA, for the subtypes carrying a PMSC in inlA, and for all L. monocytogenes isolates regardless of subtype, respectively. Our study provides further quantitative evidence that L. monocytogenes subtypes vary in abilities and relative likelihoods of causing human disease, which were mechanistically related to defined genetic markers.

Presence of antibiotic-resistant commensal bacteria in samples from agricultural, city, and national park environments evaluated by standard culture and real-time PCR methods.

This study examined the presence of antibiotic-resistant commensal bacteria among cattle operations representing areas heavily affected by agriculture, city locations representing areas affected by urban activities and indirectly affected by agriculture, and a national park representing an area not affected by agriculture. A total of 288 soil, fecal floor, and water samples were collected from cattle operations, from the city of Fort Collins, and from Rocky Mountain National Park (RMNP) in Colorado. In addition, a total of 42 new and unused feed, unused bedding, compost, and manure samples were obtained from the cattle operations. Total, tetracycline-resistant, and ceftiofur-resistant bacterial populations were enumerated by both standard culture plating and real-time PCR methods. Only wastewater samples from the cattle operations demonstrated both higher tetracycline-resistant bacterial counts (enumerated by the culture plating method) and tetracycline resistance gene copies (quantified by real-time PCR) compared to water samples collected from non-farm environments. The ceftiofur resistance gene, blaCMY-2, was not detectable in any of the samples, while the tetracycline resistance genes examined in this study, tet(B), tet(C), tet(W), and tet(O), were detected in all types of tested samples, except soil samples from RMNP. Tetracycline resistance gene pools quantified from the tet(O) and tet(W) genes were bigger than those from the tet(B) and tet(C) genes in fecal and water samples. Although only limited resistance genes, instead of a full set, were selected for real-time PCR quantification in this study, our results point to the need for further studies to determine natural and urban impacts on antibiotic resistance.

Characterization and transferability of class 1 integrons in commensal bacteria isolated from farm and nonfarm environments.

This study assessed the distribution of class 1 integrons in commensal bacteria isolated from agricultural and nonfarm environments, and the transferability of class 1 integrons to pathogenic bacteria. A total of 26 class 1 integron-positive isolates were detected in fecal samples from cattle operations and a city park, water samples from a beef ranch and city lakes, and soil, feed (unused), manure, and compost samples from a dairy farm. Antimicrobial susceptibility testing of class 1 integron-positive Enterobacteriaceae isolates from city locations displayed multi-resistance to 12-13 out of the 22 antibiotics tested, whereas class 1 integron-positive Enterobacteriaceae isolates from cattle operations only displayed tetracycline resistance. Most class 1 integrons had one gene cassette belonging to the aadA family that confers resistance to streptomycin and spectinomycin. One isolate from a dog fecal sample collected from a city dog park transferred its class 1 integron to a strain of Escherichia coli O157:H7 at a frequency of 10(-7) transconjugants/donor by in vitro filter mating experiments under the stated laboratory conditions. Due to the numerous factors that may affect the transferability testing, further investigation using different methodologies may be helpful to reveal the transferability of the integrons from other isolates. The presence of class 1 integrons among diverse commensal bacteria from agricultural and nonfarm environments strengthens the possible role of environmental commensals in serving as reservoirs of antibiotic resistance genes.

Escherichia coli O157:H7 strains that persist in feedlot cattle are genetically related and demonstrate an enhanced ability to adhere to intestinal epithelial cells.

A longitudinal study was conducted to investigate the nature of Escherichia coli O157:H7 colonization of feedlot cattle over the final 100 to 110 days of finishing. Rectal fecal grab samples were collected from an initial sample population of 788 steers every 20 to 22 days and microbiologically analyzed to detect E. coli O157:H7. The identities of presumptive colonies were confirmed using a multiplex PCR assay that screened for gene fragments unique to E. coli O157:H7 (rfbE and fliC(h7)) and other key virulence genes (eae, stx(1), and stx(2)). Animals were classified as having persistent shedding (PS), transient shedding (TS), or nonshedding (NS) status if they consecutively shed the same E. coli O157:H7 genotype (based on the multiplex PCR profile), exhibited variable E. coli O157 shedding, or never shed morphologically typical E. coli O157, respectively. Overall, 1.0% and 1.4% of steers were classified as PS and NS animals, respectively. Characterization of 132 E. coli O157:H7 isolates from PS and TS animals by pulsed-field gel electrophoresis (PFGE) typing yielded 32 unique PFGE types. One predominant PFGE type accounted for 53% of all isolates characterized and persisted in cattle throughout the study. Isolates belonging to this predominant and persistent PFGE type demonstrated an enhanced (P < 0.0001) ability to adhere to Caco-2 human intestinal epithelial cells compared to isolates belonging to less common PFGE types but exhibited equal virulence expression. Interestingly, the attachment efficacy decreased as the genetic divergence from the predominant and persistent subtype increased. Our data support the hypothesis that certain E. coli O157:H7 strains persist in feedlot cattle, which may be partially explained by an enhanced ability to colonize the intestinal epithelium.

Complete Genome Sequences of Four Salmonella enterica Strains (Including Those of Serotypes Montevideo, Mbandaka, and Lubbock) Isolated from Peripheral Lymph Nodes of Healthy Cattle.

Salmonella enterica serotype Lubbock emerged most likely from a Salmonella enterica serotype Mbandaka ancestor that acquired by recombination the fliC operon from Salmonella enterica serotype Montevideo. Here, we report the complete genome sequence of two S. Lubbock, one S. Montevideo, and one S. Mbandaka strain isolated from bovine lymph nodes.

A Systematic Approach to Identify and Characterize the Effectiveness and Safety of Novel Probiotic Strains to Control Foodborne Pathogens.

A total of 44 lactic acid bacteria (LAB) strains originally isolated from cattle feces and different food sources were screened for their potential probiotic features. The antimicrobial activity of all isolates was tested by well-diffusion assay and competitive exclusion on broth against Salmonella Montevideo, Escherichia coli O157:H7 and Listeria monocytogenes strain N1-002. Thirty-eight LAB strains showed antagonistic effect against at least one of the pathogens tested in this study. Improved inhibitory effect was observed against L. monocytogenes with zones of inhibition up to 24 mm when LAB overnight cultures were used, and up to 21 mm when cell-free filtrates were used. For E. coli O157:H7 and Salmonella maximum inhibitions of 12 and 11.5 mm were observed, respectively. On broth, 43 strains reduced L. monocytogenes up to 9.06 log10 CFU/ml, 41 reduced E. coli O157:H7 up to 0.84 log10 CFU/ml, and 32 reduced Salmonella up to 0.94 log10 CFU/ml 24 h after co-inoculation. Twenty-eight LAB isolates that exhibited the highest inhibitory effect among pathogens were further analyzed to determine their antimicrobial resistance profile, adhesion potential, and cytotoxicity to Caco-2 cells. All LAB strains tested were susceptible to ampicillin, linezolid, and penicillin. Twenty-six were able to adhere to Caco-2 cells, five were classified as highly adhesive with > 40 bacterial cells/Caco-2 cells. Low cytotoxicity percentages were observed for the candidate LAB strains with values ranging from -5 to 8%. Genotypic identification by whole genome sequencing confirmed all as members of the LAB group; Enterococcus was the genus most frequently isolated with 21 isolates, followed by Pediococcus with 4, and Lactobacillus with 3. In this study, a systematic approach was used for the improved identification of novel LAB strains able to exert antagonistic effect against important foodborne pathogens. Our findings suggest that the selected panel of LAB probiotic strains can be used as biocontrol cultures to inhibit and/or reduce the growth of L. monocytogenes, Salmonella, and E. coli O157:H7 in different matrices, and environments.

Lineage specific recombination rates and microevolution in Listeria monocytogenes.

BACKGROUND: The bacterium Listeria monocytogenes is a saprotroph as well as an opportunistic human foodborne pathogen, which has previously been shown to consist of at least two widespread lineages (termed lineages I and II) and an uncommon lineage (lineage III). While some L. monocytogenes strains show evidence for considerable diversification by homologous recombination, our understanding of the contribution of recombination to L. monocytogenes evolution is still limited. We therefore used STRUCTURE and ClonalFrame, two programs that model the effect of recombination, to make inferences about the population structure and different aspects of the recombination process in L. monocytogenes. Analyses were performed using sequences for seven loci (including the house-keeping genes gap, prs, purM and ribC, the stress response gene sigB, and the virulence genes actA and inlA) for 195 L. monocytogenes isolates. RESULTS: Sequence analyses with ClonalFrame and the Sawyer's test showed that recombination is more prevalent in lineage II than lineage I and is most frequent in two house-keeping genes (ribC and purM) and the two virulence genes (actA and inlA). The relative occurrence of recombination versus point mutation is about six times higher in lineage II than in lineage I, which causes a higher genetic variability in lineage II. Unlike lineage I, lineage II represents a genetically heterogeneous population with a relatively high proportion (30% average) of genetic material imported from external sources. Phylograms, constructed with correcting for recombination, as well as Tajima's D data suggest that both lineages I and II have suffered a population bottleneck. CONCLUSION: Our study shows that evolutionary lineages within a single bacterial species can differ considerably in the relative contributions of recombination to genetic diversification. Accounting for recombination in phylogenetic studies is critical, and new evolutionary models that account for the possibility of changes in the rate of recombination would be required. While previous studies suggested that only L. monocytogenes lineage I has experienced a recent bottleneck, our analyses clearly show that lineage II experienced a bottleneck at about the same time, which was subsequently obscured by abundant homologous recombination after the lineage II bottleneck. While lineage I and lineage II should be considered separate species from an evolutionary viewpoint, maintaining single species name may be warranted since both lineages cause the same type of human disease.

Lineage specific recombination and positive selection in coding and intragenic regions contributed to evolution of the main Listeria monocytogenes virulence gene cluster.

The major virulence cluster of Listeria monocytogenes harbors six virulence genes that encode proteins critical for the intracellular life cycle of this human and animal pathogen. In this study, we determined the sequence (8709nt) of the virulence gene cluster (including the six main virulence genes) in 40 L. monocytogenes isolates from different source populations (human clinical cases, animal clinical cases, foods, and natural environments). An alignment of the full length cluster as well as individual gene alignments and alignments of intragenic regions were used for phylogenetic, recombination, and positive selection analyses. Initial phylogenetic analyses showed that the sequences represented two main clusters, consistent with previously defined L. monocytogenes phylogenetic lineages. The 40 sequences represented 25 distinct allelic types and the overall alignment included 592 polymorphic sites. Overall, our data show that (i) virulence genes in the main L. monocytogenes virulence gene cluster include highly conserved genes (i.e., hly and prfA) as well as diverse genes that appear to have evolved by positive selection (mpl, actA, and plcA), (ii) recombination has played an important role in the evolution of the virulence gene cluster, but is limited to lineage II isolates, and (iii) the promoter region driving the transcription of virulence genes transcribed early in intracellular infection (i.e., hly and plcA) has evolved by positive selection. The genes and intragenic regions in the L. monocytogenes virulence gene cluster thus have evolved independently, despite their close physical linkage, likely reflecting distinct selective pressures associated with expression and function of the proteins encoded in this region.

Draft Genome Sequence of Enterococcus faecium Strain J19, Isolated from Cabbage.

Herein, we report the draft genome sequence of a newly discovered probiotic strain, Enterococcus faecium J19, which was isolated from cabbage. Strain J19 has shown antagonistic effects against the human foodborne pathogen Listeria monocytogenes in coculture and in different food matrices.

Draft Genome Sequence of Lactobacillus salivarius L28 Isolated from Ground Beef.

In this report, we describe the draft genome sequence of a newly discovered probiotic strain, Lactobacillus salivarius L28. L. salivarius L28 demonstrates antagonistic effects against human foodborne pathogens, including Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes, in coculture experiments and food matrices.