Genomic analysis of inter-hospital transmission of vancomycin-resistant Enterococcus faecium sequence type 80 isolated during an outbreak in Hiroshima, Japan.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Complete genome sequence of cfr(B)-carrying Enterococcus raffinosus isolated from bile in a patient in Japan.

OBJECTIVES: Linezolid is an antibiotic used to treat infectious diseases caused by vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. Recently, Enterococcus Spp.-carrying mobile linezolid resistance genes were reported. Herein, we report the complete genome sequence of Enterococcus raffinosus JARB-HU0741, which was isolated from a bile sample of a patient in Japan on May 5, 2021, and carries a linezolid resistance gene, cfr(B). Nevertheless, this isolate was susceptible to linezolid. METHODS: Whole-genome sequencing was performed using HiSeq X FIVE (Illumina) and GridION (Oxford Nanopore Technologies). The sequence reads were assembled using Unicycler v0.4.8, and the complete genome was annotated using DFAST v1.2.18. Antimicrobial resistance genes were detected with Abricate v1.0.1, using the ResFinder database. The minimum inhibitory concentrations (MICs) were determined using broth microdilution and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute. RESULTS: E. raffinosus JARB-HU0741 contained a 3 248 808-bp chromosome and a 1 156 277-bp megaplasmid. cfr(B) was present in the Tn6218-like transposon, which was inserted into a gene encoding a PRD domain-containing protein present in the megaplasmid, but the isolate was susceptible to linezolid (MIC, 0.5 µg/mL). The Tn6218-like transposon was similar to the Tn6218 of Clostridioides difficile Ox3196 and the Tn6218-like transposon of Enterococcus faecium UW11733; however, three genes encoding a topoisomerase, an S-adenosylmethionine-dependent methyltransferase, and a TetR family transcriptional regulator were present in the previous Tn6218- or Tn6218-like transposon. CONCLUSION: This is the first report of the complete genome sequence of E. raffinosus carrying cfr(B). E. raffinosus carrying cfr(B) without linezolid resistance poses a threat, as it could serve as a reservoir for mobile linezolid resistance genes.

Complete genome sequence of optrA-carrying Enterococcus faecalis isolated from open pus in a Japanese patient.

OBJECTIVES: The occurrence of linezolid resistance in enterococci has recently increased. Here, we report the genomic characterization of Enterococcus faecalis strain JARB-HU0796-isolated from the open pus of a patient in Hiroshima, Japan-which shows nonsusceptibility to linezolid (MIC of 4 µg/mL). METHODS: JARB-HU0796 whole-genome sequencing was performed using short-read sequencing with Illumina Hiseq X Five and long-read sequencing using GridION. These reads were collected using the assembly pipeline Unicycler and annotated with DFAST. Antimicrobial resistance genes were detected using the Abricate and ResFinder databases, and the sequence type identified using PubMLST. The antimicrobial susceptibility of JARB-HU0796 was determined with the Eiken dry-plate QH02 system. RESULTS: The JARB-HU0796 complete genome contained a circular chromosome (2 722 585 bp) and two circular plasmids (85 996 bp and 58 872 bp). The chromosome harbours the optrA gene, which confers resistance to oxazolidinones and phenicols. JARB-HU0796 showed nonsusceptibility to linezolid and multidrug resistance to other antibiotics. MLST analysis identified JARB-HU0796 as ST476, similar to the optrA-positive E. faecalis ST476 isolates from swine (South Korea, 2020) and pet food (Switzerland, 2022). The optrA region of JARB-HU0796 is nearly identical to that of ST476 E. faecalis strain TZ2, isolated from humans (China, 2013). CONCLUSIONS: To the best of our knowledge, this is the first report of the complete genome sequence of E. faecalis ST476 carrying optrA on a chromosome isolated from a patient in Japan. The strain may have originated in animals, suggesting that the organisms acquired resistance to linezolid because the optrA gene may be closely spread between animals and humans.

Complete Genome Sequences of Three Streptococcus ruminantium Strains Obtained from Endocarditis Lesions of Cattle in Japan.

Streptococcus ruminantium is a close relative of Streptococcus suis, an important zoonotic pathogen that causes various diseases in pigs and humans. Here, we report the complete genome sequences of three S. ruminantium strains isolated from bovine endocarditis in Japan.

Complete Genome Sequences of Four Streptococcus parasuis Strains Obtained from Saliva of Domestic Pigs in Japan.

Streptococcus parasuis is a close relative of Streptococcus suis, an important zoonotic pathogen that causes various diseases in pigs and humans. Here, we report the complete genome sequences of four strains, including the type strain of S. parasuis, isolated from the saliva of healthy pigs in Japan.

Basis of the persistence of capsule-negative Streptococcus suis in porcine endocarditis inferred from comparative genomics.

The capsule (cap) of Streptococcus suis is an anti-phagocytic element and is one of the major virulence factors. However, we have found cap-positive and cap-negative isolates in porcine endocarditis. Here, we compared genome sequences of multiple cap-negative isolates with those of a cap-positive isolate from a single endocarditis. Cap-positive and cap-negative isolates from the same pig were phylogenetically closest compared with those from other pigs. Some of cap-negative isolates from the same pig showed different mutations in capsular polysaccharide synthesis (cps) genes, suggesting that these isolates arisen in pigs after infection. Different mutations in whole-genomes were also found among isolates with identical mutations in cps genes, indicating that mutations in cps genes and the whole-genome occurred independently. Since cap-negative isolates are rarely found in lesions of other diseases, these results suggest that endocarditis lesions may simply favored cap-negative mutants to survive the niches, leading to their persistence in the lesions.

Transition of microbiota in chicken cecal droppings from commercial broiler farms.

BACKGROUND: Chickens are major sources of human nutrition worldwide, but the chicken intestinal microbiota can be a source of bacterial infection. The microbiota has potential to regulate the colonization of pathogens by competitive exclusion, production of antimicrobial compounds, and stimulation of the mucosal immune system. But information on the microbiota in commercial broiler chickens is limited because of the difficulty of conducting studies at commercial farms. To obtain fundamental information that can be used to control pathogens in chickens, we determined the 6-week dynamics of microbiota in chicken cecal droppings from commercial broiler farms. RESULTS: Cecal droppings from four chickens were collected once a week from 1 to 6 weeks of age at three commercial broiler farms. A total of 168 samples were collected from 7 flocks and subjected to 16S rRNA amplicon sequencing. Despite the farms have distinctly different climate conditions, the microbiota in the same growth stages were similar among farms. Moreover, as the chickens grew and the feed types were switched, the richness and diversity of the microbiota gradually increased and convergence of the composition of the microbiota was apparent. Notably, minor bacterial taxa (i.e. OTUs with relative abundance < 0.05%) within the microbiota were changed by the chicken age, switching of feed types, and presence of Campylobacter. In particular, the effects of switching of feed types on the microbiota were larger than the effects of age and Campylobacter. CONCLUSIONS: Irrespective of the locations of the farms, the microbiota of chicken cecum, especially minor bacteria, was successively changed more affected by feed types than by ages. Switching of feed types inducing the alteration of the microbiota may be associated with the colonization of pathogens in the chicken gut. These results will also help with extrapolation of studies in experimental animals to those in the commercial farms.

Complete Genome Sequences of Two Streptococcus suis Strains Isolated from Asymptomatic Pigs.

Streptococcus suis is an important zoonotic pathogen that causes major economic problems in the pig industry worldwide and serious infections in humans, including meningitis and septicemia. Here, we report the complete genome sequences of two strains isolated from asymptomatic pigs.

16S rRNA Gene Amplicon Sequence Data from Feces of Wild Deer (Cervus nippon) in Japan.

We report 16S rRNA amplicon sequence data from feces of 109 wild deer in Japan. The dominant bacterial taxa in fecal microbiota of wild deer hunted between village and mountainous areas and those living on Miyajima Island and in Nara Park were similar but differed in abundance.

16S rRNA Gene Amplicon Sequence Data from Feces of Five Species of Wild Animals in Japan.

We report 16S rRNA amplicon sequence data from feces from 58 wild boars, 60 feral raccoons, 9 wild Japanese badgers, 21 wild masked palm civets, and 8 wild raccoon dogs in Japan. The predominant bacterial taxa in the fecal microbiota were similar in part but varied among the animal species.

Characterization of pig saliva as the major natural habitat of Streptococcus suis by analyzing oral, fecal, vaginal, and environmental microbiota.

It is generally difficult to specify the sources of infection by which domestic animals may acquire pathogens. Through 16S rRNA gene amplicon sequencing, we compared the composition of microbiota in the saliva, vaginal mucus, and feces of pigs, and in swabs of feeder troughs and water dispensers collected from pig farms in Vietnam. The composition of the microbiota differed between samples in each sample group. Streptococcus, Actinobacillus, Moraxella, and Rothia were the most abundant genera and significantly discriminative in saliva samples, regardless of the plasticity and changeability of the composition of microbiota in saliva. Moreover, species assignment of the genus Streptococcus revealed that Streptococcus suis was exceptional in the salivary microbiota, due to being most abundant among the streptococcal species and sharing estimated proportions of 5.7%-9.4% of the total bacteria in saliva. Thus, pig oral microbiota showed unique characteristics in which the major species was the pig pathogen. On the other hand, ß-diversity analysis showed that the microbiota in saliva was distinct from those in the others. From the above results, pig saliva was shown to be the major natural habitat of S. suis, and is suggested to be the most probable source of S. suis infection.

Assessment of pig saliva as a Streptococcus suis reservoir and potential source of infection on farms by use of a novel quantitative polymerase chain reaction assay.

OBJECTIVE To evaluate colonization of Streptococcus suis and Streptococcus parasuis on pig farms in Japan and to identify sources of infections. SAMPLE Saliva, feces, and vaginal swab samples from 84 healthy pigs of several growth stages on 4 farms and swab samples of feed troughs and water dispensers at those farms. PROCEDURES Samples were collected from August 2015 to June 2016. Two quantitative PCR (qPCR) assays (one for S suis and the other for S parasuis) were designed for use in the study. The novel qPCR assays were used in combination with previously described qPCR assays for S suis serotype 2 or 1/2 and total bacteria. Relative abundance of bacteria in each sample was evaluated. RESULTS Streptococcus suis was detected in all saliva samples and some of the other samples, whereas S parasuis was not detected in any of the samples, including saliva samples, which indicated a difference in colonization preference. The ratio of S suis to total bacteria in saliva appeared to increase with age of pigs. Streptococcus suis serotype 2 or 1/2 was detected in a few saliva samples and feed trough swab samples at 2 farms where S suis infections were prevalent. CONCLUSIONS AND CLINICAL RELEVANCE Saliva, especially that of sows, appeared to be a reservoir and source of S suis infection for pigs. The qPCR assay described here may provide an effective way to monitor for S suis in live pigs, which could lead to effective disease control on pig farms.

Development of PCR for identifying Streptococcus parasuis, a close relative of Streptococcus suis.

Streptococcus parasuis has recently been removed taxonomically from Streptococcus suis, a zoonotic pathogen. S. parasuis has been detected in healthy pigs and in diseased pigs, which suggests that S. parasuis is involved in the normal microbiota of pigs and has potential pathogenicity. However, the pathogenicity of S. parasuis in pigs is unclear because of the lack of appropriate detection methods that discriminate S. parasuis from S. suis. In this study, we developed a PCR method that is specific for S. parasuis. The detection limit of the PCR was 350 CFU per reaction. Bacteria isolated from the saliva of eight pigs were collected and examined by PCR. Sixty-four isolates positive for PCR were obtained from the samples of all pigs. Thirteen of the 64 isolates were genetically confirmed as S. parasuis, and biologically and biochemically had nearly the same features of known S. parasuis strains, which suggested that strains positive for PCR were S. parasuis. Among the 64 isolates, 28 isolates were serotypes 20, 22, or 26 in the S. suis serotyping scheme. The remaining 36 isolates were untypeable, which suggested the presence of novel serotypes or a capsule-negative form. Therefore, the PCR method described in this study is a useful tool for identifying S. parasuis, and can be used in etiological studies on this bacterium.

Defining the taxonomic status of Streptococcus suis serotype 33: the proposal for Streptococcus ruminantium sp. nov.

To clarify the taxonomic classification of Streptococcus suis serotype 33, we performed biochemical and molecular genetic analyses using isolates (GUT-183, GUT-184, GUT-185, GUT-186, GUT-187T, GUT-188, GUT-189, GUT-190, GUT-191, GUT-192 and GUT-193) from bovine endocarditis. A comparative sequence analysis showed 99.2-100 % sequence similarity among the reference strain of S. suis serotype 33 and our isolates for the 16S rRNA gene. These similarities were higher than those between the isolate GUT-187T and S. suis and other streptococci. Comparison of sodA genes also showed high degrees of similarities among the reference strain of S. suis serotype 33 and our isolates (99.7-100 %), which were higher than those between the GUT-187T and S. suis and other streptococci. DNA-DNA relatedness among three isolates (GUT-186, GUT-187T, the reference strain of S. suis serotype 33) was over 76.7 %. In contrast, the relatedness between GUT-187T and the other streptococcal species (S. suis, Streptococcus parasuis, Streptococcus acidominimus and Streptococcus porci) was 8.4-24.9 %. Phylogenetic analyses showed that the isolates did not affiliate closely to any known species of the genus Streptococcus. Moreover, GUT-187T could be distinguished from S. suis and other closely related species of genus Streptococcus using biochemical tests. On the basis of the phenotypic and molecular genetic data, we propose that the isolates of S. suis serotype 33 should be classified into the genus Streptococcus, Streptococcus ruminantium sp. nov. with the type strain GUT-187T (=DSM 104980T=JCM 31869T).