Corynebacterium ulcerans 0102 carries the gene encoding diphtheria toxin on a prophage different from the C. diphtheriae NCTC 13129 prophage

BACKGROUND: Corynebacterium ulcerans can cause a diphtheria-like illness, especially when the bacterium is lysogenized with a tox gene-carrying bacteriophage that produces diphtheria toxin. Acquisition of toxigenicity upon phage lysogenization is a common feature of C. ulcerans and C. diphtheriae. However, because of a lack of C. ulcerans genome information, a detailed comparison of prophages has not been possible between these two clinically important and closely related bacterial species. RESULTS: We determined the whole genome sequence of the toxigenic C. ulcerans 0102 isolated in Japan. The genomic sequence showed a striking similarity with that of Corynebacterium pseudotuberculosis and, to a lesser extent, with that of C. diphtheriae. The 0102 genome contained three distinct prophages. One of these, ΦCULC0102-I, was a tox-positive prophage containing genes in the same structural order as for tox-positive C. diphtheriae prophages. However, the primary structures of the individual genes involved in the phage machinery showed little homology between the two counterparts. CONCLUSION: Taken together, these results suggest that the tox-positive prophage in this strain of C. ulcerans has a distinct origin from that of C. diphtheriae NCTC 13129

Simple and rapid detection method for qepA1 by loop-mediated isothermal amplification

Although fluoroquinolone (FQ) has been used for the treatment of various bacterial infectious diseases, its continued use has been problematic given the appearance of FQ-resistant bacteria. However, the recent discovery of four plasmid-mediated quinolone resistance (PMQR) genes comprising qnr, aac(6\u27)Ib-cr, qepA and OqxAB since 1998 has provided insights in the area of FQ-resistance. For practical detection of qepA in microbiology laboratory, a specific, simple, rapid and cost-effective isothermal amplification method designated as LAMP is the good candidate to use. In this study, the development of a new detection method using LAMP to identify qepA1, one variant of the qepA gene, was tried. As the results, the LAMP method using a qepA1-specific LAMP primer set comprising five primerscould detect all four qepA1-positive strains in addition to 17 qepA1-negative strains. The LAMP method is clearly much more advantageous for use in clinical laboratories. Furthermore, the time and accuracy benefits allow for the selection of antibiotics in a clinical setting

Nationwide multicenter questionnaire surveys on countermeasures against antimicrobial resistance and infections in hospitals

[Background] The goals of the National Action Plan on Antimicrobial Resistance (AMR) of Japan include “implementing appropriate infection prevention and control” and “appropriate use of antimicrobials, ” which are relevant to healthcare facilities. Specifically, linking efforts between existing infection control teams and antimicrobial stewardship programs was suggested to be important. Previous studies reported that human resources, such as full-time equivalents of infection control practitioners, were related to improvements in antimicrobial stewardship. [Methods] We posted questionnaires to all teaching hospitals (n = 1017) regarding hospital countermeasures against AMR and infections. To evaluate changes over time, surveys were conducted twice (1st survey: Nov 2016, 2nd survey: Feb 2018). A latent transition analysis (LTA) was performed to identify latent statuses, which refer to underlying subgroups of hospitals, and effects of the number of members in infection control teams per bed on being in the better statuses. [Results] The number of valid responses was 678 (response rate, 66.7%) for the 1st survey and 559 (55.0%) for the 2nd survey. More than 99% of participating hospitals had infection control teams, with differences in activity among hospitals. Roughly 70% had their own intervention criteria for antibiotics therapies, whereas only about 60 and 50% had criteria established for the use of anti-methicillin-resistant Staphylococcus aureus antibiotics and broad-spectrum antibiotics, respectively. Only 50 and 40% of hospitals conducted surveillance of catheter-associated urinary tract infections and ventilator-associated pneumonia, respectively. Less than 50% of hospitals used maximal barrier precautions for central line catheter insertion. The LTA identified five latent statuses. The membership probability of the most favorable status in the 2nd study period was slightly increased from the 1st study period (23.6 to 25.3%). However, the increase in the least favorable status was higher (26.3 to 31.8%). Results of the LTA did not support a relationship between increasing the number of infection control practitioners per bed, which is reportedly related to improvements in antimicrobial stewardship, and being in more favorable latent statuses. [Conclusions] Our results suggest the need for more comprehensive antimicrobial stewardship programs and increased surveillance activities for healthcare-associated infections to improve antimicrobial stewardship and infection control in hospitals

Molecular epidemiology of Bordetella pertussis in the Philippines in 2012–2014

SummaryObjectivesThe present study was designed to determine the genotypes of circulating Bordetella pertussis in the Philippines by direct molecular typing of clinical specimens.MethodsNasopharyngeal swabs (NPSs) were collected from 50 children hospitalized with pertussis in three hospitals during 2012–2014. Multilocus variable-number tandem repeat analysis (MLVA) was performed on the DNA extracts from NPSs. B. pertussis virulence-associated allelic genes (ptxA, prn, and fim3) and the pertussis toxin promoter, ptxP, were also investigated by DNA sequence-based typing.ResultsTwenty-six DNA extracts yielded a complete MLVA profile, which were sorted into 10 MLVA types. MLVA type 34 (MT34), which is rare in Australia, Europe, Japan, and the USA, was the predominant strain (50%). Seven MTs (MT29, MT32, MT33, and MT283–286, total 42%) were single-locus variants of MT34, while two (MT141 and MT287, total 8%) were double-locus variants of MT34. All MTs had the combination of virulence-associated allelic genes, ptxP1–ptxA1–prn1–fim3A.ConclusionsThe B. pertussis population in the Philippines comprises genetically related strains. These strains are markedly different from those found in patients from other countries where acellular pertussis vaccines are used. The differences in vaccine types between these other countries and the Philippines, where the whole-cell vaccine is still used, may select for distinct populations of B. pertussis

Transmission of Bordetella holmesii during Pertussis Outbreak, Japan

We describe the epidemiology of a pertussis outbreak in Japan in 2010–2011 and Bordetella holmesii transmission. Six patients were infected; 4 patients were students and a teacher at the same junior high school. Epidemiologic links were found between 5 patients. B. holmesii may have been transmitted from person to person

16S rRNA Methylase–producing, Gram-Negative Pathogens, Japan

To investigate the exact isolation frequency of 16S rRNA methylase–producing, gram-negative pathogenic bacteria, we tested 87,626 clinical isolates from 169 hospitals. Twenty-six strains from 16 hospitals harbored 16S rRNA methylase genes, which suggests sparse but diffuse spread of pan-aminoglycoside–resistant microbes in Japan

Genotyping of Mycoplasma pneumoniae strains isolated in Japan during 2019 and 2020: spread of p1 gene type 2c and 2j variant strains

We characterized 118 Mycoplasma pneumoniae strains isolated from three areas of Japan (Saitama, Kanagawa, and Osaka) during the period of 2019 and 2020. Genotyping of the p1 gene in these strains revealed that 29 of them were type 1 lineage (29/118, 24.6%), while 89 were type 2 lineage (89/118, 75.4%), thereby indicating that type 2 lineage was dominant in this period. The most prevalent variant of type 2 lineage was type 2c (57/89, 64%), while the second-most was type 2j, a novel variant identified in this study (30/89, 33.7%). Type 2j p1 is similar to type 2 g p1, but cannot be distinguished from reference type 2 (classical type 2) using the standard polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) with HaeIII digestion. Thus, we used MboI digestion in the PCR-RFLP analysis and re-examined the data from previous genotyping studies as well. This revealed that most strains reported as classical type 2 after 2010 in our studies were actually type 2j. The revised genotyping data showed that the type 2c and 2j strains have been spreading in recent years and were the most prevalent variants in Japan during the time-period of 2019 and 2020. We also analyzed the macrolide-resistance (MR) mutations in the 118 strains. MR mutations in the 23S rRNA gene were detected in 29 of these strains (29/118, 24.6%). The MR rate of type 1 lineage (14/29, 48.3%) was still higher than that of type 2 lineage (15/89, 16.9%); however, the MR rate of type 1 lineage was lower than that found in previous reports published in the 2010s, while that of type 2 lineage strains was slightly higher. Thus, there is a need for continuous surveillance of the p1 genotype and MR rate of M. pneumoniae clinical strains, to better understand the epidemiology and variant evolution of this pathogen, although M. pneumoniae pneumonia cases have decreased significantly since the COVID-19 pandemic

Prevalence and Genetic Characterization of Pertactin-Deficient Bordetella pertussis in Japan

The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn−) B. pertussis was observed in Japan. The Prn− isolate was first discovered in 1997, and 33 (27%) Prn− isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn− isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn− isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn− isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn− isolates belong to MLVA-186, and 6 and 3 Prn− isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn− clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn− isolates have a higher growth potential than the Prn+ back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn− strains have arisen and that Prn expression is not essential for fitness under these conditions

Expansion of KPC-producing Enterobacterales in four large hospitals in Hanoi, Vietnam

Objectives : The incidence of carbapenem resistance among nosocomial Gram-negative bacteria in Vietnam is high and increasing, including among Enterobacterales. In this study, we assessed the presence of one of the main carbapenemase genes, blaKPC, among carbapenem-resistant Enterobacterales (CRE) from four large hospitals in Hanoi, Vietnam, between 2010 and 2015, and described their key molecular characteristics. Methods : KPC-producing Enterobacterales were detected using conventional PCR and were further analysed using S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting and whole-genome sequencing (WGS) for sequence typing and genetic characterisation. Results : blaKPC genes were detected in 122 (20.4%) of 599 CRE isolates. blaKPC-carrying plasmids were diverse in size. Klebsiella pneumoniae harbouring blaKPC genes belonged to ST15 and ST11, whereas KPC-producing Escherichia coli showed more diverse sequence types including ST3580, ST448, ST709 and ST405. Genotypic relationships supported the hypothesis of circulation of a population of ‘resident’ resistant bacteria in one hospital through the years and of transmission among these hospitals via patient transfer. WGS results revealed co-carriage of several other antimicrobial resistance genes and three different genetic contexts of blaKPC-2. Among these, the combination of ISEcp1–blaCTX-M and ISKpn27–blaKPC–ΔISKpn6 on the same plasmid is reported for the first time. Conclusion : We describe the dissemination of blaKPC-expressing Enterobacterales in four large hospitals in Hanoi, Vietnam, since 2010, which may have started earlier, along with their resistance patterns, sequence types, genotypic relationship, plasmid sizes and genetic context, thereby contributing to the overall picture of the antimicrobial resistance situation in Enterobacterales in Vietnam