Detection of Respiratory Viruses in SARS-CoV-2-Negative Specimens from January to March 2020 in Fukuoka, Japan.

Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first confirmed in Japan on January 15, 2020. The Fukuoka Institute of Health and Environmental Sciences conducted testing using polymerase chain reaction (PCR) for SARS-CoV-2 from January 31 to March 4, 2020. Samples (n = 119) were collected from 81 patients suspected of having SARS-CoV-2 infection, presenting with fever, cough, fatigue, pneumonia, and other symptoms; all the samples tested during that period were negative. To identify the pathogens responsible for these symptoms, we conducted multiplex PCR. Respiratory viruses, human metapneumovirus (hMPV) was detected in 10 patients (12%), human rhinovirus (HRV) in 3 patients (4%), and influenza B virus in 1 patient (1%). In addition, the patients who had the viruses were significantly older than those who did not. Infections with hMPV and HRV have been associated with a risk of severe illness and death among older adults. Therefore, differentiating SARS-CoV-2 from other respiratory viruses, such as hMPV and HRV, is necessary to prevent and control the spread of infection, especially in older adults.

Molecular epidemiology of coxsackievirus A6 derived from hand, foot, and mouth disease in Fukuoka between 2013 and 2017.

Coxsackievirus (CV)-A6 has been the primary causative agent of hand, foot, and mouth disease (HFMD) in Japan since 2011. In Fukuoka, CV-A6-associated HFMD caused epidemics in 2013, 2015, and 2017. This paper reports the genetic characteristics of the CV-A6 entire viral protein 1 (VP1) derived from patients with HFMD in Fukuoka between 2013 and 2017. CV-A6 was detected in 105 of 280 clinical specimens, and the entire VP1 sequences could be analyzed for 90 of the 105 specimens. Phylogenetic analysis revealed that the CV-A6 strains were classified into clade A and subgrouped into subclade A3 or subclade A4. Each subclade strain carried amino acid substitutions in the presumed DE and GH loops of the VP1, and no amino acid substitutions were identified as deleterious to the protein function. No significant difference was found in the clinical symptoms between the genetic subclades using statistical analyses. In conclusion, this study clarified the genetic diversity of CV-A6 in Fukuoka from 2013 to 2017. The emergence of the CV-A6 strains was classified into derived new subclades based on phylogenetic analysis of the VP1 gene that may cause CV-A6-associated HFMD epidemics approximately every 2 years.

Shiga toxin 2f-producing Escherichia albertii from a symptomatic human.

The previously identified Shiga toxin (Stx) 2f-producing Escherichia coli O115:HNM strain F08/101-31, isolated from a symptomatic human, was confirmed to be E. albertii in the present study by whole genome DNA-DNA hybridizations, by sequencing (cpn60, dnaJ, and 16S rRNA genes), and by multi-locus sequence typing. The F08/101-31 strain was originally identified as E. coli rather than the relatively new bacterial species E. albertii, which was first described in 2003, because it did not display any of the biochemical characteristics of E. albertii. This new classification will impact public health management strategies in Japan because the present study showed that some E. albertii strains, which are often misidentified as E. coli, produce Stx and likely cause diarrhea in humans. Therefore, further guidelines for the management and identification of Stx-producing E. albertii are required in Japan.

Evolutionary model of the divergence of enterohemorrhagic Escherichia coli O157 lineage I/II clades reconstructed from high resolution melting and Shiga-like toxin 2 analyses.

We have studied 167 epidemiologically unlinked strains of enterohemorrhagic Escherichia coli O157 (O157) isolated from patients with hemorrhagic colitis (HC), 87 strains from patients not with HC and 35 asymptomatic carriers (the not HC group), and differentiated these strains into clades using high resolution melting analysis. In addition, lineage analysis was carried out using lineage-specific polymorphism assay-6 and analysis of the distribution of IS629 insertion sites was carried out using IS-printing. Most strains were correctly clustered by minimum spanning tree analysis, and strains in the major clades showed linkage disequilibrium, confirming the clade differentiation in this study. The number of O157 strains in the different clades isolated from HC patients and the not HC group was significantly different (Chi square test, P<0.05), indicating that strains in different clades had different pathogenicities for hemorrhagic colitis. Pairwise comparison of the number of strains in different clades isolated from HC patients indicated that clade 12 strains were weakly pathogenic for HC. Stx2 titers and the number of strains carrying an stx2 gene were significantly different for different clades (Kruscal-Wallis test and Chi square test, P<0.05). Pairwise comparison of the Stx2 titer and the number of strains with an stx2 gene in different clades showed that the weak HC pathogenicity of clade 12 strains would be related to the low number of clade 12 strains with an stx2 gene and the low Stx2 production in those strains. Interestingly, the Stx2 titer and the prevalence of strains with an stx2 gene were significantly higher among clade 6 and 8 strains compared to clade 7 strains, although clades 6, 7, and 8 were all in lineage I/II. These results were discordant with the O157 evolutionary model, suggesting that insertion of an stx2 gene in lineage I/II strains after divergence of each clade.

Isolation and characteristics of Shiga toxin 2f-producing Escherichia coli among pigeons in Kyushu, Japan.

An increasing number of Shiga toxin 2f-producing Escherichia coli (STEC2f) infections in humans are being reported in Europe, and pigeons have been suggested as a reservoir for the pathogen. In Japan, there is very little information regarding carriage of STEC2f by pigeons, prompting the need for further investigation. We collected 549 samples of pigeon droppings from 14 locations in Kyushu, Japan, to isolate STEC2f and to investigate characteristics of the isolates. Shiga toxin stx 2f gene fragments were detected by PCR in 16 (2.9%) of the 549 dropping samples across four of the 14 locations. We obtained 23 STEC2f-isolates from seven of the original samples and from three pigeon dropping samples collected in an additional sampling experiment (from a total of seven locations across both sampling periods). Genotypic and phenotypic characteristics were then examined for selected isolates from each of 10 samples with pulsed-field gel electrophoresis profiles. Eight of the stx 2f gene fragments sequenced in this study were homologous to others that were identified in Europe. Some isolates also contained virulence-related genes, including lpfA O26, irp 2, and fyuA, and all of the 10 selected isolates maintained the eae, astA, and cdt genes. Moreover, five of the 10 selected isolates contained sfpA, a gene that is restricted to Shiga toxin-producing E. coli O165:H2 and sorbitol-fermenting Shiga toxin-producing E. coli O157:NM. We document serotypes O152:HNM, O128:HNM, and O145:H34 as STEC2f, which agrees with previous studies on pigeons and humans. Interestingly, O119:H21 was newly described as STEC2f. O145:H34, with sequence type 722, was described in a German study in humans and was also isolated in the current study. These results revealed that Japanese zoonotic STEC2f strains harboring several virulence-related factors may be of the same clonal complexes as some European strains. These findings provide useful information for public health-related disease management strategies in Japan.

Phylogenetic Clades 6 and 8 of Enterohemorrhagic Escherichia coli O157:H7 With Particular stx Subtypes are More Frequently Found in Isolates From Hemolytic Uremic Syndrome Patients Than From Asymptomatic Carriers.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection causes severe diseases such as bloody diarrhea and hemolytic uremic syndrome (HUS). Although EHEC O157:H7 strains have exhibited high genetic variability, their abilities to cause human diseases have not been fully examined. METHODS: Clade typing and stx subtyping of EHEC O157:H7 strains, which were isolated in Japan during 1999-2011 from 269 HUS patients and 387 asymptomatic carriers (ACs) and showed distinct pulsed-field gel electrophoresis patterns, were performed to determine relationships between specific lineages and clinical presentation. RESULTS: Clades 6 and 8 strains were more frequently found among the isolates from HUS cases than those from ACs (P = .00062 for clade 6, P < .0001 for clade 8). All clade 6 strains isolated from HUS patients harbored stx2a and/or stx2c, whereas all clade 8 strains harbored either stx2a or stx2a/stx2c. However, clade 7 strains were predominantly found among the AC isolates but less frequently found among the HUS isolates, suggesting a significant association between clade 7 and AC (P < .0001). Logistic regression analysis revealed that 0-9 year old age is a significant predictor of the association between clade 8 and HUS. We also found an intact norV gene, which encodes for a nitric oxide reductase that inhibits Shiga toxin activity under anaerobic condition, in all clades 1-3 isolates but not in clades 4-8 isolates. CONCLUSIONS: Early detection of EHEC O157:H7 strains that belonged to clades 6/8 and harbored specific stx subtypes may be important for defining the risk of disease progression in EHEC-infected 0- to 9-year-old children.

Clinical significance of Escherichia albertii.

Discriminating Escherichia albertii from other Enterobacteriaceae is difficult. Systematic analyses showed that E. albertii represents a substantial portion of strains currently identified as eae-positive Escherichia coli and includes Shiga toxin 2f-producing strains. Because E. albertii possesses the eae gene, many strains might have been misidentified as enterohemorrhagic or enteropathogenic E. coli.

Emergence of enterohemorrhagic Escherichia coli serovar O157 strains in clade 8 with highly similar pulsed-field gel electrophoresis patterns.

Enterohemorrhagic Escherichia coli serovar O157 (O157) strains with highly similar pulsed-field gel electrophoresis (PFGE) patterns were isolated in Japan during 2007 and 2008. Several genetic features related to O157 evolution were investigated to indicate whether homoplasy might have contributed to the highly similar PFGE patterns in these strains. The O157 strains were classified in lineage I/II, as defined by a lineage-specific polymorphism assay-6 with an atypical allele in Z5935 (code: 231111). Analysis of the insertion sites of stx(2) phage in these strains showed that the sites were "occupied" in yehV and "intact" in wrbA, indicating that the strains were derived from "Cluster 1" of "Subgroup C." When a specific single-nucleotide polymorphism in ECs2357 in clade 8 strains was investigated, all of the strains in the present study were confirmed to be clade 8 strains. These results indicated that the O157 strains in this study had common genetic features, suggesting that the highly similar PFGE patterns of these strains were not due to homoplasy. Because no common source of these strains could be identified in 2007 to 2008 in Japan, these strains may have emerged from a unique O157 clade 8 clone and then spread by dissemination in Japan.

Evaluation of intestinal microbiotas of healthy Japanese adults and effect of antibiotics using the 16S ribosomal RNA gene based clone library method.

Intestinal microbiotas of human subjects and effect of antibiotic treatment on them have been reported with cultivation independent methods. However, Japanese fecal microbiotas have not been studied enough. We have constructed a clone library method to obtain results within 3 d. In this study, intestinal microbiotas of 29 healthy Japanese adults, whose fecal samples were collected twice at 5 month intervals from each subject, were analyzed with our clone library method, and using those data as a benchmark effect of antibiotic treatment on intestinal microbiotas was evaluated. The fifty-eight fecal microbiotas were assessed based on percentages at genus level, and the variability was analyzed with a principal component analysis (PCA). PCA showed that the microbiotas divided into three groups depending on the large eigenvectors (genera Ruminococcus, Bacteroides, and Prevotella), and the dual samples from the twenty-two individuals have belonged to the same PCA group. It suggests that almost Japanese adults have own stable intestinal microbiota. The genera Ruminococcus and Bacteroides were present in almost subjects, while the genus Prevotella was found only in nine subjects (approximately 30%) which was preserved with 5 months intervals. Next, the microbiotas before and after antibiotic treatment were evaluated comparing with the 58 healthy adult microbiotas. The results showed that beta-lactams influenced profoundly on intestinal microbiotas and the effect of macrolides depended on the cases. It suggests that our clone library method could show overview of intestinal microbiota and would give us useful information about the effect of antibiotic treatment for daily clinical diagnosis.

Biased distribution of IS629 among strains in different lineages of enterohemorrhagic Escherichia coli serovar O157.

The distribution of insertion sequence (IS) 629 among strains of enterohemorrhagic Escherichia coli serovar O157 (O157) was investigated and compared with the strain lineages defined by lineage specific polymorphism assay-6 (LSPA-6) to demonstrate the effectiveness of IS629 analysis for population genetics analysis. Using pulsed-field gel electrophoresis and variable-number tandem repeat typing, 140 strains producing both VT1 and VT2 and 98 strains producing only VT2 were selected from a total of 592 strains isolated from patients and asymptomatic carriers in Chiba Prefecture, Japan, during 2003-2008. By LSPA-6 analysis, six strains had atypical amplicon sizes in their Z5935 loci and five strains had atypical amplicon sizes in their arp-iclR intergenic regions. Sequence analyses of PCR amplified DNAs showed that five of the six loci used for LSPA-6 analysis had tandem repeats and the allele changes were due to changes in the number of tandem repeats. Subculturing and long-term incubation was found to have no detectable effect on the lineages defined by LSPA-6 analysis, demonstrating the robustness of LSPA-6 analysis. Minimum spanning tree analysis reconstruction revealed that strains in lineage I, I/II, and II clustered on separate branches, indicating that the distribution of IS629 was biased among O157 strains in different lineages. Strains with LSPA-6 codes 231111, 211113, and 211114 had atypical amplicon sizes and were clustered in lineage I/II branch, and strains with LSPA-6 codes 212114, 221123, 221223, 222123, 222224, 242123, 252123, and 242222 had atypical amplicon sizes and clustered in lineage II branches. Linkage disequilibrium was observed in strains in every lineage when the standardized index of association was calculated using IS629 distribution data. Therefore, the distribution analysis of IS629 may be effective for population genetics analysis of O157 due to the biased IS629 distribution among strains in the three O157 lineages.

Isolation of Shiga toxin 2f-producing Escherichia coli (O115:HNM) from an adult symptomatic patient in Fukuoka Prefecture, Japan.

Shiga toxin 2f-producing Escherichia coli (O115:HNM) with eae was isolated from a symptomatic patient in Fukuoka Prefecture, Japan. The patient was a 23-year-old male and his symptoms were diarrhea, abdominal pain, headaches and a fever (37.7 degrees C). He had eaten raw chicken meat, raw chicken eggs, cooked chicken meat and raw vegetables about 13 h prior to the onset of the symptoms. The patient's specimen was examined, and no diarrheagenic agents were detected except for Shiga toxin 2f-producing E. coli (STEC(2f)) with eae. This is the first report of the serotype O115:HNM possessing stx(2f). We discuss the necessity of routinely using stx(2f)-detecting PCR primers for detection of this enteric pathogen.