Complete Genome Sequence of a Macrolide-Resistant Bordetella pertussis Isolated in Japan.

We report the complete genome sequence of macrolide-resistant Bordetella pertussis BP616, which was first isolated in 2018 in Japan. The BP616 genome can serve as a valuable specific reference for genomic and epidemiological studies of this resistant bacterium.

Limitations of Ribotyping as Genotyping Method for Corynebacterium ulcerans.

We conducted molecular typing of a Corynebacterium ulcerans isolate from a woman who died in Japan in 2016. Genomic DNA modification might have affected the isolate's ribotyping profile. Multilocus sequence typing results (sequence type 337) were more accurate. Whole-genome sequencing had greater ability to discriminate lineages at high resolution.

Periodic Genotype Shifts in Clinically Prevalent Mycoplasma pneumoniae Strains in Japan.

Nationwide increases in Mycoplasma pneumoniae pneumonia cases in Japan were reported in 2011, 2012, 2015, and 2016. In this study, we isolated 554 M. pneumoniae strains in 4 areas in Japan (Kanagawa, Okayama, Osaka, and Saitama) between 2006 and 2019, and performed genotyping analysis. More than 80% of the strains isolated in 2011 and 2012 harbored type 1 p1 adhesin gene; however, strains harboring type 2 or its variant p1 gene increased in 2015 and 2016 and dominated after 2017. These findings suggested that a shift in the prevalent genotype of M. pneumoniae clinical strains occurred recently in Japan. More than 90% of the type 1 strains isolated after 2010 harbored macrolide-resistance mutations in their 23S rRNA gene, whereas most type 2 lineage strains had no such mutations. Consequently, the increase in type 2 lineage strains in Japan has reduced the macrolide resistance rate of clinical M. pneumoniae strains. During this analysis, we also identified M. pneumoniae strains carrying a novel variant type 1 p1 gene, and we classified it as type 1b. We then sequenced the genomes of 81 selected M. pneumoniae strains that we collected between 1976 and 2017 in Japan, and compared them with 156 M. pneumoniae genomes deposited in public databases to provide insights into the interpretation of M. pneumoniae genotyping methods, including p1 typing, multiple-locus variable-number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST), and typing by 8 single-nucleotide polymorphism markers (SNP-8). As expected, p1 typing, MLST, and SNP-8 results exhibited good correlation with whole-genome SNP analysis results in terms of phylogenetic relationships; however, MLVA typing results were less comparable to those of the other methods. MLVA may be useful for the discrimination of strains derived from a single outbreak within a limited area; however, is not reliable for classification of strains collected from distantly separated areas at different time points. This study showed the usefulness of genome-based comparison of M. pneumoniae for molecular epidemiology. Genome sequencing of more strains will improve our understanding of global propagation routes of this pathogen and evolutionary aspects of M. pneumoniae strains.

The First Report of Macrolide-Resistant Bordetella pertussis Isolation in Japan.

We report the first detection of a macrolide-resistant Bordetella pertussis strain in Japan. The isolate was highly resistant to the macrolides (minimum inhibitory concentrations for erythromycin and clarithromycin: > 256 µg/ml, for azithromycin: 32 µg/ml) and A2047G mutation was identified in the 23S rRNA. The Multilocus Sequence Typing and Multilocus Variable Number of Tandem Repeat Analysis genotypes of this isolate were MT195 and ptxP1/ptxA1/prn1/fim3A/fhaB3, respectively, suggesting a relationship with the macrolide-resistant B. pertussis lineage currently found in China. This raises the possibility that macrolide-resistant B. pertussis has already fully spread in Japan. For a better control of B. pertussis infections, the surveillance for macrolide-resistant B. pertussis is essential in not only Japan, but also other Asian countries.

Genetic characterization of Mycoplasma pneumoniae isolated in Osaka between 2011 and 2017: Decreased detection rate of macrolide-resistance and increase of p1 gene type 2 lineage strains.

We characterized 419 Mycoplasma pneumoniae isolates collected between 2011 and 2017 in Osaka prefecture of Japan. This analysis revealed high prevalence of macrolide-resistant M. pneumoniae (MRMP) in Osaka during 2011 and 2014 with annual detection rates of MRMP strains between 71.4% and 81.8%. However, in 2015 and after, the detection rate of MRMP decreased significantly and did not exceed 50%. Genotyping of the p1 gene of these isolates showed that most of MRMP strains harbored type 1 p1 gene. In contrast, strains expressing p1 gene type 2 or its variant were largely macrolide-susceptible M. pneumoniae (MSMP) strains. There was a strong correlation between p1 gene genotype and the presence of mutations conferring macrolide resistance in M. pneumoniae isolated in Osaka. These results indicate that lower incidence of MRMP strains in Osaka from 2015 was associated with the relative increase of p1 gene type 2 lineage strains. During these experiments, we also isolated three M. pneumoniae strains that showed irregular typing pattern in the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the p1 gene. Two of these strains harbored new variants of type 2 p1 gene and were designated as type 2f and 2g. The remaining strain with an irregular typing pattern had a large deletion in the p1 operon.

Foodborne Outbreak of Group G Streptococcal Pharyngitis in a School Dormitory in Osaka, Japan.

In September 2016, 140 patients with primary symptoms of sore throat and fever were identified in a school dormitory in Osaka, Japan. Epidemiological and laboratory investigations determined that these symptomatic conditions were from a foodborne outbreak of group G streptococcus (GGS), with GGS being isolated from samples from patients, cooks, and foods. The strain of GGS was identified as Streptococcus dysgalactiae subsp. equisimilis of two emm types (stG652.0 and stC36.0). The causative food, a broccoli salad, was contaminated with the two types of S. dysgalactiae subsp. equisimilis, totaling 1.3 × 104 CFU/g. Pulsed-field gel electrophoresis (PFGE) of samples from patients, cooks, and foods produced similar band patterns among samples with the same emm type. This result suggested the possibility of exposure from the contaminated food. The average onset time was 44.9 h and the prevalence rate was 62%. This is the first report to identify the causative food of a foodborne outbreak by Streptococcus dysgalactiae subsp. equisimilis.

Toxigenic Corynebacterium ulcerans isolated from a wild bird (ural owl) and its feed (shrew-moles): comparison of molecular types with human isolates.

BACKGROUND: Corynebacterium ulcerans is a pathogen causing diphtheria-like illness to humans. In contrast to diphtheria by Corynebacterium diphtheriae circulating mostly among humans, C. ulcerans infection is zoonotic. The present study aimed to clarify how a zoonotic pathogen C. ulcerans circulates among wild birds and animals. RESULTS: By screening 380 birds, a single strain of toxigenic C. ulcerans was isolated from a carnivorous bird, ural owl (Strix uralensis). The bacterium was also isolated from two individuals of Japanese shrew-mole (Urotrichus talpoides), a food preference of the owl. Analysis by ribotyping showed that the owl and mole isolates were classified in a group, suggesting that C. ulcerans can be transmissible among wild birds and their prey animals. Moreover, our isolates were found to belong to a group of previously reported C. ulcerans isolates from dogs and a cat, which are known to serve as sources for human infection. CONCLUSION: The findings suggest that the shrew-mole may be a potential reservoir of a zoonotic pathogen C. ulcerans.

Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

Evaluation of streptococcal toxic shock-like syndrome caused by group B streptococcus in adults in Japan between 2009 and 2013.

Infection with Streptococcus agalactiae has long been recognized in infants. In recent years, S. agalactiae is an important cause of morbidity and mortality among adults and among those with underlying medical condition. Several cases of GBS infection and more fulminant disease similar to streptococcal toxic shock syndrome have recently been reported. We report here that 19 S. agalactiae strains were isolated from streptococcal toxic shock-like syndrome cases involving adult patients in Japan between 2009 and 2013. The average age of the patients was 66.3 years. At least one underlying disease was present in 47.4% (9/19) of the patients. The most prevalent serotype among these strains was Ib. All serotype Ib strains belonged to clonal complex 10 and were ciprofloxacin resistant. In contrast, all strains were susceptible to penicillin G, ampicillin, cefazolin, cefotaxime, imipenem, panipenem, and linezolid. The characteristic type distributions of streptococcal toxic shock-like syndrome isolates differed between isolates obtained from vaginal swabs of women and infants with invasive infections.

Isolation and characterization of toxigenic Corynebacterium ulcerans from 2 closed colonies of cynomolgus macaques (Macaca fascicularis) in Japan.

The aim of the present study was to determine the prevalence of infection by toxigenic Corynebacterium ulcerans in cynomolgus macaques (Macaca fascicularis) housed in an animal facility in Japan. Samples from the pharynges of animals from 2 closed colonies (colony A, n = 47; colony B, n = 21) were cultured. C. ulcerans grew from 43% and 47% of the samples from colonies A and B, respectively. The toxigenicity of these isolates was assessed by using PCR analysis for the diphtheria toxin gene and the Elek test and Vero cytotoxicity assay to detect diphtheria toxin. The proportion of macaques harboring toxigenic C. ulcerans was 6% in colony A and 29% in colony B. Analysis of diphtheria antitoxin neutralization titers in the sera revealed that 23% and 33% of macaques from colonies A and B, respectively, had a history of infection with toxigenic C. ulcerans. Pulsed-field gel electrophoresis of the toxigenic isolates showed that all of those recovered from macaques in colony B showed an identical genotype, suggesting that transmission of the organism occurred within the colony. However, isolates from colony A macaques showed 3 different genotypes, one of which was identical to the isolate from colony B. Additional studies evaluating the prevalence and transmission of toxigenic C. ulcerans within colonies of nonhuman primates are necessary to help control the spread of the infection. The current study is the first description of the isolation and characterization of toxigenic C. ulcerans from nonhuman primates in Japan.

Bronchitis caused by Bordetella holmesii in a child with asthma misdiagnosed as mycoplasmal infection.

We report a case of a bronchitis caused by Bordetella holmesii in a 2-year-old girl with asthma. The patient had a moderate fever and productive cough, and her condition was initially diagnosed as mycoplasmal bronchitis on the basis of her clinical symptoms and rapid serodiagnosis of mycoplasmal infection. She was treated with a bronchodilator and clarithromycin, which resulted in complete recovery. However, after the initial diagnosis, nucleic acid amplification tests of her sputum showed the absence of both Mycoplasma pneumoniae and Bordetella pertussis infections. Sputum culture showed the presence of a slow-growing, gram-negative bacillus in pure culture on Bordetella agar plates; the bacillus was later identified as B. holmesii. B. holmesii infection is rare in immunocompetent children; however, the organism is a true pathogen that can cause bronchitis in young children with asthma.

Prevalence of Corynebacterium ulcerans in dogs in Osaka, Japan.

Diphtheria-like human illness caused by Corynebacterium ulcerans is an emerging threat in developed countries, with incidence sometimes higher than that of diphtheria caused by Corynebacterium diphtheriae. Companion animals are considered a potential source of human infections. In order to determine the prevalence of C. ulcerans among dogs, we performed a screening for the bacterium in 583 dogs in the custody of the Osaka Prefectural government. Forty-four dogs (7.5 %) were positive for the bacterium, although they did not show any clinical symptoms. All bacterial isolates showed resistance or decreased sensitivity to clindamycin, and some showed decreased sensitivity to levofloxacin. Comparative analysis of isolates using PFGE, toxin gene typing and antibiotic sensitivities suggests that transmission between asymptomatic dogs might have occurred.

Surveillance of severe invasive group G streptococcal infections in Japan during 2002-2008.

Group G Streptococcus strains isolated from patients with severe invasive infections in the period 2002-2008 were surveyed and their prevalence compared with that observed in the period 1995-2001 in Japan. Strains with genotypes stg485, stg6792, stc36, stg6, and stg652 were isolated in both periods, whereas various new genotypes appeared in 2002-2008 and some genotypes found in 1995-2001 were not found subsequently, thus indicating a change in the prevalent genotyped strains causing severe invasive streptococcal infections.

Genetic features of clinical isolates of Streptococcus dysgalactiae subsp. equisimilis possessing Lancefield's group A antigen.

Thirteen Streptococcus dysgalactiae subsp. equisimilis isolates possessing Lancefield's group A antigen recovered from people in Japan during 2000 to 2004 were genotyped. The results indicate that a conserved clone has persisted and spread within Japan, and two different emm types were observed within members of this clone.

[Evaluation of rapid diagnostic kits for the detection of group A streptococcus to Streptococcus pyogenes and Streptococcus spp. with Lancefield's group A antigen].

We studied the basic performance of eight rapid diagnostic kits for the detection of Group A streptococcus by immunochromatography under the same conditions. Kits were the; QuickVue Dipstick Strep A (Sumitomo Seiyaku Biomedical Co., Ltd.), TESTPACK Plus STREP A (ABBOTT JAPAN Co., Ltd), CLEAVIEW STREP A (Nihon Schering K. K.), QuickVue STREP A (Wako Pure Chemical Industries, Ltd), ImmunoCard STAT! STREP A (TFB, INC.), DIPSTICK 'Eiken' STREP A (Eiken Chemical Co., Ltd.), Rapid Testa Strep A (Daiichi Pure Chemical Co., Ltd.), and StatCheck Strep A (KAINOS Laboratories, Inc.). Four of these kits, i.e. QuickVue Dipstick Strep A, TESTPACK Plus STREP A, Rapid Testa Strep A, and StatCheck Strep A showed sensitivity at 1.0 x 10(5) CFU/mL (1.0 x 10(4)CFU/test) with all of S. pyogenes tested, while the Anginosus group and S. dysgalactiae subsp. equisimilis with Lancefield' s group A antigen showed sensitivity very similar to S. pyogenes. Of these strains, S. dysgalactiae subsp. equisimilis formed a beta-hemolytic colony resembling that of S. pyogenes on sheep blood agar, and was sensitive to bacitracin. It is thus indispensable to identify the colony using biochemical tests such as the PYR (pyrrolidonylarylamidase production) test. In using rapid diagnostic kits for the detection of Group A streptococcus, it is important to rule out the possibility of Group A streptococcus other than S. pyogenes in throats. Severe invasive group-G streptococcal infections are increasing recently. Concerning S. dysgalactiae subsp. equisimilis, it is especially important to conduct these identification tests.

[A case of Mycobacterium goodii infection wifh isolation from blood and a pacemaker lead].

We report a case of blood stream infection due to Mycobacterium goodii in a patient who had an implanting pacemaker. The patient injured left thorax where the pacemaker was implanted several days before septicemia. The microorganism was isolated from both blood cultures and leads of the pacemaker. The serial isolates were identified as M. goodii by conventional biochemical methods, tobramycin susceptibility test and 16Sr-RNA sequencing. This is the first reported case of M. goodii septicemia in Japan. M. goodii is regarded as an environmental bacterium and its pathogenicity has been recognized recently. The present case suggests that its ability as a primary invader should not be underestimated, especially in a patient with indwelling devices.