鈴木 匡弘

Klebsiella pneumoniae is a Gram-negative bacterium that causes both community- and healthcare-associated infections. Although various virulence factors and highly pathogenic phenotypes have been reported, the pathogenicity of K. pneumoniae is still not fully understood. In this study, we utilized whole-genome sequencing data of 168 clinical K. pneumoniae strains to assess pathogenicity. This work was based on the concept that the genetic composition of individual genomes (referred to as holistic gene content) of the strains may contribute to their pathogenicity. Holistic gene content analysis revealed two distinct groups of K. pneumoniae strains ('major group' and 'minor group'). The minor group included strains with known highly pathogenic clones (ST23, ST375, ST65 and ST86). The minor group had higher rates of capsular genotype K1 and presence of nine specific virulence genes (rmpA, iucA, iutA, irp2, fyuA, ybtS, iroN, allS and clbA) compared to the major group. Pathogenicity was assessed using Galleria mellonella larvae. Infection experiments revealed lower survival rates of larvae infected with strains from the minor group, indicating higher virulence. In addition, the minor group had a higher string test positivity rate than the major group. Holistic gene content analysis predicted possession of virulence genes, string test positivity and pathogenicity as observed in the G. mellonella infection model. Moreover, the findings suggested the presence of as yet unrecognized genomic elements that are either involved in the acquisition of virulence genes or associated with pathogenicity.

BACKGROUND: Stenotrophomonas maltophilia is a carbapenem-resistant Gram-negative pathogen increasingly responsible for difficult-to-treat nosocomial infections. OBJECTIVES: To describe the contemporary clinical characteristics and genome epidemiology of patients colonized or infected by S. maltophilia in a multicentre, prospective cohort. METHODS: All patients with a clinical culture growing S. maltophilia were enrolled at six tertiary hospitals across Japan between April 2019 and March 2022. The clinical characteristics, outcomes, antimicrobial susceptibility and genomic epidemiology of cases with S. maltophilia were investigated. RESULTS: In total, 78 patients were included representing 34 infection and 44 colonization cases. The median age was 72.5 years (IQR, 61-78), and males accounted for 53 cases (68%). The most common comorbidity was localized solid malignancy (39%). Nearly half of the patients (44%) were immunosuppressed, with antineoplastic chemotherapy accounting for 31%. The respiratory tract was the most common site of colonization (86%), whereas bacteraemia accounted for most infection cases (56%). The 30 day all-cause mortality rate was 21%, which was significantly higher in infection cases than colonization cases (35% versus 9%; adjusted HR, 3.81; 95% CI, 1.22-11.96). Susceptibility rates to ceftazidime, levofloxacin, minocycline and sulfamethoxazole/trimethoprim were 14%, 65%, 87% and 100%, respectively. The percentage of infection ranged from 13% in the unclassified group to 86% in genomic group 6A. The percentage of non-susceptibility to ceftazidime ranged from 33% in genomic group C to 100% in genomic groups 6 and 7 and genomic group geniculate. CONCLUSIONS: In this contemporary multicentre cohort, S. maltophilia primarily colonized the respiratory tract, whereas patients with bacteraemia had the highest the mortality from this pathogen. Sulfamethoxazole/trimethoprim remained consistently active, but susceptibility to levofloxacin was relatively low. The proportions of cases representing infection and susceptibility to ceftazidime differed significantly based on genomic groups.

Carbapenemase-producing Enterobacterales (CPEs) are one of the top priority antimicrobial-resistant pathogens. Among CPEs, those producing acquired metallo-β-lactamases (MBLs) are considered particularly problematic as few agents are active against them. Imipenemase (IMP) is the most frequently encountered acquired MBL in Japan, but comprehensive assessment of clinical and microbiological features of IMP-producing Enterobacterales infection remains scarce. Here, we retrospectively evaluated 62 patients who were hospitalized at a university hospital in Japan and had IMP-producing Enterobacterales from a clinical culture. The isolates were either Enterobacter cloacae complex or Klebsiella pneumoniae, and most of them were isolated from sputum. The majority of K. pneumoniae, but not E. cloacae complex isolates, were susceptible to aztreonam. Sequence type (ST) 78 and ST517 were prevalent for E. cloacae complex and K. pneumoniae, respectively, and all isolates carried blaIMP-1. Twenty-four of the patients were deemed infected with IMP-producing Enterobacterales. Among the infected patients, therapy varied and largely consisted of conventional β-lactam agents, fluoroquinolones, or combinations. Three (13%), five (21%), and nine (38%) of them died by days 14, 30, and 90, respectively. While incremental mortality over 90 days was observed in association with underlying comorbidities, active conventional treatment options were available for most patients with IMP-producing Enterobacterales infections, distinguishing them from more multidrug-resistant CPE infections associated with globally common MBLs, such as New Delhi metallo-β-lactamase (NDM) and Verona integron-encoded metallo-β-lactamase (VIM).

UNLABELLED: Streptococcus pyogenes causes a variety of human infections, and hospital outbreaks with this pathogen have also been reported. The purpose of this study is to describe the clinical characteristics of an outbreak of S. pyogenes involving 15 patients and four healthcare workers (HCWs), as well as the molecular characteristics of the causative isolates. The course and response to the outbreak were reviewed, and information on the characteristics of the patients was extracted retrospectively from the medical records. Whole-genome sequencing of the 16 causative isolates (14 from patients and two from HCWs) was also performed. All 15 patients were postoperative of head and neck cancer with tracheotomy, and 12 had invasive infections, primarily surgical site infections, all of which resolved without causing serious illness. All but the first case was detected more than 7 days after admission. S. pyogenes was detected in two patients after empiric antimicrobial administration was performed on all inpatients and HCWs, and the outbreak was finally contained in approximately 2 months. All isolates detected in patients and HCWs belonged to emm89/clade 3, a hypervirulent clone that has emerged worldwide and was classified as sequence type 646. These isolates had single nucleotide polymorphism (SNP) differences of zero to one, indicating clonal transmission. This study demonstrated an outbreak of S. pyogenes emm89/clade 3 in a ward of patients with head and neck cancer. The global emergence of hypervirulent isolates may increase the risk of outbreaks among high-risk patients. IMPORTANCE: This study describes an outbreak of Streptococcus pyogenes that occurred in a ward caring for patients with head and neck cancer and tracheostomies. Many cases of invasive infections occurred in a short period, and extensive empiric antimicrobial administration on patients and healthcare workers was performed to control the outbreak. Whole-genome sequencing analysis of the causative strains confirmed that it was a monoclonal transmission of strains belonging to emm89/clade 3. The epidemiology and clinical characteristics of S. pyogenes infections have changed with the replacement of the prevalent clones worldwide. In the 1980s, there was a reemergence of S. pyogenes infections in high-income countries due to the spread of hypervirulent emm1 strains. emm89/clade 3 has recently been spreading worldwide and shares common features with emm1, including increased production of two toxins, NADase, and streptolysin O. The outbreak reported here may reflect the high spreading potential and virulence of emm89/clade 3.

ABSTRACT Streptococcus mitis/oralis group isolates with reduced carbapenem susceptibility have been reported, but its isolation rate in Japan is unknown. We collected 356 clinical α-hemolytic streptococcal isolates and identified 142 of them as S. mitis/oralis using partial sodA sequencing. The rate of meropenem non-susceptibility was 17.6% (25/142). All 25 carbapenem-non-susceptible isolates harbored amino acid substitutions in/near the conserved motifs in PBP1A, PBP2B, and PBP2X. Carbapenem non-susceptibility is common among S. mitis/oralis group isolates in Japan.

β-Lactamases can accumulate stepwise mutations that increase their resistance profiles to the latest β-lactam agents. CMY-185 is a CMY-2-like β-lactamase and was identified in an Escherichia coli clinical strain isolated from a patient who underwent treatment with ceftazidime-avibactam. CMY-185, possessing four amino acid substitutions of A114E, Q120K, V211S, and N346Y relative to CMY-2, confers high-level ceftazidime-avibactam resistance, and accumulation of the substitutions incrementally enhances the level of resistance to this agent. However, the functional role of each substitution and their interplay in enabling ceftazidime-avibactam resistance remains unknown. Through biochemical and structural analysis, we present the molecular basis for the enhanced ceftazidime hydrolysis and impaired avibactam inhibition conferred by CMY-185. The substituted Y346 residue is a major driver of the functional evolution as it rejects primary avibactam binding due to the steric hindrance and augments oxyimino-cephalosporin hydrolysis through a drastic structural change, rotating the side chain of Y346 and then disrupting the H-10 helix structure. The other substituted residues E114 and K120 incrementally contribute to rejection of avibactam inhibition, while S211 stimulates the turnover rate of the oxyimino-cephalosporin hydrolysis. These findings indicate that the N346Y substitution is capable of simultaneously expanding the spectrum of activity against some of the latest β-lactam agents with altered bulky side chains and rejecting the binding of β-lactamase inhibitors. However, substitution of additional residues may be required for CMY enzymes to achieve enhanced affinity or turnover rate of the β-lactam agents leading to clinically relevant levels of resistance.IMPORTANCECeftazidime-avibactam has a broad spectrum of activity against multidrug-resistant Gram-negative bacteria including carbapenem-resistant Enterobacterales including strains with or without production of serine carbapenemases. After its launch, emergence of ceftazidime-avibactam-resistant strains that produce mutated β-lactamases capable of efficiently hydrolyzing ceftazidime or impairing avibactam inhibition are increasingly reported. Furthermore, cross-resistance towards cefiderocol, the latest cephalosporin in clinical use, has been observed in some instances. Here, we clearly demonstrate the functional role of the substituted residues in CMY-185, a four amino-acid variant of CMY-2 identified in a patient treated with ceftazidime-avibactam, for high-level resistance to this agent and low-level resistance to cefiderocol. These findings provide structural insights into how β-lactamases may incrementally alter their structures to escape multiple advanced β-lactam agents.

OBJECTIVES: Taxonomic assignment based on whole-genome sequencing data facilitates clear demarcation of species within a complex genus. Here, we applied a unique pan-genome phylogenetic method, open reading frame (ORF)-based binarized structure network analysis (OSNA), for taxonomic inference of Aeromonas spp., a complex taxonomic group consisting of 30 species. METHODS: Data from 335 publicly available Aeromonas genomes, including the reference genomes of 30 species, were used to build a phylogenetic tree using OSNA. In OSNA, whole-genome structures are expressed as binary sequences based on the presence or absence of ORFs, and a tree is generated using neighbor-net, a distance-based method for constructing phylogenetic networks from binary sequences. The tree built by OSNA was compared to that constructed by a core-genome single-nucleotide polymorphism (SNP)-based analysis. Furthermore, the orthologous average nucleotide identity (OrthoANI) values of the sequences that clustered in a single clade in the OSNA-based tree were calculated. RESULTS: The phylogenetic tree constructed with OSNA successfully delineated the majority of species of the genus Aeromonas forming conspecific clades for individual species, which was corroborated by OrthoANI values. Moreover, the OSNA-based phylogenetic tree demonstrated high compositional similarity to the core-genome SNP-based phylogenetic tree, supported by the Fowlkes-Mallows index. CONCLUSIONS: We propose that OSNA is a useful tool in predicting the taxonomic classification of complex bacterial genera.

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Carbapenem-resistant bacteria in patients with cancer are concerning due to the high risk of infection and mortality rates; however, the characteristics and prognoses of carbapenem-resistant infections in this population are currently unknown in Japan. We hence investigated the features and outcomes of carbapenem-resistant bacterial infections (CRBI) in patients with cancer in Japan.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>From April 1, 2019, to March 31, 2022, patients with CRBI who either had cancer or no cancer were prospectively enrolled at eight centers as part of the Multi-Drug Resistant Organisms Clinical Research Network (MDRnet). The primary outcome was the 30-day all-cause mortality rates in patients with and without cancer. Two secondary outcomes were evaluated: 1) composite outcomes including mortality, worsening of clinical course after culture collection, intensive care unit stay, intubation, new dialysis from the date of culture collection to the end of antimicrobial therapy, and readmission within 90 days after discharge; and 2) the length of hospital stay after CRBI excluding death.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>We included a total of 167 patients, with 66 (39.5%) in the cancer group and 101 (60.5%) in the non-cancer group. The 30-day mortality rates in the cancer and non-cancer groups were 18.2% (12/66) and 14.0% (14/101), respectively (p = 0.45), while the composite outcomes in the cancer and non-cancer groups were 56.1% (37/66) and 43.6% (44/101), respectively (p = 0.12). Average duration of hospitalization was not significantly different between the two groups (cancer group, 44.6 days; non-cancer group, 51.0 days; p = 0.55). Propensity score analysis using inverse probability weighting also showed no significant difference in 30-day mortality and average duration of hospitalization (p = 0.22 and 0.98, respectively); however, the composite outcome was significantly higher in the cancer group than in non-cancer controls (odds ratio, 2.41; 95% confidence interval, 1.11–5.21; p = 0.03).</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>There was no difference in 30-day mortality rates between the cancer and non-cancer patient groups; however, we found a significant difference in the composite outcome. Patients with cancer who had CRBI experienced a worse clinical course that non-cancer patients.</jats:p> </jats:sec> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>Masahiro Suzuki, PhD, KANTO Chemical co., inc.: Grant/Research Support Yasufumi Matsumara, MD, PhD, Beckman Coulter: Grant/Research Support|Presicion System Science: Grant/Research Support|Toyobo: Grant/Research Support David van Duin, MD, PhD, Entasis: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Qpex: Advisor/Consultant|Roche: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Union: Advisor/Consultant|Utility: Advisor/Consultant Yohei Doi, MD, PhD, bioMerieux: Advisor/Consultant|FujiFilm: Advisor/Consultant|Gilead: Advisor/Consultant|Gilead: Honoraria|GSK: Advisor/Consultant|Meiji Seika Pharma: Advisor/Consultant|Moderna: Advisor/Consultant|Moderna: Honoraria|MSD: Advisor/Consultant|MSD: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Shionogi: Honoraria Sho Saito, MD, PhD, Shionogi &amp; Company, Limited: Grant/Research Support</jats:p> </jats:sec>

Although the frequency of community-acquired infections caused by Klebsiella pneumoniae subsp. ozaenae (K. ozaenae) is low, they are often detected in sputum specimens. In addition, lung abscesses, necrotizing pneumonia, and urinary tract infections caused by K. ozaenae have also been reported. We herein report the first detection of K. ozaenae as an etiological agent of bacterial meningitis in Japan. Cases of K. ozaenae meningitis complicated by diabetes mellitus and sinusitis have been reported elsewhere. When Klebsiella pneumoniae is detected in such cases, it is important to use other detection methods in addition to mass spectrometry for correct identification.

BACKGROUND: There is a growing interest in Klebsiella variicola as a causative pathogen in humans, though its clinical features and the impact of co-infection or secondary infection with COVID-19 remain unknown. CASE PRESENTATION: A 71-year-old man presented with fever, altered mental status and generalized weakness and was admitted to ICU due to severe COVID-19 pneumonia. He was newly diagnosed with type II diabetes mellitus upon admission. On hospital day 3, his respiratory status deteriorated, requiring invasive mechanical ventilation. On hospital day 10, superimposed bacterial pneumonia was suspected and subsequently, broad-spectrum antibiotics were administered for the associated bloodstream infection. On hospital day 13, despite administration of active antibiotics and appropriate source control, he decompensated and died. The causative organism isolated from blood cultures was initially reported as K. pneumoniae, but it was identified as K. variicola by a genetic analysis. A representative isolate (FUJ01370) had a novel multilocus sequence typing allelic profile (gapA-infB-mdh-pgi-phoE-rpoB-tonB: 16-24-21-27-52-17-152), to which sequence type 5794 was assigned (GenBank assembly accession: GCA_019042755.1). CONCLUSIONS: We report a fatal case of respiratory and bloodstream infection due to K. variicola complicating severe COVID-19. Co-infection or secondary infection of K. variicola in COVID-19 is likely under-recognized and can be fulminant as in this case.

AIMS: Phylogenetic analysis based on core genome single nucleotide polymorphisms (cgSNPs) using whole-genome sequencing (WGS) is increasingly used in epidemiological investigations of bacteria. The approach, however, is both resource intensive and time-consuming. Oxford Nanopore Technologies (ONT) sequencing is capable of real-time data analysis but the high error rate hampers its application in cgSNP-based phylogenetic analysis. Here, we developed a cgSNP-independent phylogenetic analysis method using ONT read assemblies by focusing on open reading frame (ORF) content patterns. METHODS AND RESULTS: WGS data of 66 Enterobacter hormaechei strains acquired by both ONT and Illumina sequencing and 162 strains obtained from NCBI database were converted to binary sequences based on the presence or absence of ORFs using BLASTn. Phylogenetic trees calculated from binary sequences (ORF trees) were compared with cgSNP trees derived from Illumina sequences. Clusters of closely related strains in the cgSNP trees formed comparable clusters in the ORF trees built with binary sequences, and the tree topologies between them were similar based on Fowlkes-Mallows index. CONCLUSIONS: The ORF-based phylogenetic analysis using ONT sequencing may be useful in epidemiological investigations and offer advantages over the cgSNP-based approach. SIGNIFICANCE AND IMPACT OF THE STUDY: Conversion of assembled WGS data to binary sequences based on the presence or absence of ORFs circumvents read error concerns with ONT sequencing. Since ONT sequencing generates data in real time and does not require major investment, this ORF-based phylogenetic analysis method has the potential to enable phylogenetic and epidemiological analysis at the point of care.

AIMS: Klebsiella pneumoniae is a major cause of healthcare-associated infections. In this study, we aimed to develop a rapid and simple genotyping method that can characterize strains causing nosocomial infections. METHODS AND RESULTS: The PCR-based open reading frame (ORF) typing (POT) method consists of two multiplex PCR reactions that were designed to detect 25 ORFs specific to bacterial genetic lineages, species, antimicrobial-resistant genes (blaCTX-M group-1 , blaCTX-M group-9 , blaIMP and blaKPC ), a capsular K1-specific gene and a virulence factor gene (rmpA/A2). The electrophoresis results are then digitized. A total of 192 strains (136 clinical and 8 reference strains of K. pneumoniae, 33 clinical and 1 reference strains of K. variicola and 14 clinical strains of K. quasipneumoniae) were classified into 95, 26 and 11 POT values, respectively. The distribution patterns of ORFs among K. pneumoniae correlated well with multilocus sequence typing (MLST). Furthermore, closely related species could be distinguished and key antimicrobial resistance and hypervirulence genes were identified as part of POT. CONCLUSIONS: The POT method was developed and validated for K. pneumoniae. In comparison to MLST, the POT method is a rapid and easy genotyping method for monitoring transmission events by K. pneumoniae in clinical microbiology laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: The POT method supplies clear and informative molecular typing results for K. pneumoniae. The method would facilitate molecular epidemiological analysis in infection control and hospital epidemiology investigations.

Although piperacillin-tazobactam (TZP) was shown to be less effective than carbapenems in treating bacteremia due to extended-spectrum β-lactamase-producing (ESBL)-producing organisms in a randomized controlled trial, the fact that many of the causative organisms co-produced inhibitor-resistant OXA-1 along with ESBLs may have influenced the results. In this study, we compared the therapeutic effectiveness of TZP and carbapenem in treating ESBL-producing Escherichia coli bacteremia in areas with low frequency of OXA-1 co-production. Forty patients, 14 in the TZP treatment group and 26 in the carbapenem treatment group, were included in the analysis. There were no significant differences in patient background between the two groups. Urinary tract infection or cholangitis was the source of bacteremia in 26 patients (65%), and the Pitt bacteremia score was zero or one in 35 patients (87.5%). Only four (11.4%) of the 35 causative isolates available for microbiological analysis harbored blaOXA-1, and only three (8.6%) were non-susceptible to TZP. Seventeen (48.6%) isolates carried blaCTX-M-27, none of which carried other β-lactamase genes. No significant difference in the frequency of treatment failure on day 14 of bacteremia was documented between the TZP and carbapenem treatment groups in both the crude analysis and the inverse probability of treatment weighting-adjusted analysis. This study demonstrates that TZP may be a treatment option for non-severe cases of ESBL-producing E. coli bacteremia in areas with low frequency of OXA-1 co-production. IMPORTANCE Although carbapenems are considered the drug of choice for severe infections caused by extended-spectrum β-lactamase-producing (ESBL)-producing organisms, other therapeutic options are being explored to avoid increasing the selective pressure for carbapenem-resistant organisms. In this study, it was suggested that piperacillin-tazobactam may be as effective as carbapenems for the treatment of mild bacteremia caused by ESBL-producing Escherichia coli in areas where OXA-1 co-production by ESBL-producing E. coli is rare. The genetic background of each regional epidemic clone differs even among multidrug-resistant bacteria classified under the same name (e.g., ESBL-producing organisms), resulting in possible differences in the efficacy of therapeutic agents. Exploration of treatment options for multidrug-resistant organisms according to local epidemiology is worthwhile from the perspective of antimicrobial stewardship.

Carbapenemase-producing Escherichia coli sequence type (ST) 648 strains were isolated from two international visitors without previous medical exposure from Southeast Asian countries in a hospital in Japan. One isolate, FUJ80154, carried blaNDM-5 in a complex class 1 integron on an IncFIB/FII plasmid; the other isolate, FUJ80155, carried two copies of blaOXA-48 on the chromosome flanked by IS1R on both sides. The core-genome based-phylogenetic analysis with publicly available genome data of E. coli ST648 carrying blaNDM-5 or blaOXA-48-like demonstrated high genetic similarity between FUJ80154 and NDM-5-prooducing E. coli ST648 strains isolated in South and Southeast Asian countries. On the other hand, no closely related isolates of FUJ80155 were identified. In the absence of prior hospitalization overseas, neither patient had qualified for routine screening of multidrug-resistant organisms, and the isolates were incidentally identified in cultures ordered at the discretion of the treating physician. IMPORTANCE Although patients with history of international hospitalization are often subject to screening for multidrug-resistant organisms, it is unclear whether patients who reside in countries where carbapenemase-producing Enterobacterales (CPE) is endemic but have no history of local hospitalization contribute to the transmission of CPE. In this study, NDM-5-producing and OXA-48-producing Escherichia coli sequence type (ST) 648, a recently recognized high-risk, multidrug-resistant clone, were detected from two overseas visitors without previous medical exposure. The findings of this study suggest that active surveillance culture on admission to hospital may be considered for travelers from countries with endemicity of carbapenem-resistant organisms even without history of local hospitalization and underscore the need to monitor cross-border transmission of high-risk clones, such as carbapenemase-producing E. coli ST648.

Rapid detection and reporting of carbapenemase-producing Enterobacterales (CPE) is one of the top priorities of clinical microbiology laboratories. The Clinical and Laboratory Standards Institute recommends the modified carbapenem inactivation method (mCIM) as the preferred method for this purpose, but it requires a broth incubation process which can be cumbersome. Here, we compared the performance of mCIM with three alternative rapid CPE detection methods against a collection of genetically defined CPE, with most carrying blaIMP, and non-CPE clinical isolates. The sensitivities of mCIM, simplified carbapenem inactivation method (sCIM), Rapidec Carba NP, and NG-Test Carba 5 were 98.0%, 54.9%, 90.2%, and 72.5%, whereas the specificities were 89.5%, 84.2%, 89.5%, and 100%, respectively. Modification of the interpretive criteria of sCIM increased its sensitivity to 88.2% and specificity to 89.5%. The results suggest that mCIM is currently the optimal method for CPE detection in an epidemiological setting where CPE-producing IMP group carbapenemase is predominant. While sCIM is easier to perform, it requires further validation before it can be widely adopted as an alternative to mCIM in the clinical laboratory. IMPORTANCE Simple identification methods for carbapenemase-producing Enterobacterales are required for the clinical laboratory. The simplified carbapenem inactivation method (sCIM) is a carbapenemase detection method that can be performed with less hands-on time than mCIM, but its sensitivity and specificity were suboptimal compared with other phenotypic detection methods when tested against a collection of IMP-producing CPE. Insufficient inactivation of imipenem from inadequate inoculation was suspected as the cause. While sCIM is easier to perform, it requires optimization before it can be widely adopted as an alternative to mCIM in the clinical laboratory.

Posttranslational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such a function. Here, we present the function and structure of NpmB1, whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides, including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single-amino-acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

Information regarding the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in asymptomatic carriers is scarce. In order to determine the duration of infectivity and its correlation with reverse transcription-PCR (RT-PCR) results and time since initial positive PCR test in this population, we evaluated SARS-CoV-2 cell infectivity in nasopharyngeal samples longitudinally obtained from asymptomatic carriers who disembarked from a cruise ship during a COVID-19 outbreak. Of 166 nasopharyngeal samples collected from 39 asymptomatic carriers every 48 h until two consecutive negative PCR test results were obtained, SARS-CoV-2 was successfully isolated from 9 PCR-positive samples which were obtained from 7 persons (18%; 7/39). Viable viruses were isolated predominantly within 7 days after the initial positive PCR test, except for one person who shed viable virus until day 15. The median crossing point (Cp) value of RT-PCR of culture-positive samples was 24.6 (interquartile range [IQR], 20.4 to 25.8; range, 17.9 to 30.3), and Cp values were significantly associated with isolation of viable virus (odds ratio, 0.496; 95% confidence interval [CI], 0.329 to 0.747; P value, 0.001), which was consistent with existing data for symptomatic patients. Genome sequence analysis of SARS-CoV-2 samples consecutively obtained from a person who shed viable virus for 15 days identified the emergence of two novel single nucleotide variants (C8626T transition and C18452T transition) in the sample collected on day 15, with the latter corresponding to an amino acid substitution in nonstructural protein 14. The impact of these mutations on prolonged viable-virus shedding is unclear. These findings underscore the potential role of asymptomatic carriers in transmission.IMPORTANCE A growing number of studies suggest the potential role of asymptomatic SARS-CoV-2 carriers as a major driver of the COVID-19 pandemic; however, virological assessment of asymptomatic infection has largely been limited to reverse transcription-PCR (RT-PCR), which can be persistently positive without necessarily indicating the presence of viable virus (e.g., replication-competent virus). Here, we evaluated the infectivity of asymptomatic SARS-CoV-2 carriers by detecting SARS-CoV-2-induced cytopathic effects on Vero cells using longitudinally obtained nasopharyngeal samples from asymptomatic carriers. We show that asymptomatic carriers can shed viable virus until 7 days after the initial positive PCR test, with one outlier shedding until day 15. The crossing point (Cp) value of RT-PCR was the leading predictive factor for virus viability. These findings provide additional insights into the role of asymptomatic carriers as a source of transmission and highlight the importance of universal source control measures, along with isolation policy for asymptomatic carriers.

A patient in Japan with coronavirus disease and hypervirulent Klebsiella pneumoniae K2 sequence type 86 infection died of respiratory failure. Bacterial and fungal co-infections caused by region-endemic pathogens, including hypervirulent K. pneumoniae in eastern Asia, should be included in the differential diagnosis of coronavirus disease patients with acutely deteriorating condition.

Pathogenic bacteria of the genus Bartonella can induce vasoproliferative lesions during infection. The underlying mechanisms are unclear, but involve secretion of an unidentified mitogenic factor. Here, we use functional transposon-mutant screening in Bartonella henselae to identify such factor as a pro-angiogenic autotransporter, called BafA. The passenger domain of BafA induces cell proliferation, tube formation and sprouting of microvessels, and drives angiogenesis in mice. BafA interacts with vascular endothelial growth factor (VEGF) receptor-2 and activates the downstream signaling pathway, suggesting that BafA functions as a VEGF analog. A BafA homolog from a related pathogen, Bartonella quintana, is also functional. Our work unveils the mechanistic basis of vasoproliferative lesions observed in bartonellosis, and we propose BafA as a key pathogenic factor contributing to bacterial spread and host adaptation.

We characterized 29 blaCTX-M-27-harboring plasmids of Escherichia coli ST131 sublineage C1/H30R isolates from healthy individuals and long-term care facility (LTCF) residents. Most (27/29) plasmids were of the FIA, FIB, and FII multi-replicon type with the same pMLST. Several plasmids (7/23) from LTCF residents harbored only blaCTX-M-27 as the resistance gene; however, their fundamental structures were very similar to those of previously isolated blaCTX-M-27/F1:A2:B20 plasmids, suggesting their prevalence as a newly arising public health concern.

OBJECTIVES: Systematic comparison of multiple plasmids remains challenging. We aimed to develop a new method for phylogenetic analysis of plasmids, open reading frame (ORF)-based binarized structure network analysis of plasmids (OSNAp). METHODS: With the OSNAp, the genetic structures of plasmids in a given plasmid group are expressed as binary sequences based on the presence or absence of ORFs regardless of their positions or directions. As a proof-of-concept, ORFs were collected from 101 complete I1 plasmid sequences, and their corresponding binary sequences were generated. A tree was generated using the neighbor-net, an algorithm for constructing phylogenetic networks based on distance between taxa, to visualize the plasmid phylogeny drawn from binary sequences. The results were compared with those of plasmid sequence types (pSTs) defined by plasmid multilocus sequence typing (pMLST). RESULTS: All I1 plasmids were placed on the phylogenetic tree constructed from the binary sequences. Most plasmids belonging to the same pSTs had Dice indices of ≥0.95 and were placed in the same OSNAp split. On the other hand, pST12 plasmids were distributed on separate splits due to differences in ORFs not used in pMLST, suggesting improved differentiation of the plasmids with OSNAp compared with pMLST. CONCLUSION: OSNAp is a novel holistic approach to assess relatedness of a population of plasmids in a given plasmid group based on nucleotide sequence data. It provides higher discrimination than pMLST, which may prove useful in tracing bacteria that harbor plasmids of shared origins.

INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS

Global widespread of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, especially Escherichia coli poses a greater threat in healthcare and community settings of humans. Raw meats from food animals colonized with ESBL producers may be one of important transmission routes for those bacteria in the community. This study investigated the presence of ESBL-producing E. coli in retail raw chicken and pork meats in Japan. ESBL producers were detected from the 59 of 150 (39.3%) chicken samples, but none were from all the 50 pork samples tested. The blaCTX-M-14 (17; 24.3%) was most frequently identified, followed by blaCTX-M-2 (16; 22.9%), blaSHV-12 (11; 15.7%), and blaCTX-M-55 (10; 14.3%) among a total of 70 ESBL-producing E. coli isolates from 59 chicken samples. The isolates with blaCTX-M-14 were often combined with phylogroup B1 (9/17) mainly composed of ST162 (7/9), and phylogroup F (5/17) with diverse STs. The blaCTX-M-14 was basically associated with the common elements ISEcp1 and ΔIS903 or IS903 in all 17 isolates. In 6 isolates, comprising 5 phylogroup B1-ST162 and a nontypeable-ST162 isolates, an IS26-truncated ISEcp1 was identified upstream of the blaCTX-M-14, and a fosA3 was further located downstream of ΔIS903. Furthermore, some mobile genetic elements mediating blaCTX-M-14 unique to raw chicken meat portions were identified. The blaCTX-M-2 gene was preceded by ISEcp1 or ISCR1 in 16 isolates, whereas the presence of Δorf3 downstream of blaCTX-M-2 was limited only in 6 isolates from Brazilian samples though they exhibited diverse phylogroups and STs. The blaCTX-M-55 and blaCTX-M-1 shared classical flanking structures, ISEcp1-blaCTX-M-orf477, although the length of spacer sequences between ISEcp1 and the start codon of blaCTX-M was 45 bp and 80 bp for blaCTX-M-55 and blaCTX-M-1, respectively. Among blaSHV-12-harboring isolates, ST38 was frequently detected (6/11) though their phylogroup distribution varied. In conclusion, besides transmission of bla gene-harboring E. coli lineages which have adaptability to both human and chicken, spread of mobile genetic elements associated with bla genes from E. coli lineages adapted to chicken to those adapted to human is highly suggested. Our results provide important information to gain a better understanding of the transmission risk of bla genes from retail chicken meats to human.

Purpose. The decline in methicillin-resistant Staphylococcus aureus (MRSA) isolation rates has become a general observation worldwide, including Japan. We hypothesized that some genetic shift in MRSA might cause this phenomenon, and therefore we investigated the genetic profiles among MRSA clinical isolates obtained from three different epidemic phases in Japan. Methodology. A total of 353 MRSA isolates were selected from 202 medical facilities in 1990 (pre-epidemic phase), 2004 (epidemic phase) and 2016 (post-epidemic phase). Molecular typing was performed by PCR detection of 22 genes using the polymerase chain reaction (PCR)-based ORF typing (POT) system, including an additional eight genes including small genomic islets and seven toxin genes. Results. Isolates with a POT1 of score 93, identified as presumed clonal complex (pCC)5-staphylococcal cassette chromosome mec (SCCmec) type II including ST5-SCCmec type II New York/Japan clone, represented the major epidemic MRSA lineage in 1990 and 2004. In 2016, however, a marked decrease in isolates with a POT1 score of 93, along with changes in the epidemiology of toxin genes carried, was noted, where the carriers of tst genes including the tst-sec combination were markedly reduced, and those possessing the seb gene alone were markedly increased. Rather, isolates with a POT1 score of 106, including pCC1 or pCC8 among the isolates with SCCmec type IV, which often links to communityassociated MRSA, were predominant. Interestingly, the pCC1 and pCC8 lineages were related to sea and tst-sec carriage, respectively. Conclusions. Over time, a transition in MRSA genetic profiles from a POT1 score of 93 in 1990 and 2004 to 106 in 2014 was found in Japan.

Multidrug-resistant (MDR) Acinetobacter spp. have been globally disseminated in association with the successful clonal lineage Acinetobacter baumannii international clone II (IC II). Because the prevalence of MDR Acinetobacter spp. in Japan remains very low, we characterized all Acinetobacter spp. (n = 866) from 76 hospitals between October 2012 and March 2013 to describe the entire molecular epidemiology of Acinetobacter spp. The most prevalent species was A. baumannii (n = 645; 74.5%), with A. baumannii IC II (n = 245) accounting for 28.3% of the total. Meropenem-resistant isolates accounted for 2.0% (n = 17) and carried ISAba1-blaOXA-23-like (n = 10), blaIMP (n = 4), or ISAba1-blaOXA-51-like (n = 3). Multilocus sequence typing of 110 representative A. baumannii isolates revealed the considerable prevalence of domestic sequence types (STs). A. baumannii IC II isolates were divided into the domestic sequence type 469 (ST469) (n = 18) and the globally disseminated STs ST208 (n = 14) and ST219 (n = 4). ST469 isolates were susceptible to more antimicrobial agents, while ST208 and ST219 overproduced the intrinsic AmpC β-lactamase. A. baumannii IC II and some A. baumannii non-IC II STs (e.g., ST149 and ST246) were associated with fluoroquinolone resistance. This study revealed that carbapenem-susceptible A. baumannii IC II was moderately disseminated in Japan. The low prevalence of acquired carbapenemase genes and presence of domestic STs could contribute to the low prevalence of MDR A. baumannii A similar epidemiology might have appeared before the global dissemination of MDR epidemic lineages. In addition, fluoroquinolone resistance associated with A. baumannii IC II may provide insight into the significance of A. baumannii epidemic clones.

We investigated the genetic backbones of 14 bla(CTX-M-8)-positive Escherichia coli isolates recovered from human stool samples and chicken meat. All isolates carried IncI1 plasmids with bla(CTX-M-8) (bla(CTX-M-8)/IncI1), and most (9/14) belonged to a specific genetic lineage, namely, plasmid sequence type 113 (pST113). The genetic contexts of the nine bla(CTX-M-8)/IncI1 pST113 plasmids were similar, regardless of the source. These results suggest the probable local transfer of bla(CTX-M-8)/IncI1 between humans and chickens with genetically diverse E. coli.

We report a case of rat bite fever, diagnosed based on positive cultures of Streptobacillus moniliformis from blood and synovial fluid. The patient was a 45-year-old man who presented with history of a rat bite and alcoholic liver cirrhosis. He had been bitten on his third finger by a rat, which was caught in a mousetrap installed in his house. Over the course of approximately 2 weeks after the bite, the patient developed fever, rash, and arthralgia. The patient was admitted to our hospital and treated with a combination of ampicillin-sulbactam, vancomycin (VAN), and minocycline (MIN) antibiotics. Initial culture findings from the Anaerobic/F resin blood culture were positive for gram-negative bacillus after overnight incubation. Thus, S. moniliformis infection was suspected, and administration of VAN and MIN was ceased. On hospital day 8, the treatment was switched to oral amoxicillin-clavulanic acid, and the patient was discharged from the hospital. Subsequently, the pathogen was also detected in synovial fluid and identified as S. moniliformis using 16S rRNA sequencing analysis.

We identified hypervirulent Streptococcus pyogenes in 27 and 420 isolates from patients with invasive and non-invasive diseases, respectively, in Aichi Prefecture, Japan, between 2003 and 2012, in an attempt to understand why the prevalence of streptococcal toxic shock syndrome (STSS) suddenly increased in this location during 2011. Hypervirulent strains belong to the emm1 genotype, with a mutation in the covR/S genes that regulate many other genes, encoding virulence determinants and resulting in the absence of the proteinase streptococcal exotoxin B and the production of virulence factors such as the superantigen streptococcal exotoxin A, the nuclease streptococcal DNase, the cytotoxin NAD-glycohydrolase, and the hemolysin streptolysin O. We found 1 strain from invasive disease and 1 from non-invasive disease with traits similar to those of hypervirulent strains, except that the sda1 gene was absent. We also found 1 non-emm1 strain with phenotypic and genetic traits identical to those of the emm1 hypervirulent strains except that it did not belong to emm1 genotype, from non-invasive diseases cases in 2011. These findings suggested that hypervirulent and hypervirulent-like strains from invasive and non-invasive disease cases could have at least partially contributed to the sudden increase in the number of patients with STSS in Aichi during 2011.

AimsMolecular epidemiological techniques, such as pulsed-field gel electrophoresis (PFGE), or multilocus sequence typing (MLST) have facilitated our understanding of the transmission routes of nosocomial infections by Pseudomonas aeruginosa. However, they are time consuming and technically demanding. To perform molecular epidemiological analysis in a standard microbiology laboratory, we aimed to develop a simpler and effective molecular epidemiological technique based on the open-reading frame (ORF) distribution patterns detected by PCR, which we call PCR-based ORF typing (POT). Methods and ResultsTen ORFs from genomic islets, five ORFs from genomic islands, and the metallo--lactamases (MBLs) bla(IMP) and bla(VIM) were selected by comparing the whole-genome sequences of different Ps.aeruginosa strains (PAO1, PA7, UCBPP-PA14 and LESB58). These 17 ORFs were detected, along with a Ps.aeruginosa marker, using 9-plex and 10-plex PCR systems. The genotypes in the POT were compared to those obtained by using PFGE and MLST. ConclusionsUsing the POT method, molecular epidemiological analyses of Ps.aeruginosa can be completed in 4h. Significance and Impact of the StudySince this method is very easy to perform, even in standard clinical laboratories, it could be a valuable tool for monitoring daily infection control measures.

黄色ブドウ球菌は，食中毒や院内感染型MRSA （hospital-acquired methicillin-resistant Staphylococcus aureus （HA-MRSA））の起炎菌として公衆衛生上重要な位置を占めてきた．加えて近年，市中感染型MRSA （communityacquired methicillin-resistant Staphylococcus aureus （CA-MRSA））の増加が問題になっている．しかし，その感染源は明らかにされておらず，疫学情報の報告も少ない．本稿では，市中におけるMRSAの汚染状況を把握するため，大分県下で流通する市販食肉305検体と下痢症患者便1,543検体を対象に，黄色ブドウ球菌ならびにMRSAの分離を試みた．黄色ブドウ球菌は301株分離され，うちMRSAは18株（鶏肉2株，豚肉1株，鴨肉1株，下痢症患者便14株）で，すべてPantonValentine leucocidin遺伝子陰性であった．これらMRSA株の性状比較ならびに分子遺伝学的疫学解析を行い，市販流通食肉がCA-MRSAの来源の一つとして伝播拡大に関与している可能性を明らかにした．

Antimicrobial resistance issues have become a global health concern. The rapid identification of multidrug-resistant microbes, which depends on microbial genomic information, is essential for overcoming growing antimicrobial resistance challenges. However, genotyping methods, such as multilocus sequence typing (MLST), for identifying international epidemic clones of Acinetobacter baumannii are not easily performed as routine tests in ordinary clinical laboratories. In this study, we aimed to develop a novel genotyping method that can be performed in ordinary microbiology laboratories. Several open reading frames (ORFs) specific to certain bacterial genetic lineages or species, together with their unique distribution patterns on the chromosomes showing a good correlation with the results of MLST, were selected in A. baumannii and other Acinetobacter spp. by comparing their genomic data. The distribution patterns of the ORFs were visualized by agarose gel electrophoresis after multiplex PCR amplification and digitized. A. baumannii sequence types (STs) corresponding to international clones I and II were successfully discriminated from other STs and Acinetobacter species by detecting the distribution patterns of their ORFs using the multiplex PCR developed here. Since bacterial STs can be easily expressed as digitized numeric data with plus (-) expressed as 1 and minus (+) expressed as 0, the results of the method can be easily compared with those obtained by different tests or laboratories. This PCR-based ORF typing (POT) method can easily and rapidly identify international epidemic clones of A. baumannii and differentiate this microbe from other Acinetobacter spp. Since this POT method is easy enough to be performed even in ordinary clinical laboratories, it would also contribute to daily infection control measures and surveillance.

The aim of this study was to examine the link between Campylobacter jejuni isolates obtained from chicken meat (n = 7) and gastroenteritis patients (n = 744). In total, 751 isolates were subjected to Lior serotyping. All the isolates from chicken meats were serotyped as Lior serotype 76 (LIO76). Among 23 of the identified LIO76 strains, 13 strains (6 from chicken meat and 7 from clinical specimens) were indistinguishable by Penner serotyping, antimicrobial susceptibility testing, and pulsedfield gel electrophoresis. These strains were isolated in 2 different Japanese prefectures in 2004-2005, suggesting that chicken meat is an etiological agent of Campylobacter gastroenteritis and that a diffuse outbreak occurred during this time. Therefore, a continuous surveillance program should be established in Japan in order to prevent Campylobacter gastroenteritis, especially large-scale food-borne outbreaks.