Rapid identification of enteric bacteria from whole genome sequences using average nucleotide identity metrics.

Identification of enteric bacteria species by whole genome sequence (WGS) analysis requires a rapid and an easily standardized approach. We leveraged the principles of average nucleotide identity using MUMmer (ANIm) software, which calculates the percent bases aligned between two bacterial genomes and their corresponding ANI values, to set threshold values for determining species consistent with the conventional identification methods of known species. The performance of species identification was evaluated using two datasets: the Reference Genome Dataset v2 (RGDv2), consisting of 43 enteric genome assemblies representing 32 species, and the Test Genome Dataset (TGDv1), comprising 454 genome assemblies which is designed to represent all species needed to query for identification, as well as rare and closely related species. The RGDv2 contains six Campylobacter spp., three Escherichia/Shigella spp., one Grimontia hollisae, six Listeria spp., one Photobacterium damselae, two Salmonella spp., and thirteen Vibrio spp., while the TGDv1 contains 454 enteric bacterial genomes representing 42 different species. The analysis showed that, when a standard minimum of 70% genome bases alignment existed, the ANI threshold values determined for these species were ≥95 for Escherichia/Shigella and Vibrio species, ≥93% for Salmonella species, and ≥92% for Campylobacter and Listeria species. Using these metrics, the RGDv2 accurately classified all validation strains in TGDv1 at the species level, which is consistent with the classification based on previous gold standard methods.

Characterization of a Nonagglutinating Toxigenic Vibrio cholerae Isolate.

Toxigenic Vibrio cholerae serogroup O1 is the etiologic agent of the disease cholera, and strains of this serogroup are responsible for pandemics. A few other serogroups have been found to carry cholera toxin genes-most notably, O139, O75, and O141-and public health surveillance in the United States is focused on these four serogroups. A toxigenic isolate was recovered from a case of vibriosis from Texas in 2008. This isolate did not agglutinate with any of the four different serogroups' antisera (O1, O139, O75, or O141) routinely used in phenotypic testing and did not display a rough phenotype. We investigated several hypotheses that might explain the recovery of this potential nonagglutinating (NAG) strain using whole-genome sequencing analysis and phylogenetic methods. The NAG strain formed a monophyletic cluster with O141 strains in a whole-genome phylogeny. Furthermore, a phylogeny of ctxAB and tcpA sequences revealed that the sequences from the NAG strain also formed a monophyletic cluster with toxigenic U.S. Gulf Coast (USGC) strains (O1, O75, and O141) that were recovered from vibriosis cases associated with exposures to Gulf Coast waters. A comparison of the NAG whole-genome sequence showed that the O-antigen-determining region of the NAG strain was closely related to those of O141 strains, and specific mutations were likely responsible for the inability to agglutinate. This work shows the utility of whole-genome sequence analysis tools for characterization of an atypical clinical isolate of V. cholerae originating from a USGC state. IMPORTANCE Clinical cases of vibriosis are on the rise due to climate events and ocean warming (1, 2), and increased surveillance of toxigenic Vibrio cholerae strains is now more crucial than ever. While traditional phenotyping using antisera against O1 and O139 is useful for monitoring currently circulating strains with pandemic or epidemic potential, reagents are limited for non-O1/non-O139 strains. With the increased use of next-generation sequencing technologies, analysis of less well-characterized strains and O-antigen regions is possible. The framework for advanced molecular analysis of O-antigen-determining regions presented herein will be useful in the absence of reagents for serotyping. Furthermore, molecular analyses based on whole-genome sequence data and using phylogenetic methods will help characterize both historical and novel strains of clinical importance. Closely monitoring emerging mutations and trends will improve our understanding of the epidemic potential of Vibrio cholerae to anticipate and rapidly respond to future public health emergencies.

CLONAL SPREAD OF YERSINIA ENTEROCOLITICA 1B/O:8 IN MULTIPLE ZOO SPECIES.

Yersinia enterocolitica (YE) bioserotype 1B/O:8 (YE 1B/O:8) was identified in routine culture of a variety of zoo species housed at Omaha's Henry Doorly Zoo and Aquarium (OHDZA) from April to July 2011. Animal cases representing 12 species had YE detected from 34 cases during routine fecal monitoring and/or during postmortem examination: Coquerel's sifakas (Propithecus coquereli, two cases), black & white (BW) ruffed lemurs (Varecia variegata variegata, six cases), red ruffed lemurs (Varecia rubra, seven cases), white handed gibbon (Hylobates lar albimana, one case), black lemurs (Eulemur macaco, three cases), mongoose lemurs (Eulemur mongoz, two cases), African hunting dogs (Lycaon pictus, five cases), agile gibbons (Hylobates agilis, three cases), siamangs (Hylobates syndactylus, two cases), colobus monkey (Colobus angolensis palliates, one case), argus pheasant (Argusianus argus, one case), and orangutan (Pongo pygmaeus, one case). Most species were not symptomatic; however, three symptomatic cases in Coquerel's sifakas (two) and a white handed gibbon (one) showed clinical signs of diarrhea and lethargy that resulted in death for the Coquerel's sifakas. One unexpected death also occurred in a BW ruffed lemur. To the authors' knowledge, this is the first report of YE 1B/O:8 in such a large variety of zoo species. The source of the YE could not be identified, prompting the initiation of a diseases surveillance program to prevent further cases for the species that are sensitive to YE. To date, no additional cases have been identified, thus suggesting a single introduction of the YE 1B/O:8 strain into the zoo environment.

A Multistate Outbreak of E Coli O157:H7 Infections Linked to Soy Nut Butter.

BACKGROUND: In 2017, we conducted a multistate investigation to determine the source of an outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 infections, which occurred primarily in children. METHODS: We defined a case as infection with an outbreak strain of STEC O157:H7 with illness onset between January 1, 2017, and April 30, 2017. Case patients were interviewed to identify common exposures. Traceback and facility investigations were conducted; food samples were tested for STEC. RESULTS: We identified 32 cases from 12 states. Twenty-six (81%) cases occurred in children <18 years old; 8 children developed hemolytic uremic syndrome. Twenty-five (78%) case patients ate the same brand of soy nut butter or attended facilities that served it. We identified 3 illness subclusters, including a child care center where person-to-person transmission may have occurred. Testing isolated an outbreak strain from 11 soy nut butter samples. Investigations identified violations of good manufacturing practices at the soy nut butter manufacturing facility with opportunities for product contamination, although the specific route of contamination was undetermined. CONCLUSIONS: This investigation identified soy nut butter as the source of a multistate outbreak of STEC infections affecting mainly children. The ensuing recall of all soy nut butter products the facility manufactured, totaling >1.2 million lb, likely prevented additional illnesses. Prompt diagnosis of STEC infections and appropriate specimen collection aids in outbreak detection. Child care providers should follow appropriate hygiene practices to prevent secondary spread of enteric illness in child care settings. Firms should manufacture ready-to-eat foods in a manner that minimizes the risk of contamination.

Characterization of clinical and environmental isolates of Vibrio cidicii sp. nov., a close relative of Vibrio navarrensis.

Four Vibrio spp. isolates from the historical culture collection at the Centers for Disease Control and Prevention, obtained from human blood specimens (n=3) and river water (n=1), show characteristics distinct from those of isolates of the most closely related species, Vibrio navarrensis and Vibrio vulnificus, based on phenotypic and genotypic tests. They are specifically adapted to survival in both freshwater and seawater, being able to grow in rich media without added salts as well as salinities above that of seawater. Phenotypically, these isolates resemble V. navarrensis, their closest known relative with a validly published name, but the group of isolates is distinguished from V. navarrensis by the ability to utilize l-rhamnose. Average nucleotide identity and percent DNA-DNA hybridization values obtained from the pairwise comparisons of whole-genome sequences of these isolates to V. navarrensis range from 95.4-95.8 % and 61.9-64.3 %, respectively, suggesting that the group represents a different species. Phylogenetic analysis of the core genome, including four protein-coding housekeeping genes (pyrH, recA, rpoA and rpoB), places these four isolates into their own monophyletic clade, distinct from V. navarrensis and V. vulnificus. Based on these differences, we propose these isolates represent a novel species of the genus Vibrio, for which the name Vibrio cidicii sp. nov. is proposed; strain LMG 29267T (=CIP 111013T=2756-81T), isolated from river water, is the type strain.

Determination of Evolutionary Relationships of Outbreak-Associated Listeria monocytogenes Strains of Serotypes 1/2a and 1/2b by Whole-Genome Sequencing.

We used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates of Listeria monocytogenes serotypes 1/2a and 1/2b. Isolates from 6 of 11 outbreaks fell outside the clonal groups or "epidemic clones" that have been previously associated with outbreaks, suggesting that epidemic potential may be widespread in L. monocytogenes and is not limited to the recognized epidemic clones. Pairwise comparisons between epidemiologically related isolates within clonal complexes showed that genome-level variation differed by 2 orders of magnitude between different comparisons, and the distribution of point mutations (core versus accessory genome) also varied. In addition, genetic divergence between one closely related pair of isolates from a single outbreak was driven primarily by changes in phage regions. The evolutionary analysis showed that the changes could be attributed to horizontal gene transfer; members of the diverse bacterial community found in the production facility could have served as the source of novel genetic material at some point in the production chain. The results raise the question of how to best utilize information contained within the accessory genome in outbreak investigations. The full magnitude and complexity of genetic changes revealed by genome sequencing could not be discerned from traditional subtyping methods, and the results demonstrate the challenges of interpreting genetic variation among isolates recovered from a single outbreak. Epidemiological information remains critical for proper interpretation of nucleotide and structural diversity among isolates recovered during outbreaks and will remain so until we understand more about how various population histories influence genetic variation.

Genome Sequences of Vibrio navarrensis, a Potential Human Pathogen.

Vibrio navarrensis is an aquatic bacterium recently shown to be associated with human illness. We report the first genome sequences of three V. navarrensis strains obtained from clinical and environmental sources. Preliminary analyses of the sequences reveal that V. navarrensis contains genes commonly associated with virulence in other human pathogens.

Molecular and phenotypic characterization of Vibrio navarrensis isolates associated with human illness.

We characterized 18 Vibrio isolates, including 15 recovered from human clinical specimens, and found that they clustered with two previously characterized Vibrio navarrensis isolates in a phylogenetic analysis. Four of the 18 strains may represent a new Vibrio species, distinct from V. navarrensis. The potential role of V. navarrensis in human disease needs further investigation.

Evolutionary dynamics of Vibrio cholerae O1 following a single-source introduction to Haiti.

UNLABELLED: Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics. IMPORTANCE: Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain.