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.

Oral and Rectal Colonization by Antimicrobial-Resistant Gram-Negative Bacteria and Their Association with Death among Residents of Long-Term Care Facilities: A Prospective, Multicenter, Observational, Cohort Study.

INTRODUCTION: The prevalence of antimicrobial-resistant bacteria (ARB) in long-term care facilities (LTCFs) remains unclear. Furthermore, the effect of ARB colonization on the clinical outcomes of LTCF residents has not been explored. METHODS: We conducted a prospective multicenter cohort study and investigated the residents (N = 178) of six Japanese LTCFs (three Welfare Facilities for the Elderly Requiring Long-term Care and three Geriatric Health Service Facilities) for oral and rectal carriage of ARB. The clinical outcomes of the residents were evaluated based on isolating bacterial strains and subjecting them to whole-genome sequencing. RESULTS: Of the 178 participants, 32 belonging to Geriatric Health Service Facilities with no information on their clinical outcome were excluded, and the remaining 146 were followed up for at most 21 months. Extended-spectrum ß-lactamases (ESBL)-producing Enterobacterales and Pseudomonas aeruginosa were detected in 42.7% (n = 76) and 2.8% (n = 5) of the rectal swabs and 5.6% (n = 10) and 3.4% (n = 6) of the oral swabs, respectively. Detection of ARB in the oral and rectal cavities showed remarkable association with enteral nutrition. Further, P. aeruginosa was significantly associated with an increase in mortality of the residents, but there were not significant association between ESBL-producing Enterobacterales and mortality. Core-genome phylogeny of P. aeruginosa revealed a wide-spread distribution of the isolated strains across the phylogeny, which included a cluster of ST235 strains with substantially higher biofilm formation ability than the other isolated P. aeruginosa strains. DISCUSSION/CONCLUSION: This study is the first to investigate the carriage of both oral and rectal ARB, genomic relatedness and determinants of antimicrobial resistance in isolated strains, and clinical outcomes of LTCF residents. Our study provides the first direct evidence for the burden of antimicrobial resistance in LTCFs.

In Vitro Activity and Clinical Efficacy of Faropenem against Third-Generation Cephalosporin-Resistant Escherichia coli and Klebsiella pneumoniae.

Faropenem (FRPM) is active against extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales, but evidence for its efficacy is lacking. This study determined the correlation between the susceptibility by disk diffusion method and the MIC of FRPM for third-generation cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae, and the effectiveness of FRPM for the treatment of urinary tract infection (UTI) caused by these two bacteria in a retrospective cohort analysis. Of the 48 third-generation cephalosporin-resistant clinical isolates tested, 44 isolates produced ESBL, and 8 isolates produced AmpC, including 4 isolates produced both ESBL and AmpC. Thirty-seven isolates had an FRPM MIC of ≤1 mg/L, and seven had an FRPM MIC of 2 mg/L. An FRPM MIC of >2 mg/L was observed with four isolates. In a retrospective cohort analysis, 63 patients with UTI treated with FRPM were identified. All isolates of ESBL-producing E. coli (n = 54) and K. pneumoniae (n = 9) treated with FRPM showed disk diffusion zone diameters larger than 16.0 mm (estimated MIC, 2.2 mg/L). All patients completed the scheduled treatment courses with FRPM, but 28- and 90-day relapses happened in 10 patients (16%) and 16 patients (25%), respectively. No significant risk factors for the 28- and 90-day relapses were found. FRPM can be used according to disk diffusion susceptibility testing in UTI. Further investigations are necessary to assess the clinical breakpoint of FRPM for ESBL-producing Enterobacterales and the candidates most likely to benefit from using FRPM.

PCR-based ORF typing of Klebsiella pneumoniae for rapid identification of global clones and transmission events.

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.

Vegetable-Derived Carbapenemase-Producing High-Risk Klebsiella pneumoniae ST15 and Acinetobacter baumannii ST2 Clones in Japan: Coexistence of blaNDM-1, blaOXA-66, blaOXA-72, and an AbaR4-Like Resistance Island in the Same Sample.

This study was conducted to characterize carbapenemase-producing Klebsiella pneumoniae and Acinetobacter baumannii isolated from fresh vegetables in Japan. Two K. pneumoniae isolates (AO15 and AO22) and one A. baumannii isolate (AO22) were collected from vegetables in the city of Higashihiroshima, Japan, and subjected to antimicrobial susceptibility testing, conjugation experiments, and complete genome sequencing using Illumina MiniSeq and Oxford Nanopore MinION sequencing platforms. The two K. pneumoniae isolates were clonal, belonging to sequence type 15 (ST15), and were determined to carry 19 different antimicrobial resistance genes, including blaNDM-1 Both the isolates carried blaNDM-1 on a self-transmissible IncFII(K):IncR plasmid of 122,804 bp with other genes conferring resistance to aminoglycosides [aac(6')-Ib, aadA1, and aph(3')-VI], ß-lactams (blaCTX-M-15, blaOXA-9, and blaTEM-1A), fluoroquinolones [aac(6')-Ib-cr], and quinolones (qnrS1). A. baumannii AO22 carried blaOXA-66 on the chromosome, while blaOXA-72 was found as two copies on a GR2-type plasmid of 10,880 bp. Interestingly, A. baumannii AO22 harbored an AbaR4-like genomic resistance island (GI) of 41,665 bp carrying genes conferring resistance to tetracycline [tet(B)], sulfonamides (sul2), and streptomycin (strAB). Here, we identified Japanese carbapenemase-producing Gram-negative bacteria isolated from vegetables, posing a food safety issue and a public health concern. Additionally, we reported a GR2-type plasmid carrying two copies of blaOXA-72 and an AbaR4-like resistance island from a foodborne A. baumannii isolate.IMPORTANCE Carbapenemase-producing Gram-negative bacteria (CPGNB) cause severe health care-associated infections and constitute a major public health threat. Here, we investigated the genetic features of CPGNB isolated from fresh vegetable samples in Japan and found CPGNB, including Klebsiella pneumoniae and Acinetobacter baumannii, with dissimilar carbapenemases. The NDM carbapenemase, rarely described in Japan, was detected in two K. pneumoniae isolates. The A. baumannii isolate identified in this study carried blaOXA-66 on the chromosome, while blaOXA-72 was found as two copies on a GR2-type plasmid. This study indicates that even one fresh ready-to-eat vegetable sample might serve as a significant source of genes (blaNDM-1, blaOXA-72, blaCTX-M-14b, and blaCTX-M-15) encoding resistance to frontline and clinically important antibiotics (carbapenems and cephalosporins). Furthermore, the detection of these organisms in fresh vegetables in Japan is alarming and poses a food safety issue and a public health concern.

Aeromonas dhakensis is not a rare cause of Aeromonas bacteremia in Hiroshima, Japan.

Aeromonas dhakensis, a newly recognized species, is often misidentified as A. hydrophila, A. veronii, or A. caviae by commercial phenotypic tests. Limited data about A. dhakensis are available in Japan. We retrospectively analyzed the patients with monomicrobial Aeromonas bacteremia at Hiroshima University Hospital from January 2011 to December 2017, and species re-identification was conducted using rpoD and gyrB gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. Of the 19 strains from blood isolates, A. caviae (n = 9, 47.4%), A. dhakensis (n = 4, 21.1%), A. hydrophila (n = 3, 15.8%), and A. veronii (n = 3, 15.8%) were re-identified. A. dhakensis was phenotypically misidentified as A. hydrophila (n = 3, 75%) or A. sobria (n = 1, 25%). A. dhakensis was also misidentified as A. caviae (n = 2, 50%), A. hydrophila (n = 1, 25%), and A. jandaei (n = 1, 25%) in MALDI-TOF MS system. Malignancies (n = 12, 63.2%) and liver cirrhosis (n = 7, 36.8%) were common comorbidities. Biliary tract infection was the most frequent source of Aeromonas bacteremia (n = 11, 57.9%). The major source of A. dhakensis bacteremia was also biliary tract infection (n = 3, 75%), and the 14-day infection-related mortality of A. dhakensis was 25%. A. dhakensis isolates showed similar clinical characteristics, antimicrobial susceptibility, and mortality with those of other Aeromonas species isolates. This study demonstrated that A. dhakensis is not a rare cause of Aeromonas bacteremia, but is often misidentified as A. hydrophila in Hiroshima, Japan. Further studies should be conducted to identify the geographical distribution and clinical impact of A. dhakensis in Japan.

Complete Nucleotide Sequence of pKOI-34, an IncL/M Plasmid Carrying blaIMP-34 in Klebsiella oxytoca Isolated in Japan.

We determined the complete nucleotide sequence of a self-transmissible IncL/M plasmid, pKOI-34, from a Klebsiella oxytoca isolate. pKOI-34 possessed the core structure of an IncL/M plasmid found in Erwinia amylovora, pEL60, with two mobile elements inserted, a transposon carrying the arsenic resistance operon and a Tn21-like core module (tnp and mer modules) piggybacking blaIMP-34 as a class 1 integron, In808, where blaIMP-34 confers a resistance to carbapenems in K. oxytoca and Klebsiella pneumoniae.

Inhibitory effects of antibiofilm compound 1 against Staphylococcus aureus biofilms.

A novel benzimidazole molecule that was identified in a small-molecule screen and is known as antibiofilm compound 1 (ABC-1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 µM ABC-1 was tested in various biofilm-forming strains of S. aureus. It was demonstrated that ABC-1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall-associated protein dependent or cell wall- associated extracellular DNA (eDNA). Of note, ABC-1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC-1 treated strains, implying that ABC-1 affects not only SpA but also other factors. Indeed, ABC-1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC-1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.

Complete nucleotide sequence of the IncN plasmid encoding IMP-6 and CTX-M-2 from emerging carbapenem-resistant Enterobacteriaceae in Japan.

We have determined the DNA sequence of Klebsiella pneumoniae multidrug resistance plasmid pKPI-6, which is a self-transmissible IncN-type plasmid. pKPI-6 harboring blaIMP-6 and blaCTX-M-2 confers a stealth-type carbapenem resistance phenotype on members of the family Enterobacteriaceae that is not detectable with imipenem. pKPI-6 is already epidemic in Japan, favoring the dissemination of IMP-6 and CTX-M-2 in members of the family Enterobacteriaceae.

Persistence and epidemic propagation of a Pseudomonas aeruginosa sequence type 235 clone harboring an IS26 composite transposon carrying the blaIMP-1 integron in Hiroshima, Japan, 2005 to 2012.

A 9-year surveillance for multidrug-resistant (MDR) Pseudomonas aeruginosa in the Hiroshima region showed that the number of isolates harboring the metallo-ß-lactamase gene bla(IMP-1) abruptly increased after 2004, recorded the highest peak in 2006, and showed a tendency to decline afterwards, indicating a history of an epidemic. PCR mapping of the variable regions of the integrons showed that this epidemic was caused by the clonal persistence and propagation of an MDR P. aeruginosa strain harboring the bla(IMP-1) gene and an aminoglycoside 6'-N-acetyltransferase gene, aac(6')-Iae in a class I integron (In113), whose integrase gene intl1 was disrupted by an IS26 insertion. Sequence analysis of the representative strain PA058447 resistance element containing the In113-derived gene cassette array showed that the element forms an IS26 transposon embedded in the chromosome. It has a Tn21 backbone and is composed of two segments sandwiched by three IS26s. In Japan, clonal nationwide expansion of an MDR P. aeruginosa NCGM2.S1 harboring chromosomally encoded In113 with intact intl1 is reported. Multilocus sequence typing and genomic comparison strongly suggest that PA058447 and NCGM2.S1 belong to the same clonal lineage. Moreover, the structures of the resistance element in the two strains are very similar, but the sites of insertion into the chromosome are different. Based on tagging information of the IS26 present in both resistance elements, we suggest that the MDR P. aeruginosa clone causing the epidemic in Hiroshima for the past 9 years originated from a common ancestor genome of PA058447 and NCGM2.S1 through an IS26 insertion into intl1 of In113 and through IS26-mediated genomic rearrangements.

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.

In vitro activity of cefiderocol against carbapenemase-producing and meropenem-non-susceptible Gram-negative bacteria collected in the Japan Antimicrobial Resistant Bacterial Surveillance.

OBJECTIVES: The treatment options available for infections caused by multidrug-resistant Gram-negative pathogens are often limited. Cefiderocol (CFDC) is a novel siderophore cephalosporin that exhibits activity against these pathogens. Several studies have reported the in vitro activity of CFDC against isolates from Europe, the United States, and China, but the activity against carbapenem-resistant bacteria with IMP-type carbapenemase has not been extensively studied. We, therefore, studied the in vitro activities of CFDC against carbapenem-resistant bacteria with available genomic backgrounds based on whole-genome sequencing (WGS) in Japan, where the IMP-type is the predominant carbapenemase produced by Gram-negative rods. METHODS: We selected 603 isolates (528 Enterobacterales, 18 Pseudomonas aeruginosa, and 57 Acinetobacter spp.) from a collection of Gram-negative clinical isolates collected during a Japan Antimicrobial Resistance Bacterial Surveillance program and evaluated the antimicrobial activities of CFDC, ceftolozane/tazobactam (CTLZ/TAZ), imipenem-relebactam (IPM/REL), and ceftazidime/avibactam (CAZ/AVI) against carbapenemase-producing Enterobacterales, carbapenemase-non-producing meropenem-non-susceptible Enterobacterales, and carbapenemase-producing nonfermentative bacteria. RESULTS: Among these, 97.7% of carbapenemase-producing Enterobacterales (99.2% of IMP-type carbapenemase-producing Enterobacterales), 100% of carbapenemase-producing P. aeruginosa, and 91.2% of carbapenemase-producing Acinetobacter spp. were susceptible to CFDC, showing better antimicrobial activity than the other antimicrobial agents evaluated in this study. CFDC was highly effective against class A-, B-, and D ß-lactamase-harbouring isolates when compared to the other antimicrobial agents. In addition, the relationship between CFDC resistance and three genetic factors involved in resistance was discussed. CONCLUSIONS: This is the first large-scale study to systematically demonstrate the efficacy of CFDC against IMP-type carbapenemase-producing strains with known genomic backgrounds.

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.

Emergence of Staphylococcus aureus carrying multiple drug resistance genes on a plasmid encoding exfoliative toxin B.

We report the complete nucleotide sequence and analysis of pETBTY825, a Staphylococcus aureus TY825 plasmid encoding exfoliative toxin B (ETB). S. aureus TY825 is a clinical isolate obtained from an impetigo patient in 2002. The size of pETBTY825, 60.6 kbp, was unexpectedly larger than that of the archetype pETBTY4 (∼30 kbp). Genomic comparison of the plasmids shows that pETBTY825 has the archetype pETBTY4 as the backbone and has a single large extra DNA region of 22.4 kbp. The extra DNA region contains genes for resistance to aminoglycoside [aac(6')/aph(2″)], macrolide (msrA), and penicillin (blaZ). A plasmid deletion experiment indicated that these three resistance elements were functionally active. We retrospectively examined the resistance profile of the clinical ETB-producing S. aureus strains isolated in 1977 to 2007 using a MIC determination with gentamicin (GM), arbekacin (ABK), and erythromycin (EM) and by PCR analyses for aac(6')/aph(2″) and msrA using purified plasmid preparations. The ETB-producing S. aureus strains began to display high resistance to GM, which was parallel with the detection of aac(6')/aph(2″) and mecA, after 1990. Conversely, there was no significant change in the ABK MIC during the testing period, although it had a tendency to slightly increase. After 2001, isolates resistant to EM significantly increased; however, msrA was hardly detected in ETB-producing S. aureus strains, and only five isolates were positive for both aac(6')/aph(2″) and msrA. In this study, we report the emergence of a fusion plasmid carrying the toxin gene etb and drug resistance genes. Prevalence of the pETBTY825 carrier may further increase the clinical threat, since ETB-producing S. aureus is closely related to more severe impetigo or staphylococcal scalded-skin syndrome (SSSS), which requires a general antimicrobial treatment.

Meropenem resistance in imipenem-susceptible meropenem-resistant Klebsiella pneumoniae isolates not detected by rapid automated testing systems.

Klebsiella pneumoniae showing high resistance to all ß-lactams except imipenem, designated as ISMRK (imipenem-susceptible meropenem-resistant Klebsiella) is emerging in Japan. The carbapenem resistance of ISMRK cannot be screened by the Vitek and the RAISUS rapid automated susceptibility test systems, which may lead to inappropriate antimicrobial therapy, resulting in compromised patient outcomes.

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.

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 Analysis of Bacteriocins Produced by the Hypermucoviscous Klebsiella pneumoniae Species Complex.

Klebsiella pneumoniae produces several kinds of bacteriocins that have antimicrobial effects against closely related species, but few studies have comprehensively reported bacteriocin distribution among the Klebsiella population. In this study, we identified bacteriocin genes in 180 K. pneumoniae species complex genomes, including 170 hypermucoviscous isolates, and investigated the antibacterial activity against 50 strains, including antimicrobial-resistant organisms, belonging to multiple species, namely, Klebsiella spp., Escherichia coli, Pseudomonas spp., Acinetobacter spp., Enterobacter cloacae, Stenotrophomonas maltophilia, Chryseobacterium indologenes, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus mutans. Our study determined that 32.8% (59/180) of isolates carried at least one bacteriocin type. Different types of bacteriocin were usually present in different specific sequence types (STs); meanwhile, bacteriocins were not detected in certain STs. Microcin E492 was the most prevalent bacteriocin (14.4%), mostly in ST23 isolates, and displayed a wide spectrum of activity, including against Klebsiella spp., E. coli, Pseudomonas spp., and Acinetobacter spp. Cloacin-like bacteriocin was detected in 7.2% of strains, all of which were non-ST23 isolates, and exhibited inhibitory activity against closely related species, mainly Klebsiella spp. Klebicin B-like bacteriocin was detected at a rate of 9.4%, although 82.4% of these strains carried a disrupted bacteriocin gene, and an inhibitory effect could not be observed from the intact-gene-carrying isolates. Other bacteriocins, such as microcin S-like, microcin B17, and klebicin C-like, were detected at lower rates and had limited inhibitory activity. Our findings suggested that Klebsiella strains that carry different bacteriocin types may affect the composition of the surrounding bacterial community. IMPORTANCE Klebsiella pneumoniae is a Gram-negative commensal bacterium that asymptomatically colonizes human mucosal membranes, such as the intestinal tract, but it is also a leading cause of health care- and community-associated infections. Additionally, multidrug-resistant K. pneumoniae has been continuously evolving, which significantly challenges the available chemotherapeutic treatment for its infections. K. pneumoniae produces several kinds of antimicrobial peptides known as bacteriocins, which have antibacterial activity against closely related species. This work was the first comprehensive report of bacteriocin distribution among the hypermucoviscous K. pneumoniae species complex population and the inhibitory activity of each bacteriocin type against various species, including multidrug-resistant strains. Our findings provide a foundation for future studies on the K. pneumoniae species complex, including studies on the competition within the microflora and the potential applications of bacteriocins in treating multidrug-resistant bacteria.