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Background: The transmission of carbapenemase-producing Enterobacterales (CPE) in the external environment, especially through food, presents a significant public health risk. Objectives: To investigate the prevalence and genetic characteristics of CPE in food markets of Dhaka, Bangladesh, using WGS. Methods: CPE isolates were obtained from different food and water samples collected from food markets in the southern part of Dhaka, Bangladesh. The isolates subsequently underwent molecular typing, WGS employing both short- and long-read sequencers, and plasmid analysis. Results: This study unveiled an extensive spread of CPE, with no significant difference in contamination rates observed in samples (Nâ=â136), including meat (nâ=â8), fish (nâ=â5), vegetables (nâ=â36) or various food-washed water (nâ=â65) from markets near hospitals or residential areas. Thirty-eight Enterobacterales from 33 samples carried carbapenemase genes (bla NDM-1, -4, -7, bla KPC-2, bla OXA-181 or bla IMI-1). Among these, the high-risk Escherichia coli ST410 clone was the most prevalent and distributed across various locations. Furthermore, the identification of IncHI2 plasmids co-harbouring resistance genes like bla NDM-5 and mcr-1.1, without discernible epidemiological connections, is a unique finding, suggesting their widespread dissemination. Conclusions: The analysis unveils a dynamic landscape of CPE dissemination in food markets, underscored by the proliferation of novel IncHI2 hybrid plasmids carrying both colistin- and carbapenem-resistance genes. This illuminates the ever-evolving landscape of antimicrobial resistance in Dhaka, urging us to confront its emergent challenges.
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Introduction. Maternal screening tests and prophylactic antibiotics are important to prevent neonatal and infant group B streptococcal (GBS) infections.Hypothesis/Gap Statement. The performance of enrichment broth media for GBS screening that are available in Japan is unclear. Whole-genome data of GBS isolates from pregnant women in Japan is lacking.Aim. The aim of this study was to compare the protocol performance of six enrichment broths and two subculture agar plates, which were all available in Japan, for GBS detection. In addition, we showed whole-genome data of GBS isolates from pregnant women in Japan.Methodology. We collected 133 vaginal-rectal swabs from pregnant women visiting clinics and hospitals in Nagasaki Prefecture, Japan, and compared the protocol performance of 6 enrichment broths and 2 subculture agar plates. All GBS isolates collected in this study were subjected to whole-genome sequencing analysis.Results. We obtained 133 vaginal-rectal swabs from pregnant women at 35-37 weeks of gestation from 8 private clinics and 2 local municipal hospitals within Nagasaki Prefecture, Japan. The detection rate of the protocol involving the six enrichment broths and subsequent subcultures varied between 95.5 and 100â%, depending on the specific choice of enrichment broth. The GBS carriage rate among pregnant women in this region was 18.8â%. All 25 isolates derived from the swabs were susceptible to penicillin, whereas 48 and 36â% of the isolates demonstrated resistance to erythromycin and clindamycin, respectively. The distribution of serotypes was highly diverse, encompassing seven distinct serotypes among the isolates, with the predominant serotype being serotype V (n = 8). Serotype V isolates displayed a tendency towards increased resistance to erythromycin and clindamycin, with all resistant isolates containing the ermB gene.Conclusion. There was no difference in performance among the culture protocols evaluated in this study. GBS strains isolated from pregnant women appeared to have greater genomic diversity than GBS strains detected in neonates/infants with invasive GBS infections. To confirm this result, further studies with larger sample sizes are needed.
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Antibacterianos , Infecções Estreptocócicas , Streptococcus agalactiae , Vagina , Humanos , Streptococcus agalactiae/genética , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/isolamento & purificação , Streptococcus agalactiae/classificação , Feminino , Gravidez , Japão/epidemiologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/epidemiologia , Antibacterianos/farmacologia , Vagina/microbiologia , Meios de Cultura/química , Complicações Infecciosas na Gravidez/microbiologia , Complicações Infecciosas na Gravidez/epidemiologia , Reto/microbiologia , Testes de Sensibilidade Microbiana , Sequenciamento Completo do Genoma , Adulto , Clindamicina/farmacologia , Genoma BacterianoRESUMO
To prevent nosocomial infection, it is important to screen for potential vancomycin-resistant Enterococcus (VRE) among patients. In this study, we analyzed enterococcal isolates from inpatients in one hospital without any apparent outbreak of VRE. Enterococcal isolates were collected from inpatients at Hiroshima University Hospital from April 1 to June 30, 2021 using selective medium for Enterococci. Multilocus sequence typing, antimicrobial susceptibility testing, and whole-genome sequencing were performed. A total of 164 isolates, including Enterococcus faecium (41 isolates), Enterococcus faecalis (80 isolates), Enterococcus raffinosus (11 isolates), Enterococcus casseliflavus (nine isolates), Enterococcus avium (12 isolates), Enterococcus lactis (eight isolates), Enterococcus gallinarum (two isolates), and Enterococcus malodoratus (one isolate), were analyzed. We found one vanA-positive E. faecium, which was already informed when the patient was transferred to the hospital, nine vanC-positive E. casseliflavus, and two vanC-positive E. gallinarum. E. faecium isolates showed resistance to ampicillin (95.1%), imipenem (95.1%), and levofloxacin (87.8%), and E. faecalis isolates showed resistance to minocycline (49.4%). Ampicillin- and levofloxacin-resistant E. faecium had multiple mutations in penicillin-binding protein 5 (PBP5) (39/39 isolates) and ParC/GyrA (21/36 isolates), respectively. E. raffinosus showed resistance to ampicillin (81.8%), imipenem (45.5%), and levofloxacin (45.5%), and E. lactis showed resistance to ampicillin (37.5%) and imipenem (50.0%). The linezolid resistance genes optrA and cfr(B) were found only in one isolate of E. faecalis and E. raffinosus, respectively. This study, showing the status of enterococci infection in hospitalized patients, is one of the important information when considering nosocomial infection control of VRE.
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Antibacterianos , Infecções por Bactérias Gram-Positivas , Testes de Sensibilidade Microbiana , Sequenciamento Completo do Genoma , Humanos , Japão/epidemiologia , Antibacterianos/farmacologia , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/epidemiologia , Pacientes Internados , Infecção Hospitalar/microbiologia , Infecção Hospitalar/epidemiologia , Genoma Bacteriano/genética , Enterococos Resistentes à Vancomicina/genética , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/isolamento & purificação , Tipagem de Sequências Multilocus , Surtos de Doenças , Enterococcus/genética , Enterococcus/efeitos dos fármacos , Enterococcus/isolamento & purificação , Enterococcus/classificação , Vancomicina/farmacologiaRESUMO
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.
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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.
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The emergence of drug-resistant bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), has increased the need to discover novel antimicrobial agents that are effective against these species. Here, we describe the identification and purification of the mutacin BHT-B-like gene locus and bacteriocin peptide from Streptococcus ursoris, which is closely related to Streptococcus ratti; hence, we named this bacteriocin ursoricin. Ursoricin is a cationic, chromosome-encoded peptide that has potent antimicrobial effects against Gram-positive pathogens, including MRSA and VRE, with minimum inhibitory concentrations in the micromolar range. Ursoricin also inhibits the biofilm formation of high biofilm-forming S. aureus. Antibacterial activity was retained after treatment at 100°C for 60 min at a pH range of 3-9 and was partially reduced by treatment with proteinase K for 2 h (63% residual activity). The potent anti-MRSA, anti-VRE, and antibiofilm effects of ursoricin suggest that it is a possible candidate for the treatment of MRSA, VRE, and biofilm-associated infections. IMPORTANCE: The emergence of multidrug-resistant bacteria worldwide has posed a significant public health threat and economic burdens that make the identification and development of novel antimicrobial agents urgent. Bacteriocins are promising new agents that exhibit antibacterial activity against a wide range of human pathogens. In this study, we report that the bacteriocin produced by Streptococcus ursoris showed good antibacterial activity against a wide range of Staphylococcus aureus and enterococcus strains, particularly methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and high biofilm-forming S. aureus. Interestingly, this bacteriocin had a stronger effect on S. aureus than on Staphylococcus epidermidis, which is a major commensal bacterium in human skin; this result is important when considering the disturbance of bacterial flora, especially on the skin, mediated by the application of antibacterial agents.
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Antibacterianos , Bacteriocinas , Biofilmes , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Streptococcus , Enterococos Resistentes à Vancomicina , Bacteriocinas/farmacologia , Bacteriocinas/genética , Antibacterianos/farmacologia , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Streptococcus/efeitos dos fármacosRESUMO
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.
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Antibacterianos , Proteínas de Bactérias , Carbapenêmicos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Japão/epidemiologia , Carbapenêmicos/farmacologia , Antibacterianos/farmacologia , Humanos , Proteínas de Bactérias/genética , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/tratamento farmacológico , beta-Lactamases/genética , Genoma Bacteriano/genética , Combinação Piperacilina e Tazobactam/uso terapêutico , Combinação Piperacilina e Tazobactam/farmacologia , Sequenciamento Completo do Genoma , Meropeném/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Amicacina/farmacologiaRESUMO
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.
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Surtos de Doenças , Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Tipagem de Sequências Multilocus , Filogenia , Plasmídeos , Enterococos Resistentes à Vancomicina , Sequenciamento Completo do Genoma , Enterococcus faecium/genética , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/isolamento & purificação , Japão/epidemiologia , Humanos , Enterococos Resistentes à Vancomicina/genética , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/isolamento & purificação , Plasmídeos/genética , Infecções por Bactérias Gram-Positivas/transmissão , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecção Hospitalar/microbiologia , Infecção Hospitalar/transmissão , Infecção Hospitalar/epidemiologia , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Carbono-Oxigênio Ligases/genética , Testes de Sensibilidade Microbiana , Polimorfismo de Nucleotídeo Único , Hospitais , Vancomicina/farmacologia , Genoma Bacteriano/genéticaRESUMO
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.
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Carbapenêmicos , Escherichia coli , Ralstonia , Humanos , Idoso , Carbapenêmicos/farmacologia , Carbapenêmicos/uso terapêutico , Meropeném , Doripenem , Escherichia coli/genética , Escherichia coli/metabolismo , Japão , beta-Lactamases/genética , beta-Lactamases/metabolismo , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Imipenem , Testes de Sensibilidade MicrobianaRESUMO
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.
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Antibacterianos , Farmacorresistência Bacteriana , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Proteínas de Bactérias/genética , beta-Lactamases/genética , Escherichia coli , Plasmídeos/genética , Genômica , Carbapenêmicos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
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.
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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.
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Colistina , Enterobacter cloacae , Colistina/farmacologia , Enterobacter cloacae/genética , Antibacterianos/farmacologia , Verduras/microbiologia , Japão , Farmacorresistência Bacteriana/genética , Plasmídeos/genética , Transferases/genética , Testes de Sensibilidade MicrobianaRESUMO
Background: Escherichia coli is the most common cause of urinary tract infections and has fluoroquinolone (FQ)-resistant strains, which are a worldwide concern. Objectives: To characterize FQ-resistant determinants among 103 carbapenem-resistant E. coli (CREc) urinary isolates using WGS. Methods: Antimicrobial susceptibility, biofilm formation, and short-read sequencing were applied to these isolates. Complete genome sequencing of five CREcs was conducted using short- and long-read platforms. Results: ST410 (50.49%) was the predominant ST, followed by ST405 (12.62%) and ST361 (11.65%). Clermont phylogroup C (54.37%) was the most frequent. The genes NDM-5 (74.76%) and CTX-M-15 (71.84%) were the most identified. Most CREcs were resistant to ciprofloxacin (97.09%) and levofloxacin (94.17%), whereas their resistance rate to nitrofurantoin was 33.98%. Frequently, the gene aac(6')-Ib (57.28%) was found and the coexistence of aac(6')-Ib and blaCTX-M-15 was the most widely predominant. All isolates carried the gyrA mutants of S83L and D87N. In 12.62% of the isolates, the coexistence was detected of gyrA, gyrB, parC, and parE mutations. Furthermore, the five urinary CREc-complete genomes revealed that blaNDM-5 or blaNDM-3 were located on two plasmid Inc types, comprising IncFI (60%, 3/5) and IncFI/IncQ (40%, 2/5). In addition, both plasmid types carried other resistance genes, such as blaOXA-1, blaCTX-M-15, blaTEM-1B, and aac(6')-Ib. Notably, the IncFI plasmid in one isolate carried three copies of the blaNDM-5 gene. Conclusions: This study showed FQ-resistant determinants in urinary CREc isolates that could be a warning sign to adopt efficient strategies or new control policies to prevent further spread and to help in monitoring this microorganism.
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Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Escherichia coli , Humanos , Escherichia coli/genética , Fluoroquinolonas/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Tailândia/epidemiologia , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , beta-Lactamases/genéticaRESUMO
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.
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Carbapenêmicos , beta-Lactamases , beta-Lactamases/genética , Japão , Plasmídeos/genética , Carbapenêmicos/farmacologia , NucleotídeosRESUMO
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.
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Aeromonas caviae , Aeromonas caviae/genética , Águas Residuárias , beta-Lactamases/genética , Proteínas de Bactérias/genética , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologiaRESUMO
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.
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Introduction. Carbapenemase-producing Enterobacteriaceae (CPE) have emerged as a global threat to public health and clinical practice.Hypothesis/Gap Statement. In Thailand, reports describing CPEs carrying bla NDM and bla OXA-48-like genes have been increasing recently; however, data on detailed plasmid analysis and temporal shift of sequence type and carbapenemase type are limited.Aim. In this study, we analysed whole-genome sequencing (WGS) data of clinically isolated carbapenemase-producing Klebsiella pneumoniae (CPKP) to reveal the molecular epidemiology of CPKP in a tertiary-care hospital in Bangkok, Thailand.Methodology. Seventy-seven non-duplicated CPKP isolates collected during 2013-2016 were examined for their drug-resistance genes, sequence types and phylogenetic relationships.Results. All the tested isolates possessed carbapenemase gene(s), and the major type of carbapenemase gene in 2014-2015 was bla NDM-1, whereas isolates in 2016 harboured more bla OXA-232 than bla NDM-1. Other carbapenemase gene variants, such as bla NDM-4, bla NDM-5, bla OXA-48, bla OXA-181 and bla IMP-14 were detected in some CPKP isolates. Furthermore, this study revealed that CPKP co-harbouring two genes, bla NDM-1 and bla OXA-232 or bla OXA-181, emerged during this period. Notably, such isolates co-carrying the two carbapenemase genes emerged in three different sequence types, even in a single hospital, and then spread clonally. The WGS of CPKP revealed a temporal shift of the predominant carbapenemase genes from bla NDM-1 to bla OXA-232 along with a variation in other carbapenemase gene types within a span of 4 years.Conclusion. Our findings suggest that a substantial change in CPE types occurred in Thailand and potentially in Southeast Asian countries.
Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Enterobacteriaceae , Humanos , Klebsiella pneumoniae/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Tailândia/epidemiologia , Filogenia , Infecções por Enterobacteriaceae/epidemiologia , beta-Lactamases/genética , Proteínas de Bactérias/genética , Testes de Sensibilidade MicrobianaRESUMO
OBJECTIVES: Linezolid is an antibiotic used to treat infectious diseases caused by vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. Recently, Enterococcus Spp.-carrying mobile linezolid resistance genes were reported. Herein, we report the complete genome sequence of Enterococcus raffinosus JARB-HU0741, which was isolated from a bile sample of a patient in Japan on May 5, 2021, and carries a linezolid resistance gene, cfr(B). Nevertheless, this isolate was susceptible to linezolid. METHODS: Whole-genome sequencing was performed using HiSeq X FIVE (Illumina) and GridION (Oxford Nanopore Technologies). The sequence reads were assembled using Unicycler v0.4.8, and the complete genome was annotated using DFAST v1.2.18. Antimicrobial resistance genes were detected with Abricate v1.0.1, using the ResFinder database. The minimum inhibitory concentrations (MICs) were determined using broth microdilution and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute. RESULTS: E. raffinosus JARB-HU0741 contained a 3 248 808-bp chromosome and a 1 156 277-bp megaplasmid. cfr(B) was present in the Tn6218-like transposon, which was inserted into a gene encoding a PRD domain-containing protein present in the megaplasmid, but the isolate was susceptible to linezolid (MIC, 0.5 µg/mL). The Tn6218-like transposon was similar to the Tn6218 of Clostridioides difficile Ox3196 and the Tn6218-like transposon of Enterococcus faecium UW11733; however, three genes encoding a topoisomerase, an S-adenosylmethionine-dependent methyltransferase, and a TetR family transcriptional regulator were present in the previous Tn6218- or Tn6218-like transposon. CONCLUSION: This is the first report of the complete genome sequence of E. raffinosus carrying cfr(B). E. raffinosus carrying cfr(B) without linezolid resistance poses a threat, as it could serve as a reservoir for mobile linezolid resistance genes.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Humanos , Linezolida/farmacologia , Japão , Bile , Enterococcus/genéticaRESUMO
Epidemiological surveys have shown that carbapenem resistance is mainly transmitted across species by carbapenemase genes located on conjugative plasmids. As chromosomal integration of carbapenemase genes has rarely been identified, only a few studies have investigated their advantages to the carbapenem-resistant bacterial community. Here, we confirmed the increased stability of blaIMP-6 on a chromosome-integrated plasmid in an Escherichia coli isolate compared with that on original plasmids in the absence of antibiotic pressure. Although plasmids carrying carbapenemase genes are supposedly lost in successive generations, we found that the complete plasmid backbone was retained in bacterial cells even after the occasional loss of their antibiotic-resistance cassettes. This backbone structure has been observed worldwide to carry various antimicrobial resistance genes. Although the chromosomally integrated plasmid carrying blaIMP-6 could not be transmitted by conjugation, we found that meropenem treatment for 1 wk allowed the plasmid to be released from the chromosome and spread among E. coli strains that were susceptible to meropenem. The copy number of blaIMP-6 on the plasmid was amplified eight times, resulting in enhanced resistance. Although the carbapenemase producers that carry chromosomal carbapenemase genes comprised of small subpopulations, they functioned as stable, long-term reservoirs of carbapenem resistance that could be disseminated via plasmids with amplified resistance upon meropenem stimulation. Although plasmids occasionally lose their resistance cassettes as a scaffold for the acquisition of another resistance gene, chromosomal integration may contribute to the effective sharing of carbapenem resistance within a population, complicating the development of a strategy to avoid the dissemination of antimicrobial resistance. IMPORTANCE Although carbapenem antibiotics are the last resort for combating multidrug-resistant organisms, global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens public health. Carbapenemases, which are enzymes responsible for carbapenem resistance, are mainly encoded by genes on plasmids that can be transmitted across bacterial species. Owing to the rarity of chromosomally encoded carbapenemase genes, studies investigating their properties in bacterial communities are lacking. In our study, we revealed the stability of carbapenemase genes on chromosomes compared with those on plasmids, which can be lost through the loss of antimicrobial resistance cassettes despite robust retention of plasmid backbones. Following exposure to meropenem, the carbapenemase gene integrated into the chromosome was released as a plasmid, restarting the dissemination of enhanced carbapenem resistance through amplified copy numbers of carbapenemase genes. Chromosomally encoded carbapenemase genes may function as a reservoir of resistance genes within the bacterial community and challenge infection control against CRE dissemination.