The importance of meropenem resistance, rather than imipenem resistance, in defining carbapenem-resistant Enterobacterales for public health surveillance: an analysis of national population-based surveillance.

BACKGROUND: In Japan, carbapenem-resistant Enterobacterales (CRE) infections were incorporated into the National Epidemiological Surveillance of Infectious Diseases (NESID) in 2014, necessitating mandatory reporting of all CRE infections cases. Subsequently, pathogen surveillance was initiated in 2017, which involved the collection and analysis of CRE isolates from reported cases to assess carbapenemase gene possession. In this surveillance, CRE is defined as (i) minimum inhibitory concentration (MIC) of meropenem ≥2 mg/L (MEPM criteria) or (ii) MIC of imipenem ≥2 mg/L and MIC of cefmetazole ≥64 mg/L (IPM criteria). This study examined whether the current definition of CRE surveillance captures cases with a clinical and public health burden. METHODS: CRE isolates from reported cases were collected from the public health laboratories of local governments, which are responsible for pathogen surveillance. Antimicrobial susceptibility tests were conducted on these isolates to assess compliance with the NESID CRE definition. The NESID data between April 2017 and March 2018 were obtained and analyzed using antimicrobial susceptibility test results. RESULTS: In total, 1681 CRE cases were identified during the study period, and pathogen surveillance data were available for 740 (44.0%) cases. Klebsiella aerogenes and Enterobacter cloacae complex were the dominant species, followed by Klebsiella pneumoniae and Escherichia coli. The rate of carbapenemase gene positivity was 26.5% (196/740), and 93.4% (183/196) of these isolates were of the IMP type. Meanwhile, 315 isolates were subjected to antimicrobial susceptibility testing. Among them, 169 (53.7%) fulfilled only the IPM criteria (IPM criteria-only group) which were susceptible to meropenem, while 146 (46.3%) fulfilled the MEPM criteria (MEPM criteria group). The IPM criteria-only group and MEPM criteria group significantly differed in terms of carbapenemase gene positivity (0% vs. 67.8%), multidrug resistance rates (1.2% vs. 65.8%), and mortality rates (1.8% vs 6.9%). CONCLUSION: The identification of CRE cases based solely on imipenem resistance has had a limited impact on clinical management. Emphasizing resistance to meropenem is crucial in defining CRE, which pose both clinical and public health burden. This emphasis will enable the efficient allocation of limited health and public health resources and preservation of newly developed antimicrobials.

Identification of a mecA/mecC-positive MRSA ST1-t127 isolate from a racehorse in Japan.

OBJECTIVES: MRSA is a known pathogen that affects horses. We investigated an equine MRSA isolate for potential antimicrobial resistance genes, classified the staphylococcal cassette chromosome mec (SCCmec) and identified the strain-specific dissemination in the horse community based on WGS. METHODS: WGS, using short-read sequencing, and subsequent long-read sequencing by hybrid assembly, was conducted to obtain a complete genome sequence. Pairwise sequence alignment of relative SCCmec sequences and core-genome phylogenetic analysis were performed to highlight transmission routes of the SCCmec and MRSA strain-specific lineages. RESULTS: In 2018, we isolated the MRSA JRA307 strain from the pus of a wound on a racehorse and the complete genome sequence suggests that it is a clinically relevant pvl-negative ST1-t127 MRSA that harbours both mecA and mecC on SCCmec-307. SCCmec-307 exhibited marked sequence identity to the previously reported SCCmec-mecC in the Staphylococcus sciuri GVGS2 strain isolated from cattle. The JRA307 mecC gene was classified as a mecC allotype of S. sciuri rather than that of Staphylococcus aureus. CONCLUSIONS: We demonstrated the complete genome sequence of equine isolate JRA307, which is a clinically relevant MRSA harbouring mecA and mecC on SCCmec-307. The finding of mecC MRSA suggests a possible SCCmec transmission between distinct staphylococcal species. To the best of our knowledge, this is the first report of mecC detection in Japan.

Clade II Candida auris possess genomic structural variations related to an ancestral strain.

Candida auris is an invasive and multidrug-resistant ascomycetous yeast that is under global surveillance. All clinical cases of C. auris infection diagnosed from 1997 to 2019 in Japan were non-invasive and sporadic otitis media cases. In the present study, we performed whole-genome sequencing of seven C. auris strains isolated from patients with otitis media in Japan, all of which belonged to clade II. Comparative genome analysis using the high-quality draft genome sequences JCM 15448T revealed that single nucleotide variations (SNVs), clade-specific accessory genes, and copy number variations (CNVs) were identified in each C. auris clade. A total of 61 genes involved in cell wall and stress response-related functions was absent in clade II, and the pattern of conserved CNVs in each clade was more stable in clade II than in other clades. Our data suggest that the genomic structural diversity is stable in C. auris isolated from each biogeographic location, and Japanese strains isolated from patients with otitis media might belong to an ancestral type of C. auris. One Japanese strain, TWCC 58362, with reduced susceptibility to fluconazole, exhibited no mutation in ergosterol biosynthesis-related genes (ERG). However, TWCC 58362-specific variations, including SNVs, indels, and CNVs were detected, suggesting that gene duplication events in C. auris might contribute to antifungal drug resistance. Taken together, we demonstrated that genomic structural variations in C. auris could correlate to geographical dissemination, epidemiology, lesions in the host, and antifungal resistance.

Aseptic meningitis caused by torque teno virus in an infant: a case report.

BACKGROUND: Torque teno virus-induced aseptic meningitis has not been documented, although torque teno virus infections still remain under consideration for etiological agents. This study identified a torque teno virus sequence using next generation sequencing and immunoglobulin M response to the torque teno virus antigen, therefore, that would be a comprehensive diagnosis for torque teno virus infection. CASE PRESENTATION: A 2-month-old Japanese boy was brought to our hospital because he was irritable, drowsy, and lethargic. He was admitted based on his test results which indicated the possibility of septic meningitis. He was started on treatment with high-dose antibiotics and steroids. On the third day of hospitalization, he became afebrile with improvement in his general status and was discharged on the sixth day. He had no developmental problems for up to 1 year after discharge. Metagenomic ribonucleic acid-Seq pathogen detection using next generation sequencing of a sample of his cerebrospinal fluid, which was collected at admission, revealed three short reads homologous to those in torque teno virus out of a total of 1,708,516 reads. This finding indicated that our patient was positive compared to the torque teno virus-negative cerebrospinal fluid samples (controls) from 13 other patients. The torque teno virus has been shown to have a whole genome sequence of 2810 nt by polymerase chain reaction. We prepared a recombinant GP2 antigen from torque teno virus and used it to study our patient's anti-torque teno virus immune response. An anti-GP2 serum immunoglobulin M response was detected, providing further supportive evidence of torque teno virus infection. CONCLUSIONS: This case speculates that torque teno virus-induced aseptic meningitis has a good course. New technologies like next generation sequencing can help in the identification of such cases, and an accumulation of future cases is expected.

Characterization of NDM-5- and CTX-M-55-coproducing Escherichia coli GSH8M-2 isolated from the effluent of a wastewater treatment plant in Tokyo Bay.

PURPOSE: New Delhi metallo-ß-lactamase (NDM)-5-producing Enterobacteriaceae have been detected in rivers, sewage, and effluents from wastewater treatment plants (WWTPs). Environmental contamination due to discharged effluents is of particular concern as NDM variants may be released into waterways, thereby posing a risk to humans. In this study, we collected effluent samples from a WWTP discharged into a canal in Tokyo Bay, Japan. METHODS: Testing included the complete genome sequencing of Escherichia coli GSH8M-2 isolated from the effluent as well as a gene network analysis. RESULTS: The complete genome sequencing of GSH8M-2 revealed that it was an NDM-5-producing E. coli strain sequence type ST542, which carries multiple antimicrobial resistance genes for ß-lactams, quinolone, tetracycline, trimethoprim-sulfamethoxazole, florfenicol/chloramphenicol, kanamycin, and fosfomycin. The bla NDM-5 gene was found in the IncX3 replicon plasmid pGSH8M-2-4. Gene network analysis using 142 IncX3 plasmid sequences suggested that pGSH8M-2-4 is related to both clinical isolates of  E. coli and Klebsiella species in Eastern Asia. GSH8M-2 also carries the bla CTX-M-55 gene in IncX1 plasmid pGSH8M-2-3. CONCLUSION: This is the first report of environmental NDM-5-producing E. coli isolated from a WWTP in Japan. NDM-5 detection is markedly increasing in veterinary and clinical settings, suggesting that dual ß-lactamases, such as NDM-5 and CTX-M-55, might be acquired through multiple steps in environment settings. Environmental contamination through WWTP effluents that contain producers of NDM variants could be an emerging potential health hazard. Thus, regular monitoring of WWTP effluents is important for the detection of antimicrobial-resistant bacteria that may be released into the waterways and nearby communities.

Potential KPC-2 carbapenemase reservoir of environmental Aeromonas hydrophila and Aeromonas caviae isolates from the effluent of an urban wastewater treatment plant in Japan.

Aeromonas hydrophila and Aeromonas caviae adapt to saline water environments and are the most predominant Aeromonas species isolated from estuaries. Here, we isolated antimicrobial-resistant (AMR) Aeromonas strains (A. hydrophila GSH8-2 and A. caviae GSH8M-1) carrying the carabapenemase blaKPC-2 gene from a wastewater treatment plant (WWTP) effluent in Tokyo Bay (Japan) and determined their complete genome sequences. GSH8-2 and GSH8M-1 were classified as newly assigned sequence types ST558 and ST13, suggesting no supportive evidence of clonal dissemination. The strains appear to have acquired blaKPC-2 -positive IncP-6-relative plasmids (pGSH8-2 and pGSH8M-1-2) that share a common backbone with plasmids in Aeromonas sp. ASNIH3 isolated from hospital wastewater in the United States, A. hydrophila WCHAH045096 isolated from sewage in China, other clinical isolates (Klebsiella, Enterobacter and Escherichia coli), and wastewater isolates (Citrobacter, Pseudomonas and other Aeromonas spp.). In addition to blaKPC-2 , pGSH8M-1-2 carries an IS26-mediated composite transposon including a macrolide resistance gene, mph(A). Although Aeromonas species are opportunistic pathogens, they could serve as potential environmental reservoir bacteria for carbapenemase and AMR genes. AMR monitoring from WWTP effluents will contribute to the detection of ongoing AMR dissemination in the environment and might provide an early warning of potential dissemination in clinical settings and communities.

Complete Genome Sequence of a blaKPC-2-Positive Klebsiella pneumoniae Strain Isolated from the Effluent of an Urban Sewage Treatment Plant in Japan.

Antimicrobial resistance genes (ARGs) and the bacteria that harbor them are widely distributed in the environment, especially in surface water, sewage treatment plant effluent, soil, and animal waste. In this study, we isolated a KPC-2-producing Klebsiella pneumoniae strain (GSU10-3) from a sampling site in Tokyo Bay, Japan, near a wastewater treatment plant (WWTP) and determined its complete genome sequence. Strain GSU10-3 is resistant to most ß-lactam antibiotics and other antimicrobial agents (quinolones and aminoglycosides). This strain is classified as sequence type 11 (ST11), and a core genome phylogenetic analysis indicated that strain GSU10-3 is closely related to KPC-2-positive Chinese clinical isolates from 2011 to 2017 and is clearly distinct from strains isolated from the European Union (EU), United States, and other Asian countries. Strain GSU10-3 harbors four plasmids, including a blaKPC-2-positive plasmid, pGSU10-3-3 (66.2 kb), which is smaller than other blaKPC-2-positive plasmids and notably carries dual replicons (IncFII [pHN7A8] and IncN). Such downsizing and the presence of dual replicons may promote its maintenance and stable replication, contributing to its broad host range with low fitness costs. A second plasmid, pGSU10-3-1 (159.0 kb), an IncA/C2 replicon, carries a class 1 integron (containing intI1, dfrA12, aadA2, qacEΔ1, and sul1) with a high degree of similarity to a broad-host-range plasmid present in the family Enterobacteriaceae The plasmid pGSU10-3-2 (134.8 kb), an IncFII(K) replicon, carries the IS26-mediated ARGs [aac(6')Ib-cr,blaOXA-1, catB4 (truncated), and aac(3)-IId], tet(A), and a copper/arsenate resistance locus. GSU10-3 is the first nonclinical KPC-2-producing environmental Enterobacteriaceae isolate from Japan for which the whole genome has been sequenced.IMPORTANCE We isolated and determined the complete genome sequence of a KPC-2-producing K. pneumoniae strain from a sampling site in Tokyo Bay, Japan, near a wastewater treatment plant (WWTP). In Japan, the KPC type has been very rarely detected, while IMP is the most predominant type of carbapenemase in clinical carbapenemase-producing Enterobacteriaceae (CPE) isolates. Although laboratory testing thus far suggested that Japan may be virtually free of KPC-producing Enterobacteriaceae, we have detected it from effluent from a WWTP. Antimicrobial resistance (AMR) monitoring of WWTP effluent may contribute to the early detection of future AMR bacterial dissemination in clinical settings and communities; indeed, it will help illuminate the whole picture in which environmental contamination through WWTP effluent plays a part.

Asymptomatic malaria, growth status, and anaemia among children in Lao People's Democratic Republic: a cross-sectional study.

BACKGROUND: Asymptomatic malaria can be observed in both stable endemic areas and unstable transmission areas. However, although much attention has been given to acute malaria infections, relatively little attention has been paid to asymptomatic malaria. Nonetheless, because the asymptomatic host serves as a reservoir for the malaria parasite, asymptomatic malaria is now recognized as an important obstacle to malaria elimination. Asymptomatic malaria is also associated with anaemia, a global public health problem with serious consequences on human health as well as social and economic development. In Lao People's Democratic Republic (Lao PDR), malaria, anaemia, and malnutrition are serious public health concerns. However, few studies have focused on the relationship between these variables. Therefore, this study investigated the relationship between asymptomatic malaria, growth status, and the prevalence of anaemia among children aged 120 months old or younger in rural villages in Lao PDR. METHODS: In December 2010 and March 2011, data were collected from five villages in Savannakhet province. Anthropometric measurements, blood samples, and malaria rapid diagnostic tests were conducted. The presence of malaria was confirmed with polymerase chain reaction assays for Plasmodium falciparum. Underweight status, stunting, and anaemia were defined according to World Health Organization standards. RESULTS: The mean age of participants (n = 319) was 88.3 months old (Standard Deviation: 20.6, ranged from 30-119 months old), and 20 participants (6.3 %) had an asymptomatic malaria infection, 92 (28.8 %) were anaemic, 123 (38.6 %) were underweight, and 137 (42.9 %) were stunted. Stunted children were more likely to be infected with asymptomatic malaria [odds ratio (OR) 3.34, 95 % confidence interval (CI) 1.25-8.93], and asymptomatic malaria was associated with anaemia [OR 5.17, 95 % CI 1.99-13.43]. CONCLUSIONS: These results suggest a significant association between asymptomatic malaria and anaemia in children. Furthermore, stunted children were more likely to have lower Hb levels and to be infected with asymptomatic malaria than children without stunting. However, further studies examining the impact of asymptomatic malaria infection on children's nutritional and development status are necessary.

Association between serum zinc concentration and the Plasmodium falciparum antibody titer among rural villagers of Attapeu Province, Lao People's Democratic Republic.

Experimental studies have indicated that low serum zinc levels affect immune responses. However, few studies have evaluated the impact of serum zinc levels on antibody responses in the field in developing countries. We investigated an association between the anti-Plasmodium falciparum (Pf) antibody (immunoglobulin G) titer and serum zinc concentration among villagers in rural areas of the Lao People's Democratic Republic. Blood samples were collected to detect Pf infection. An enzyme-linked immunosorbent assay (ELISA) was used to measure the anti-PfIgG antibody titer. Each serum sample was assayed to measure the concentration of zinc. Pearson's correlation coefficient was applied to the association between zinc concentration and anti-PfIgG antibody titers. Multiple linear regression analysis was used to assess the association between zinc concentration and anti-PfIgG antibody titers, controlling for age and albumin level. Of 71 blood samples, 40 were Pf positive and 31 were Pf negative. The median serum zinc concentrations were 56.0 µg/dl in the Pf-positive group and 62.5 µg/dl in the Pf-negative group. The median anti-Pf titers were 833.4 in the positive group and 1237.2 in the negative group. Unexpectedly, there was a negative correlation between serum zinc and anti-Pf IgG antibody titers; the correlation coefficient were -0.453 and (p=0.003) in the positive group and -0.461 (p=0.009) in the negative group. The results of this study indicated sustained antibody responses among the villagers, who had likely been exposed to malaria periodically throughout their lives. Further studies are necessary to determine the conditions in which zinc could be effective against malaria.

Protective function of an unconventional γδ T cell subset against malaria infection in apoptosis inhibitor deficient mice.

Various subsets of T-cell receptor (TCR) γδ⁺ T cells expressing distinct TCR-Vγ chains are found in many different anatomical locations. These TCR γδ⁺ T cells perform multiple functions as an essential part of the immune system. In particular, protection against malaria infection by TCR γδ⁺ T cells is of special interest. In the present study, we confirmed that the Vγ7⁺ γδ T cells, which are an unconventional subset usually localized within the intestine, are recruited to the liver and spleen at the late stage of malaria infection, and contribute to protection against malaria infection via a multifunctional approach in apoptosis inhibitor-deficient mice.