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

Carbapenem-Resistant Bacteria Recovered from Faeces of Dairy Cattle in the High Plains Region of the USA

OBJECTIVE:A study was conducted to recover carbapenem-resistant bacteria from the faeces of dairy cattle and identify the underlying genetic mechanisms associated with reduced phenotypic susceptibility to carbapenems. METHODS:One hundred and fifty-nine faecal samples from dairy cattle were screened for carbapenem-resistant bacteria. Phenotypic screening was conducted on two media containing ertapenem. The isolates from the screening step were characterised via disk diffusion, Modified Hodge, and Carba NP assays. Carbapenem-resistant bacteria and carbapenemase-producing isolates were subjected to Gram staining and biochemical testing to include Gram-negative bacilli. Whole genome sequencing was performed on bacteria that exhibited either a carbapenemase-producing phenotype or were not susceptible to ertapenem and were presumptively Enterobacteriaceae. RESULTS:Of 323 isolates collected from the screening media, 28 were selected for WGS; 21 of which were based on a carbapenemase-producing phenotype and 7 were presumptively Enterobacteriaceae and not susceptible to ertapenem. Based on analysis of WGS data, isolates included: 3 Escherichia coli harbouring blaCMY-2 and truncated ompF genes; 8 Aeromonas harbouring blacphA-like genes; 1 Acinetobacter baumannii harbouring a novel blaOXA gene (blaOXA-497); and 6 Pseudomonas with conserved domains of various carbapenemase-producing genes. CONCLUSIONS:Carbapenem resistant bacteria appear to be rare in cattle. Nonetheless, carbapenem-resistant bacteria were detected across various genera and were found to harbour a variety of mechanisms conferring reduced susceptibility. The development and dissemination of carbapenem-resistant bacteria in livestock would have grave implications for therapeutic treatment options in human medicine; thus, continued monitoring of carbapenem susceptibility among enteric bacteria of livestock is warranted

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 stx 2-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

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

Two Draft Genome Sequences of a New Serovar of \u3ci\u3eSalmonella enterica\u3c/i\u3e, Serovar Lubbock

Salmonella enterica is principally a foodborne pathogen that shows considerable serovar diversity. In this report, we present two draft genome sequences of Salmonella enterica subsp. enterica serovar Lubbock, a novel serovar

Reduction of Pathogens in Feces and Lymph Nodes Collected from Beef Cattle Fed <i>Lactobacillus salivarius</i> (L28), <i>Lactobacillus acidophilus</i> (NP51) and <i>Propionibacterium freudenreichii</i> (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

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

Substantial within-Animal Diversity of \u3ci\u3eSalmonella\u3c/i\u3e 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

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 stx 2-positive sequence type 29 (ST29) human O26:H11 strains, while stx 1 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