Emergence of an IncX3 plasmid co-harbouring the carbapenemase genes blaNDM-5 and blaOXA-181.

Background: The spread of transmissible plasmids with carbapenemase genes has contributed to a global increase in carbapenemase-producing Enterobacterales over the past two decades, with blaNDM and blaOXA among the most prevalent carbapenemase genes. Objectives: To characterize an Escherichia coli isolate co-carrying blaNDM-5 and blaOXA-181 (JBEHAAB-19-0176) that was isolated in the Japan Antimicrobial Resistant Bacterial Surveillance in 2019-20, and to evaluate the functional advantage of carrying both genes as opposed to only one. Methods: The whole-genome sequence of the isolate was determined using long- and short-read sequencing. Growth assay and co-culture experiments were performed for phenotypic characterization in the presence of different ß-lactam antibiotics. Results: WGS analysis showed that blaNDM-5 and blaOXA-181 were carried by the same IncX3 plasmid, pJBEHAAB-19-0176_NDM-OXA. Genetic characterization of the plasmid suggested that the plasmid emerged through the formation of a co-integrate and resolution of two typical IncX3 plasmids harbouring blaNDM-5 and blaOXA-181, which involved two recombination events at the IS3000 and IS26 sequences. When cultured in the presence of piperacillin or cefpodoxime, the growth rate of the transformant co-harbouring blaNDM-5 and blaOXA-181 was significantly higher than the transformant with only blaNDM-5. Furthermore, in co-culture where the two blaNDM-5-harbouring transformants were allowed to compete directly, the strain additionally harbouring blaOXA-181 showed a marked growth advantage. Conclusions: The additional carriage of blaOXA-181 confers a selective advantage to bacteria in the presence of piperacillin and cefpodoxime. These findings may explain the current epidemiology of carbapenemase-producing Enterobacterales, in which bacteria carrying both blaNDM-5 and blaOXA-48-like genes have emerged independently worldwide.

Genomic characterization of Staphylococcus aureus isolated from patients admitted to intensive care units of a tertiary care hospital: epidemiological risk of nasal carriage of virulent clone during admission.

We conducted a molecular epidemiological study of Staphylococcus aureus using whole-genome sequence data and clinical data of isolates from nasal swabs of patients admitted to the intensive care unit (ICU) of Hiroshima University hospital. The relationship between isolate genotypes and virulence factors, particularly for isolates that caused infectious diseases during ICU admission was compared with those that did not. The nasal carriage rates of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in patients admitted to the ICU were 7.0% and 20.1%, respectively. The carriage rate of community-acquired (CA)-MRSA was 2.3%, accounting for 32.8% of all MRSA isolates. Whole-genome sequencing analysis of the MRSA isolates indicated that most, including CA-MRSA and healthcare-associated (HA)-MRSA, belonged to clonal complex (CC) 8 [sequence type (ST) 8] and SCCmec type IV. Furthermore, results for three disease foci (pneumonia, skin and soft tissue infection, and deep abscess) and the assessment of virulence factor genes associated with disease conditions [bacteremia, acute respiratory distress syndrome (ARDS), disseminated intravascular coagulopathy (DIC), and septic shock] suggested that nasal colonization of S. aureus clones could represent a risk for patients within the ICU. Particularly, MRSA/J and MSSA/J may be more likely to cause deep abscess infection; ST764 may cause ventilation-associated pneumonia, hospital-acquired pneumonia and subsequent bacteremia, and ARDS, and tst-1-positive isolates may cause DIC onset.IMPORTANCENasal colonization of MRSA in patients admitted to the intensive care unit (ICU) may predict the development of MRSA infections. However, no bacteriological data are available to perform risk assessments for Staphylococcus aureus infection onset. In this single-center 2-year genomic surveillance study, we analyzed all S. aureus isolates from nasal swabs of patients admitted to the ICU and those from the blood or lesions of in-patients who developed infectious diseases in the ICU. Furthermore, we identified the virulent clones responsible for causing infectious diseases in the ICU. Herein, we report several virulent clones present in the nares that are predictive of invasive infections. This information may facilitate the design of preemptive strategies to identify and eradicate virulent MRSA strains, reducing nosocomial infections within the ICU.

Nationwide genome surveillance of carbapenem-resistant Pseudomonas aeruginosa in Japan.

Japan is a country with an approximate 10% prevalence rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Currently, a comprehensive overview of the genotype and phenotype patterns of CRPA in Japan is lacking. Herein, we conducted genome sequencing and quantitative antimicrobial susceptibility testing for 382 meropenem-resistant CRPA isolates that were collected from 78 hospitals across Japan from 2019 to 2020. CRPA exhibited susceptibility rates of 52.9%, 26.4%, and 88.0% against piperacillin-tazobactam, ciprofloxacin, and amikacin, respectively, whereas 27.7% of CRPA isolates was classified as difficult-to-treat resistance P. aeruginosa. Of the 148 sequence types detected, ST274 (9.7%) was predominant, followed by ST235 (7.6%). The proportion of urine isolates in ST235 was higher than that in other STs (P = 0.0056, χ2 test). Only 4.1% of CRPA isolates carried the carbapenemase genes: blaGES (2) and blaIMP (13). One ST235 isolate carried the novel blaIMP variant blaIMP-98 in the chromosome. Regarding chromosomal mutations, 87.1% of CRPA isolates possessed inactivating or other resistance mutations in oprD, and 28.8% showed mutations in the regulatory genes (mexR, nalC, and nalD) for the MexAB-OprM efflux pump. Additionally, 4.7% of CRPA isolates carried a resistance mutation in the PBP3-encoding gene ftsI. The findings from this study and other surveillance studies collectively demonstrate that CRPA exhibits marked genetic diversity and that its multidrug resistance in Japan is less prevailed than in other regions. This study contributes a valuable data set that addresses a gap in genotype/phenotype information regarding CRPA in the Asia-Pacific region, where the epidemiological background markedly differs between regions.

Genomic analysis of inter-hospital transmission of vancomycin-resistant Enterococcus faecium sequence type 80 isolated during an outbreak in Hiroshima, Japan.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Complete sequence of carbapenem-resistant Ralstonia mannitolilytica clinical isolate co-producing novel class D ß-lactamase OXA-1176 and OXA-1177 in Japan.

In 2020, the Ralstonia mannitolilytica strain JARB-RN-0044 was isolated from a midstream urine sample of an elderly hospitalized patient in Japan and was highly resistant to carbapenem (i.e., imipenem, meropenem, and doripenem). Whole-genome sequencing revealed that the complete genome consists of two replicons, a 3.5-Mb chromosome and a 1.5-Mb large non-chromosomal replicon which has not been reported in R. mannitolilytica, and referred to as the "megaplasmid" in this study based on Cluster of Orthologous Group of proteins functional analysis. The strain JARB-RN-0044 harbored two novel OXA-60 and OXA-22 family class D ß-lactamase genes blaOXA-1176 and blaOXA-1177 on the megaplasmid. Cloning experiments indicated that Escherichia coli recombinant clone expressing blaOXA-1176 gene showed increased minimum inhibitory concentrations (MICs) of imipenem, meropenem, and doripenem, indicating that blaOXA-1176 gene encodes carbapenemase. In contrast, E. coli recombinant clone expressing blaOXA-1177 gene showed increased MICs of piperacillin and cefazolin, but not of carbapenem. Interestingly, the 44.6 kb putative prophage region containing genes encoding phage integrase, terminase, head and tail protein was identified in the downstream region of blaOXA-1176 gene, and comparative analysis with some previously reported R. mannitolilytica isolates revealed that the prophage region was unique to strain JARB-RN-0044. The existence of a highly carbapenem-resistant R. mannitolilytica isolate may raise human health concerns in Japan, where the population is rapidly aging.IMPORTANCERalstonia mannitolilytica is an aerobic non-fermenting Gram-negative rod commonly found in aquatic environments and soil. The bacteria can occasionally cause severe hospital-acquired bloodstream infections in immunocompromised patients and it has been recently recognized as an emerging opportunistic human pathogen. Furthermore, some R. mannitolilytica isolates are resistant to various antimicrobial agents, including ß-lactams and aminoglycosides, making antimicrobial therapy challenging and clinically problematic. However, clinical awareness of this pathogen is limited. To our knowledge, in Japan, there has been only one report of a carbapenem-resistant R. mannitolilytica clinical isolate from urine by Suzuki et al. in 2015. In this study, whole-genome sequencing analysis revealed the presence and genetic context of novel blaOXA-1176 and blaOXA-1177 genes on the 1.5 Mb megaplasmid from highly carbapenem-resistant R. mannitolilytica isolate and characterized the overall distribution of functional genes in the chromosome and megaplasmid. Our findings highlight the importance of further attention to R. mannitolilytica isolate in clinical settings.

Exploring the effects of antimicrobial treatment on the gut and oral microbiomes and resistomes from elderly long-term care facility residents via shotgun DNA sequencing.

Monitoring antibiotic-resistant bacteria (ARB) and understanding the effects of antimicrobial drugs on the human microbiome and resistome are crucial for public health. However, no study has investigated the association between antimicrobial treatment and the microbiome-resistome relationship in long-term care facilities, where residents act as reservoirs of ARB but are not included in the national surveillance for ARB. We conducted shotgun metagenome sequencing of oral and stool samples from long-term care facility residents and explored the effects of antimicrobial treatment on the human microbiome and resistome using two types of comparisons: cross-sectional comparisons based on antimicrobial treatment history in the past 6 months and within-subject comparisons between stool samples before, during and 2-4 weeks after treatment using a single antimicrobial drug. Cross-sectional analysis revealed two characteristics in the group with a history of antimicrobial treatment: the archaeon Methanobrevibacter was the only taxon that significantly increased in abundance, and the total abundance of antimicrobial resistance genes (ARGs) was also significantly higher. Within-subject comparisons showed that taxonomic diversity did not decrease during treatment, suggesting that the effect of the prescription of a single antimicrobial drug in usual clinical treatment on the gut microbiota is likely to be smaller than previously thought, even among very elderly people. Additional analysis of the detection limit of ARGs revealed that they could not be detected when contig coverage was <2.0. This study is the first to report the effects of usual antimicrobial treatments on the microbiome and resistome of long-term care facility residents.

Comparison of disease and economic burden between MRSA infection and MRSA colonization in a university hospital: a retrospective data integration study.

BACKGROUND: Although there is a growing concern and policy regarding infections or colonization caused by resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), the prognosis of MRSA infections compared to that of methicillin-susceptible Staphylococcus aureus (MSSA) infections remains controversial. Moreover, there have not been any studies comparing both the burden of disease and its impact on the healthcare economy between MRSA infection and colonization while adjusting for confounding factors. These comparisons are crucial for developing effective infection control measures and healthcare policies. We aimed to compare the disease and economic burden between MRSA and MSSA infections and between MRSA infection and colonization. METHODS: We retrospectively investigated data of 496 in-patients with MRSA or MSSA infections and of 1178 in-patients with MRSA infections or MRSA colonization from a university hospital in Japan from 2016 to 2021. We compared in-hospital mortality, length of stay, and hospital charges between in-patients with MRSA and MSSA infections and those with MRSA infections and MRSA colonization using multiple regressions. We combined surveillance data, including all microbiological test results, data on patients with infections, treatment histories, and clinical outcomes, to create the datasets. RESULTS: There was no statistically significant difference in in-hospital mortality rates between matched MRSA vs. MSSA infections and MRSA infection vs. colonization. On the contrary, the adjusted effects of the MRSA infection compared to those of MSSA infection on length of stay and hospital charges were 1.21-fold (95% confidence interval [CI] 1.03-1.42, P = 0.019) and 1.70-fold (95% CI 1.39-2.07, P < 0.00001), respectively. The adjusted effects of the MRSA infection compared to those of MRSA colonization on length of stay and hospital charges were 1.41-fold (95% CI 1.25-1.58, P < 0.00001) and 1.53-fold (95% CI 1.33-1.75, P < 0.00001), respectively. Regarding confounding factors, hemodialysis or hemofiltration was consistently identified and adjusted for in the multiple regression analyses comparing MRSA and MSSA infections, as well as MRSA infection and MRSA colonization. CONCLUSIONS: MRSA infection was associated with longer length of stay and higher hospital charges than both MSSA infection and MRSA colonization. Furthermore, hemodialysis or hemofiltration was identified as a common underlying factor contributing to increased length of stay and hospital charges.

Coordination of prophage and global regulator leads to high enterotoxin production in staphylococcal food poisoning-associated lineage.

Staphylococcus species in food produce Staphylococcal enterotoxins (SEs) that cause Staphylococcal food poisoning (SFP). More than 20 SE types have been reported, among which Staphylococcal enterotoxin A (SEA) has been recognized as one of the most important SEs associated with SFP. However, the regulatory mechanisms underlying its production remain unclear. Previously, we identified a major SFP clone in Japan, CC81 subtype-1, which exhibits high SEA production. In this study, we attempted to identify the factors contributing to this phenomenon. Thus, we demonstrated that the attenuation of the activity of endogenous regulator, Staphylococcal accessory regulator S (SarS), and the lysogenization of a high SEA-producing phage contributed to this phenomenon in CC81 subtype-1. Furthermore, our results indicated that SarS could directly bind to the promoter upstream of the sea gene and suppress SEA expression; this low SarS repression activity was identified as one of the reasons for the high SEA production observed. Therefore, we revealed that both exogenous and endogenous factors may probably contribute to the high SEA production. Our results confirmed that SE production is a fundamental and critical factor in SFP and clarified the associated production mechanism while enhancing our understanding as to why a specific clone frequently causes SFP. IMPORTANCE: The importance of this study lies in its unveiling of a molecular regulatory mechanism associated with the most important food poisoning toxin and the evolution of Staphylococcal food poisoning (SFP)-associated clone. SFP is primarily caused by Staphylococcus aureus, with Staphylococcal enterotoxin A (SEA) being commonly involved in many cases. Thus, SEA has been recognized as a major toxin type. However, despite almost a century since its discovery, the complete mechanism of SEA production is as yet unknown. In this study, we analyzed an SEA-producing SFP clone isolated in East Asia and discovered that this strain, besides acquiring the high SEA-producing phage, exhibits remarkably high SEA production due to the low activity of SarS, an intrinsic regulatory factor. This is the first report documenting the evolution of the SFP clone through the coordinated action of exogenous mobile genetic factors and endogenous regulators on this notorious toxin.

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.

Oral and rectal colonization of methicillin-resistant Staphylococcus aureus in long-term care facility residents and their association with clinical status.

Staphylococcus aureus is a commensal bacterium in humans, but it sometimes causes opportunistic infectious diseases such as suppurative skin disease, pneumonia, and enteritis. Therefore, it is important to determine the prevalence of S. aureus and methicillin-resistant S. aureus (MRSA) in individuals, especially older adults. In this study, we investigated the prevalence of S. aureus and MRSA in the oral cavity and feces of residents in long-term care facilities (LTCFs). S. aureus was isolated from the oral cavity of 61/178 (34.3%) participants, including 28 MRSA-positive participants (15.7%), and from the feces of 35/127 (27.6%) participants, including 16 MRSA-positive participants (12.6%). S. aureus and MRSA were isolated from both sites in 19/127 individuals (15.0%) and 10/127 individuals (7.9%), respectively. Among 19 participants with S. aureus isolation from both sites, 17 participants showed the same sequence type (ST) type. Then, we analyzed the correlation of S. aureus and MRSA in the oral cavity and rectum with the participant's condition. S. aureus and MRSA positivity in the oral cavity was significantly related to tube feeding, while there was no correlation of rectal S. aureus/MRSA with any factors. Our findings regarding the oral inhabitation of MRSA and its risk factors indicate the importance of considering countermeasures against MRSA infection in LTCFs.

Antimicrobial use and combination of resistance phenotypes in bacteraemic Escherichia coli in primary care: a study based on Japanese national data in 2018.

BACKGROUND: Antimicrobial use (AMU) in primary care is a contributing factor to the emergence of antimicrobial-resistant bacteria. We assessed the potential effects of AMU on the prevalence of a combination of resistance phenotypes in bacteraemic Escherichia coli in outpatient care settings between primary care facilities ('clinics') and hospitals. METHODS: Population-weighted total AMU calculated from the national database was expressed as DDDs per 1000 inhabitants per day (DID). National data for all routine microbiological test results were exported from the databases of a major commercial clinical laboratory, including 16 484 clinics, and the Japan Nosocomial Infections Surveillance, including 1947 hospitals. AMU and the prevalence of combinations of resistance phenotypes in bacteraemic E. coli isolates were compared between clinics and hospitals. RESULTS: The five most common bacteria isolated from patients with bacteraemia were the same in clinics, outpatient settings and inpatient settings in hospitals, with E. coli as the most frequent. Oral third-generation cephalosporins and fluoroquinolones were the top two AMU outpatient drugs, except for macrolides, and resulted in at least three times higher AMU in clinics than in hospitals. The percentage of E. coli isolates resistant to both drugs in clinics (18.7%) was 5.6% higher than that in hospitals (13.1%) (P < 10-8). CONCLUSIONS: Significant AMU, specifically of oral third-generation cephalosporins and fluoroquinolones, in clinics is associated with a higher prevalence of E. coli isolates resistant to both drugs. This study provides a basis for national interventions to reduce inappropriate AMU in primary care settings.

Isolation of Streptococcus mutans temperate bacteriophage with broad killing activity to S. mutans clinical isolates.

Bacteriophages are expected to be therapeutic agents against infectious diseases. Streptococcus mutans are involved in dental plaque formation related to dental caries and periodontitis. In S. mutans, lytic bacteriophages have been isolated previously, but the isolation of temperate bacteriophage has not been reported although their presence in the genome has been confirmed. Here, we report the isolation of temperate bacteriophage, φKSM96, from S. mutans. φKSM96 has a circular DNA 39,820 bp long and reveals Siphoviridae morphology. φKSM96 shows a broad range of susceptibility against S. mutans strains with different serotypes. By the addition of φKSM96, S. mutans growth and biofilm formation were significantly inhibited. In cocultures of S. mutans with other bacterial species, the proportion of S. mutans significantly decreased in the presence of φKSM96. In summary, φKSM96 shows selective anti-S. mutans activity. The isolation of temperate bacteriophage is important for future genetic manipulation to create more efficient bacteriophages.

National genomic surveillance integrating standardized quantitative susceptibility testing clarifies antimicrobial resistance in Enterobacterales.

Antimicrobial resistance is a global health concern; Enterobacterales resistant to third-generation cephalosporins (3GCs) and carbapenems are of the highest priority. Here, we conducted genome sequencing and standardized quantitative antimicrobial susceptibility testing of 4,195 isolates of Escherichia coli and Klebsiella pneumoniae resistant to 3GCs and Enterobacterales with reduced meropenem susceptibility collected across Japan. Our analyses provided a complete classification of 3GC resistance mechanisms. Analyses with complete reference plasmids revealed that among the blaCTX-M extended-spectrum ß-lactamase genes, blaCTX-M-8 was typically encoded in highly similar plasmids. The two major AmpC ß-lactamase genes were blaCMY-2 and blaDHA-1. Long-read sequencing of representative plasmids revealed that approximately 60% and 40% of blaCMY-2 and blaDHA-1 were encoded by such plasmids, respectively. Our analyses identified strains positive for carbapenemase genes but phenotypically susceptible to carbapenems and undetectable by standard antimicrobial susceptibility testing. Systematic long-read sequencing enabled reconstruction of 183 complete plasmid sequences encoding three major carbapenemase genes and elucidation of their geographical distribution stratified by replicon types and species carrying the plasmids and potential plasmid transfer events. Overall, we provide a blueprint for a national genomic surveillance study that integrates standardized quantitative antimicrobial susceptibility testing and characterizes resistance determinants.

Emergence of colistin-resistant Enterobacter cloacae and Raoultella ornithinolytica carrying the phosphoethanolamine transferase gene, mcr-9, derived from vegetables in Japan.

IMPORTANCE: Plasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice. Here, we described the genetic features of two mcr-9.1-carrying Gram-negative bacteria with a colistin-resistant phenotype derived from vegetables in Japan. The colistin-resistant mcr-9.1, which has never been detected in vegetables, was located on a large plasmid in Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1, suggesting a high chance of horizontal gene transfer. To the best of our knowledge, this is the first report of mcr-9 in R. ornithinolytica. This study indicates that fresh vegetables might be a potential source for the transmission of mcr-9 genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials. The study also provides additional consideration for colistin use and the relevance of routine surveillance in epidemiological perspective to curb the continuous spread of mcr alleles.

Genetic and phenotypic characterizations of IncX3 plasmids harboring bla NDM-5 and bla NDM-16b in Japan.

IMPORTANCE: IncX3 plasmids harboring bla NDM-5 play a major role in the spread of carbapenem resistance in Asia, particularly in China, in clinical and environmental settings. In this study, we present that Enterobacterales isolates carrying IncX3 plasmids harboring bla NDM-5 have been disseminated in Japan, where their identification was previously rare. In addition, bla NDM-16b, a single-nucleotide variant of bla NDM-5, was found to be carried by an identical IncX3 plasmid. A comparative sequence analysis revealed that the bla NDM-16b gene emerged from a single nucleotide substitution on an IncX3 plasmid harboring bla NDM-5. The bla NDM-16b gene did not confer elevated carbapenem resistance compared to bla NDM-5 in our assay using transformants carrying the plasmid harboring either of these genes, although the A233V substitution was reported to confer stability to the enzyme in ion-depleted conditions. Nevertheless, vigilance regarding the emergence of novel variants is required.

Municipal wastewater monitoring revealed the predominance of bla GES genes with diverse variants among carbapenemase-producing organisms: high occurrence and persistence of Aeromonas caviae harboring the new bla GES variant bla GES-48.

IMPORTANCE: The emergence and spread of carbapenemase-producing organisms (CPOs) represent a global health threat because they are associated with limited treatment options and poor clinical outcomes. Wastewater is considered a hotspot for the evolution and dissemination of antimicrobial resistance. Thus, analyses of municipal wastewater are critical for understanding the circulation of these CPOs and carbapenemase genes in local communities, which remains scarcely known in Japan. This study resulted in several key observations: (i) the vast majority of bla GES genes, including six new bla GES variants, and less frequent bla IMP genes were carbapenemase genes encountered exclusively in wastewater influent; (ii) the most dominant CPO species were Aeromonas spp., in which a remarkable diversity of new sequence types was observed; and (iii) CPOs were detected from combined sewer wastewater, but not from separate sewer wastewater, suggesting that the load of CPOs from unrecognized environmental sources could greatly contribute to their detection in influent wastewater.

Characterization of 29 newly isolated bacteriophages as a potential therapeutic agent against IMP-6-producing Klebsiella pneumoniae from clinical specimens.

Carbapenemase-producing Enterobacteriaceae (CPE) are one of the most detrimental species of antibiotic-resistant bacteria globally. Phage therapy has emerged as an effective strategy for the treatment of CPE infections. In western Japan, the rise of Klebsiella pneumoniae strains harboring the pKPI-6 plasmid encoding bla IMP-6 is of increasing concern. To address this challenge, we isolated 29 phages from Japanese sewage, specifically targeting 31 K. pneumoniae strains and one Escherichia coli strain harboring the pKPI-6 plasmid. Electron microscopy analysis revealed that among the 29 isolated phages, 21 (72.4%), 5 (17.2%), and 3 (10.3%) phages belonged to myovirus, siphovirus, and podovirus morphotypes, respectively. Host range analysis showed that 18 Slopekvirus strains within the isolated phages infected 25-26 K. pneumoniae strains, indicating that most of the isolated phages have a broad host range. Notably, K. pneumoniae strain Kp21 was exclusively susceptible to phage øKp_21, whereas Kp22 exhibited susceptibility to over 20 phages. Upon administering a phage cocktail composed of 10 phages, we observed delayed emergence of phage-resistant bacteria in Kp21 but not in Kp22. Intriguingly, phage-resistant Kp21 exhibited heightened sensitivity to other bacteriophages, indicating a "trade-off" for resistance to phage øKp_21. Our proposed phage set has an adequate number of phages to combat the K. pneumoniae strain prevalent in Japan, underscoring the potential of a well-designed phage cocktail in mitigating the occurrence of phage-resistant bacteria. IMPORTANCE The emergence of Klebsiella pneumoniae harboring the bla IMP-6 plasmid poses an escalating threat in Japan. In this study, we found 29 newly isolated bacteriophages that infect K. pneumoniae strains carrying the pKPI-6 plasmid from clinical settings in western Japan. Our phages exhibited a broad host range. We applied a phage cocktail treatment composed of 10 phages against two host strains, Kp21 and Kp22, which displayed varying phage susceptibility patterns. Although the phage cocktail delayed the emergence of phage-resistant Kp21, it was unable to hinder the emergence of phage-resistant Kp22. Moreover, the phage-resistant Kp21 became sensitive to other phages that were originally non-infective to the wild-type Kp21 strains. Our study highlights the potential of a well-tailored phage cocktail in reducing the occurrence of phage-resistant bacteria.

Comprehensive Genomic Characterization of Staphylococcus aureus Isolated from Atopic Dermatitis Patients in Japan: Correlations with Disease Severity, Eruption Type, and Anatomical Site.

Atopic dermatitis (AD) shows frequent recurrence. Staphylococcus aureus is the primary microbial component in AD and is associated with disease activity. However, traditional typing methods have failed to characterize virulent AD isolates at the clone level. We conducted a comprehensive genomic characterization of S. aureus strains isolated from the skin of AD patients and healthy donors, comparing the whole-genome sequences of the 261 isolates with anatomical and lesional (AD-A)/nonlesional (AD-NL)/healthy sites, eruption types, clinical scores, virulence, and antimicrobial resistance gene repertoires in Japan. Sequence type (ST) diversity was lost with worsening disease activity; ST188 was the most frequently detected ST in AD-A and had the strongest correlation with AD according to the culture rate and proportion with worsening disease activity. ST188 and ST20 isolates inhabited all skin conditions, with significantly higher proportions in AD skin than in healthy skin. ST8, ST15, and ST5 proportions were equivalent for all skin conditions; ST30 was detected only in healthy skin; and ST12 was detected only in AD skin. ST97 detected in AD-A and healthy skin was clearly branched into two subclades, designated ST97A and ST97H. A comparison of two genomes led to the discovery that only ST97A possessed the complete trp operon, enabling bacterial survival without exogenous tryptophan (Trp) on AD skin, where the Trp level was significantly reduced. Primary STs showing an AD skin inhabitation trend (ST188, ST97A, ST20, and ST12) were all trp operon positive. The predominant clones (ST188 and ST97) possessed almost no enterotoxin genes, no mecA gene, and few other antimicrobial resistance genes, different from the trend observed in Europe/North America. IMPORTANCE While Staphylococcus aureus is a member of the normal human skin flora, its strong association with the onset of atopic dermatitis (AD) has been suggested. However, previous studies failed to assign specific clones relevant to disease activities. Enterotoxins produced by S. aureus have been suggested to aggravate and exacerbate the inflammation of AD skin, but their role remains ambiguous. We conducted a nuanced comprehensive characterization of isolates from AD patients and healthy donors, comparing the whole-genome sequences of the isolates with anatomical and lesional/nonlesional/healthy sites, eruption types, clinical scores, virulence, and antimicrobial resistance gene repertoires in Japan. We demonstrate that specific clones are associated with disease severity and clinical manifestations, and the dominant clones are devoid of enterotoxin genes and antimicrobial resistance genes. These findings undermine the established notion of the pathophysiological function of S. aureus associated with AD and introduce a new concept of S. aureus colonization in AD.