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.

Cronobacter sakazakii Infections in Two Infants Linked to Powdered Infant Formula and Breast Pump Equipment - United States, 2021 and 2022.

Cronobacter sakazakii, a species of gram-negative bacteria belonging to the Enterobacteriaceae family, is known to cause severe and often fatal meningitis and sepsis in young infants. C. sakazakii is ubiquitous in the environment, and most reported infant cases have been attributed to contaminated powdered infant formula (powdered formula) or breast milk that was expressed using contaminated breast pump equipment (1-3). Previous investigations of cases and outbreaks have identified C. sakazakii in opened powdered formula, breast pump parts, environmental surfaces in the home, and, rarely, in unopened powdered formula and formula manufacturing facilities (2,4-6). This report describes two infants with C. sakazakii meningitis reported to CDC in September 2021 and February 2022. CDC used whole genome sequencing (WGS) analysis to link one case to contaminated opened powdered formula from the patient's home and the other to contaminated breast pump equipment. These cases highlight the importance of expanding awareness about C. sakazakii infections in infants, safe preparation and storage of powdered formula, proper cleaning and sanitizing of breast pump equipment, and using WGS as a tool for C. sakazakii investigations.

Vibrio tarriae sp. nov., a novel member of the Cholerae clade.

A number of bacteria with close resemblance to Vibrio cholerae have been isolated over the years by the Centres for Disease Control and Prevention (CDC), which could not be assigned a proper taxonomic designation on the basis of the results from preliminary identification methods. Nine such isolates have been found to share 16S rRNA gene identity exceeding 99 % with V. cholerae, yet DNA-DNA hybridization (60.4-62.1 %) and average nucleotide identity values (94.4-95.1 %) were below the species cut-off, indicating a potentially novel species. Phylogenetic analysis of core genomes places this group of isolates in a monophyletic clade, within the 'Cholerae clade', but distinct from any other species. Extensive phenotypic characterization reveals unique biochemical properties that distinguish this novel species from V. cholerae. Comparative genomic analysis reveals a unique set of siderophore genes, indicating that iron acquisition strategies could be vital for the divergence of the novel species from a common ancestor with V. cholerae. On the basis of the genetic, phylogenetic and phenotypic differences observed, we propose that these isolates represent a novel species of the genus Vibrio, for which the name Vibrio tarriae sp. nov. is proposed. Strain 2521-89 T (= DSM 112461=CCUG 75318), isolated from lake water, is the type strain.

Population Analysis of Vibrio cholerae in Aquatic Reservoirs Reveals a Novel Sister Species (Vibrio paracholerae sp. nov.) with a History of Association with Humans.

Most efforts to understand the biology of Vibrio cholerae have focused on a single group, the pandemic-generating lineage harboring the strains responsible for all known cholera pandemics. Consequently, little is known about the diversity of this species in its native aquatic environment. To understand the differences in the V. cholerae populations inhabiting regions with a history of cholera cases and those lacking such a history, a comparative analysis of population composition was performed. Little overlap was found in lineage compositions between those in Dhaka, Bangladesh (where cholera is endemic), located in the Ganges Delta, and those in Falmouth, MA (no known history of cholera), a small coastal town on the United States east coast. The most striking difference was the presence of a group of related lineages at high abundance in Dhaka, which was completely absent from Falmouth. Phylogenomic analysis revealed that these lineages form a cluster at the base of the phylogeny for the V. cholerae species and were sufficiently differentiated genetically and phenotypically to form a novel species. A retrospective search revealed that strains from this species have been anecdotally found from around the world and were isolated as early as 1916 from a British soldier in Egypt suffering from choleraic diarrhea. In 1935, Gardner and Venkatraman unofficially referred to a member of this group as Vibrio paracholerae. In recognition of this earlier designation, we propose the name Vibrio paracholerae sp. nov. for this bacterium. Genomic analysis suggests a link with human populations for this novel species and substantial interaction with its better-known sister species. IMPORTANCE Cholera continues to remain a major public health threat around the globe. Understanding the ecology, evolution, and environmental adaptation of the causative agent (Vibrio cholerae) and tracking the emergence of novel lineages with pathogenic potential are essential to combat the problem. In this study, we investigated the population dynamics of Vibrio cholerae in an inland locality, which is known as endemic for cholera, and compared them with those of a cholera-free coastal location. We found the consistent presence of the pandemic-generating lineage of V. cholerae in Dhaka, where cholera is endemic, and an exclusive presence of a lineage phylogenetically distinct from other V. cholerae lineages. Our study suggests that this lineage represents a novel species that has pathogenic potential and a human link to its environmental abundance. The possible association with human populations and coexistence and interaction with toxigenic V. cholerae in the natural environment make this potential human pathogen an important subject for future studies.

Draft Genome Sequences of Eight Vibrio sp. Clinical Isolates from across the United States That Form a Basal Sister Clade to Vibrio cholerae.

We sequenced the genomes of eight isolates from various regions of the United States. These isolates form a monophyletic cluster clearly related to but distinct from Vibrio cholerae. Phylogenetic and genomic analyses suggest that they represent a basal lineage highly divergent from Vibrio cholerae or a novel species.

Draft Genome Sequences of Nine Vibrio sp. Isolates from across the United States Closely Related to Vibrio cholerae.

We are reporting whole-genome sequences of nine Vibrio sp. isolates closely related to the waterborne human pathogen Vibrio cholerae. These isolates were recovered from sources, including human samples, from different regions of the United States. Genome analysis suggests that this group of isolates represents a highly divergent basal V. cholerae lineage or a closely related novel species.