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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.

Campylobacter jejuni clone SA is the major cause of sheep abortion and contributes significantly to foodborne illnesses in the United States. Clone SA is hypervirulent because of its distinct ability to produce systemic infection and its predominant role in clinical sheep abortion. Despite the importance of clone SA, little is known about its distribution and epidemiological features in cattle. Here we describe a prospective study on C. jejuni clone SA prevalence in 35 feedlots in 5 different states in the United States and a retrospective analysis of clone SA in C. jejuni isolates collected by National Animal Health Monitoring System (NAHMS) dairy studies in 2002, 2007, and 2014. In feedlot cattle feces, the overall prevalence of Campylobacter organisms was 72.2%, 82.1% of which were C. jejuni . Clone SA accounted for 5.8% of the total C. jejuni isolates, but its prevalence varied by feedlot and state. Interestingly, starlings on the feedlots harbored C. jejuni in feces, including clone SA, suggesting that these birds may play a role in the transmission of Campylobacter . In dairy cattle, the overall prevalence of clone SA was 7.2%, but a significant decrease in the prevalence was observed from 2002 to 2014. Whole-genome sequence analysis of the dairy clone SA isolates revealed that it was genetically stable over the years and most of the isolates carried the tetracycline resistance gene tet (O) in the chromosome. These findings indicate that clone SA is widely distributed in both beef and dairy cattle and provide new insights into the molecular epidemiology of clone SA in ruminants. IMPORTANCE C. jejuni clone SA is a major cause of small-ruminant abortion and an emerging threat to food safety because of its association with foodborne outbreaks. Cattle appear to serve as a major reservoir for this pathogenic organism, but there is a major gap in our knowledge about the epidemiology of clone SA in beef and dairy cattle. By taking advantage of surveillance studies conducted on a national scale, we found a wide but variable distribution of clone SA in feedlot cattle and dairy cows in the United States. Additionally, the work revealed important genomic features of clone SA isolates from cattle. These findings provide critically needed information for the development of preharvest interventions to control the transmission of this zoonotic pathogen. Control of C. jejuni clone SA will benefit both animal health and public health, as it is a zoonotic pathogen causing disease in both ruminants and humans.

IncA/C plasmids are a class of plasmids from the Enterobacteriaceae that are relatively large (49 to >180 kbp), that are readily transferred by conjugation, and that carry multiple antimicrobial resistance genes. Reconstruction of the phylogeny of these plasmids has been difficult because of the high rate of remodeling by recombination-mediated horizontal gene transfer (HGT). We hypothesized that evaluation of nucleotide polymorphisms relative to the rate of HGT would help to develop a clock to show whether anthropic practices have had significant influences on the lineages of the plasmid. A system was developed to rapidly sequence up to 191 known open reading frames from each of 39 recently isolated IncA/C plasmids from a diverse panel of Salmonella enterica and Escherichia coli strains. With these data plus sequences from GenBank, we were able to distinguish six distinct lineages that had extremely low numbers of polymorphisms within each lineage, especially among the largest group designated as group 1. Two regions, each about half the plasmid in size, could be distinguished with a separate lineal pattern. The distribution of group 1 showed that it has migrated extremely rapidly with fewer polymorphisms than can be expected in 2,000 years. Remodeling by frequent HGT was evident, with a pattern that appeared to have the highest rate just upstream of the putative conjugation origin of transfer (oriT). It seems likely that when an IncA/C plasmid is transferred by conjugation there is an opportunity for plasmid remodeling adjacent to the oriT, which was also adjacent to a multiple antimicrobial resistance gene cassette.

In an attempt to develop a method to discriminate among isolates of Listeria monocytogenes , the sequences of all of the annotated genes from the fully sequenced strain L. monocytogenes EGD-e (serotype 1/2a) were compared by BLASTn to a file of the unfinished genomic sequence of L. monocytogenes ATCC 19115 (serotype 4b). Approximately 7% of the matching genes demonstrated 90% or lower identity between the two strains, and the lowest observed identity was 80%. Nine genes ( hisJ , cbiE , truB , ribC , comEA , purM , aroE , hisC , and addB ) in the 80 to 90% identity group and two genes ( gyrB and rnhB ) with approximately 97% identity were selected for multilocus sequence analysis in two sets of L. monocytogenes isolates (a 15-strain diversity set and a set of 19 isolates from a single food-processing plant). Based on concatenated sequences, a total of 33 allotypes were differentiated among the 34 isolates tested. Population genetics analyses revealed three lineages of L. monocytogenes that differed in their history of apparent recombination. Lineage I appeared to be completely clonal, whereas representatives of the other lineages demonstrated evidence of horizontal gene transfer and recombination. Although most of the gene sequences for lineage II strains were distinct from those of lineage I, a few strains with the majority of genes characteristic of lineage II had some that were characteristic of lineage I. Genes from lineage III organisms were mostly similar to lineage I genes, with instances of genes appearing to be mosaics with lineage II genes. Even though lineage I and lineage II generally demonstrated very distinct sequences, the sequences for the 11 selected genes demonstrated little discriminatory power within each lineage. In the L. monocytogenes isolate set obtained from one food-processing plant, lineage I and lineage II were found to be almost equally prevalent. While it appears that different lineages of L. monocytogenes can share habitats, they appear to differ in their histories of horizontal gene transfer.

The DNA sequence of the flaA short variable region (SVR) was used to analyze a random population of Campylobacter isolates to investigate the weakly clonal population structure of members of the genus. The SVR sequence from 197 strains of C. jejuni and C. coli isolated from humans, bovine, swine, and chickens identified a group of 43 strains containing disparate short variable region sequences compared to the rest of the population. This group contains both C. jejuni and C. coli strains but disproportionately consisted of bovine isolates. Relative synonymous codon usage analysis of the sequences identified two groups: one group typified C. jejuni , and the second group was characteristic for C. coli and the disparate alleles were not clustered. The data show that there is significant differentiation of Campylobacter populations according to the source of the isolate even without considering the disparate isolates. Even though there is significant differentiation of chicken and bovine isolates, the bovine isolates did not show any difference in ability to colonize chickens. It is possible that disparate sequences were obtained through the lateral transfer of DNA from Campylobacter species other than C. jejuni and C. coli . It is evident that recombination within the flaA SVR occurs rapidly. However, the rate of migration between populations appears to limit the distribution of sequences and results in a weakly clonal population structure.