ABSTRACT
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.
Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Carbapenems , Microbial Sensitivity Tests , Pseudomonas Infections , Pseudomonas aeruginosa , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/isolation & purification , Japan/epidemiology , Carbapenems/pharmacology , Anti-Bacterial Agents/pharmacology , Humans , Bacterial Proteins/genetics , Pseudomonas Infections/microbiology , Pseudomonas Infections/epidemiology , Pseudomonas Infections/drug therapy , beta-Lactamases/genetics , Genome, Bacterial/genetics , Piperacillin, Tazobactam Drug Combination/therapeutic use , Piperacillin, Tazobactam Drug Combination/pharmacology , Whole Genome Sequencing , Meropenem/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Amikacin/pharmacologyABSTRACT
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.
Subject(s)
Disease Outbreaks , Enterococcus faecium , Gram-Positive Bacterial Infections , Multilocus Sequence Typing , Phylogeny , Plasmids , Vancomycin-Resistant Enterococci , Whole Genome Sequencing , Enterococcus faecium/genetics , Enterococcus faecium/drug effects , Enterococcus faecium/isolation & purification , Japan/epidemiology , Humans , Vancomycin-Resistant Enterococci/genetics , Vancomycin-Resistant Enterococci/drug effects , Vancomycin-Resistant Enterococci/isolation & purification , Plasmids/genetics , Gram-Positive Bacterial Infections/transmission , Gram-Positive Bacterial Infections/microbiology , Gram-Positive Bacterial Infections/epidemiology , Cross Infection/microbiology , Cross Infection/transmission , Cross Infection/epidemiology , Bacterial Proteins/genetics , Anti-Bacterial Agents/pharmacology , Carbon-Oxygen Ligases/genetics , Microbial Sensitivity Tests , Polymorphism, Single Nucleotide , Hospitals , Vancomycin/pharmacology , Genome, Bacterial/geneticsABSTRACT
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.
Subject(s)
Anti-Bacterial Agents , Bacteriocins , Biofilms , Methicillin-Resistant Staphylococcus aureus , Microbial Sensitivity Tests , Streptococcus , Vancomycin-Resistant Enterococci , Bacteriocins/pharmacology , Bacteriocins/genetics , Anti-Bacterial Agents/pharmacology , Vancomycin-Resistant Enterococci/drug effects , Methicillin-Resistant Staphylococcus aureus/drug effects , Biofilms/drug effects , Streptococcus/drug effectsABSTRACT
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.
Subject(s)
Anti-Bacterial Agents , Gram-Positive Bacterial Infections , Microbial Sensitivity Tests , Whole Genome Sequencing , Humans , Japan/epidemiology , Anti-Bacterial Agents/pharmacology , Gram-Positive Bacterial Infections/microbiology , Gram-Positive Bacterial Infections/epidemiology , Inpatients , Cross Infection/microbiology , Cross Infection/epidemiology , Genome, Bacterial/genetics , Vancomycin-Resistant Enterococci/genetics , Vancomycin-Resistant Enterococci/drug effects , Vancomycin-Resistant Enterococci/isolation & purification , Multilocus Sequence Typing , Disease Outbreaks , Enterococcus/genetics , Enterococcus/drug effects , Enterococcus/isolation & purification , Enterococcus/classification , Vancomycin/pharmacologyABSTRACT
We have recently reported the isolation of third-generation-cephalosporin-resistant Gram-negative bacteria from the oral cavity of residents of a long-term-care facility (LTCF). Since disinfectants are often used in the oral cavity, it is important to investigate the disinfectant susceptibility of oral bacteria. Here, we evaluated the susceptibilities of Gram-negative antimicrobial-resistant bacteria (GN-ARB), including Pseudomonas, Acinetobacter, and Enterobacteriaceae, obtained from the oral cavity of residents of LTCFs to povidone-iodine (PVPI), cetylpyridinium chloride (CPC), benzalkonium chloride (BZK), and chlorhexidine chloride (CHX). We also evaluated the susceptibilities of isolates from the rectum to the same agents to compare the susceptibility profiles of oral and rectal isolates. Next, we investigated the relationship between their susceptibility and disinfectant resistance genes delineated by whole-genome sequencing of the isolates. Additionally, we evaluated the correlation between disinfectant-resistant GN-ARB and clinical information. In oral GN-ARB, the MIC of PVPI showed almost identical values across isolates, while the MICs of CPC, BZK, and CHX showed a wide range of variation among species/strains. In particular, Pseudomonas aeruginosa exhibited high-level resistance to CPC and BZK. The disinfectant susceptibility of rectal GN-ARB showed a tendency similar to that of oral GN-ARB. The presence of qacEΔ1 was correlated with CPC/BZK resistance in P. aeruginosa, while other species exhibited no correlation between qacEΔ1 and resistance. Multiple analyses showed the correlation between the presence of CPC-resistant bacteria in the oral cavity and tube feeding. In conclusion, we found that some oral GN-ARB isolates showed resistance to not only antibiotics but also disinfectants. IMPORTANCE Antibiotic-resistant bacteria (ARB) are becoming a serious concern worldwide. We previously reported the isolation of third-generation-cephalosporin-resistant Gram-negative bacteria from the oral cavity of residents of a long-term-care facility (LTCF). To prevent infection with ARB in hospitals and eldercare facilities, we must pay more attention to the use of not only antibiotics but also disinfectants. However, the effect of disinfectants on ARB is unclear. In this study, we evaluated the susceptibility of Gram-negative ARB (GN-ARB) from the oral cavity of residents of LTCFs to some disinfectants that are often used for the oral cavity; we found that some isolates showed resistance to several disinfectants. This is the first comprehensive analysis of the disinfectant susceptibility of oral GN-ARB. These results provide some important information for infection control and suggest that disinfectants should be applied carefully.
Subject(s)
Disinfectants , Angiotensin Receptor Antagonists/pharmacology , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Cephalosporins/pharmacology , Disinfectants/pharmacology , Drug Resistance, Bacterial , Gram-Negative Bacteria , Microbial Sensitivity Tests , Mouth , Povidone-Iodine/pharmacology , Pseudomonas aeruginosa , Long-Term Care , HumansABSTRACT
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.
Subject(s)
Anti-Infective Agents , Methicillin-Resistant Staphylococcus aureus , Humans , Aged , Cohort Studies , Prospective Studies , Long-Term Care , Angiotensin Receptor Antagonists , Drug Resistance, Multiple, Bacterial/genetics , Methicillin-Resistant Staphylococcus aureus/genetics , Angiotensin-Converting Enzyme Inhibitors , Gram-Negative Bacteria/geneticsABSTRACT
Despite frequent identification of plasmids carrying carbapenemase genes, the transfer of plasmids carrying carbapenemase genes is not well recognized in clinical settings because of technical limitations. To investigate the detailed mechanisms of the spread of carbapenem-resistant Enterobacteriaceae (CRE), we performed multifaceted genomic surveillance of CRE isolates in Thailand and analyzed their plasmidome. We analyzed 371 Enterobacteriaceae isolates carrying blaNDM-1 and 114 Enterobacteriaceae isolates carrying blaNDM-5 obtained from clinical samples of 473 patients in 11 representative hospitals located in six provinces in Thailand between 2012 and 2017. The complete structures of plasmids carrying blaNDM and chromosomal phylogeny were determined by combining Southern blotting hybridization analysis and our previously performed whole-genome short-read sequencing data. Dissemination of the blaNDM-5 gene among the Enterobacteriaceae isolates in Thailand was mainly owing to the nationwide clonal spread of Escherichia coli ST410 and regional clonal spreads of Escherichia coli ST361 and ST405. Analysis of blaNDM-1-carrying isolates revealed nationwide dissemination of two specific plasmids and nationwide clonal dissemination of Klebsiella pneumoniae ST16 accompanied with regional disseminations of three distinctive K. pneumoniae clones (ST231, ST14, and ST147) with different plasmids. Dissemination of CRE carrying blaNDM in Thailand is mainly based on nationwide clonal expansions of E. coli ST410 carrying blaNDM-5 and K. pneumoniae ST16 carrying blaNDM-1, nationwide dissemination of two distinctive plasmids carrying blaNDM-1, and accumulation of clonal expansions in regional areas. Although the overuse of antibiotics can promote CRE dissemination, the limited variety of transmitters highlights the importance of preventing horizontal dissemination among patients.
Subject(s)
Carbapenem-Resistant Enterobacteriaceae , Enterobacteriaceae Infections , Humans , Carbapenem-Resistant Enterobacteriaceae/genetics , Escherichia coli/genetics , Enterobacteriaceae Infections/epidemiology , Enterobacteriaceae Infections/drug therapy , Thailand/epidemiology , Microbial Sensitivity Tests , beta-Lactamases/genetics , Enterobacteriaceae/genetics , Plasmids/genetics , Klebsiella pneumoniae/genetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic useABSTRACT
The plasmid-mediated tet(X7) conferring high-level tigecycline resistance was identified in five mcr-1.1-positive Escherichia coli strains (ST10 [n = 3] and ST155 [n = 2]) isolated from chickens in Egypt. Two fosfomycin-resistant fosA4-carrying IncFII plasmids (â¼79 kb in size) were detected. Transposase ISCR3 (IS91 family) is syntenic with tet(X7) in all isolates, suggesting its role in the mobilization of tet(X7). To our knowledge, this is the first global report of ST4-IncHI2 plasmids cocarrying tet(X7) and mcr-1.1 from chickens.
Subject(s)
Escherichia coli Infections , Escherichia coli Proteins , Fosfomycin , Animals , Anti-Bacterial Agents/pharmacology , Chickens , Colistin/pharmacology , Drug Resistance, Bacterial/genetics , Egypt , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Fosfomycin/pharmacology , Plasmids/genetics , TigecyclineABSTRACT
BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are spreading in hospitals, environment and retail foods in Yangon, Myanmar. OBJECTIVES: To investigate whether CPE colonize healthy individuals living in Yangon and whether clinical-related strains are spreading in the community. METHODS: CPE was isolated from faecal samples obtained from healthy Japanese residents of Yangon with no history of hospitalization. Isolates were subjected to WGS using short- and long-read sequencers and compared with those previously isolated in Yangon. RESULTS: Six Escherichia coli strains harbouring blaNDM-1 or blaNDM-5 belonging to five different STs-ST10, ST38, ST48, ST410 and ST8453-were isolated from 69 volunteers. The ST38 isolates were related to those previously isolated from retail food in Yangon. The ST410 and ST8453 isolates were highly related to previous Yangon isolates including those of clinical and food origins. CONCLUSIONS: The analysis suggested the acquisition of blaNDM-positive E. coli, which are disseminating in a clinical setting and through retail foods, by healthy residents in Yangon.
Subject(s)
Escherichia coli Infections , Escherichia coli , Anti-Bacterial Agents/pharmacology , Escherichia coli/genetics , Escherichia coli Infections/epidemiology , Humans , Japan/epidemiology , Microbial Sensitivity Tests , Myanmar/epidemiology , beta-Lactamases/geneticsABSTRACT
Enterobacteriaceae, quinolone resistance is largely attributed to mutations in the quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE, and plasmid-italiciated quinolone resistance (PMQR) genes (e.g., qnr genes, aac(6')-Ib-cr, or qepA). .
ABSTRACT
The spread of carbapenemase-producing Enterobacteriaceae (CPE), contributing to widespread carbapenem resistance, has become a global concern. However, the specific dissemination patterns of carbapenemase genes have not been intensively investigated in developing countries, including Myanmar, where NDM-type carbapenemases are spreading in clinical settings. In the present study, we phenotypically and genetically characterized 91 CPE isolates obtained from clinical (n = 77) and environmental (n = 14) samples in Yangon, Myanmar. We determined the dissemination of plasmids harboring genes encoding NDM-1 and its variants using whole-genome sequencing and plasmid analysis. IncFII plasmids harboring blaNDM-5 and IncX3 plasmids harboring blaNDM-4 or blaNDM-7 were the most prevalent plasmid types identified among the isolates. The IncFII plasmids were predominantly carried by clinical isolates of Escherichia coli, and their clonal expansion was observed within the same ward of a hospital. In contrast, the IncX3 plasmids were found in phylogenetically divergent isolates from clinical and environmental samples classified into nine species, suggesting widespread dissemination of plasmids via horizontal transfer. Half of the environmental isolates were found to possess IncX3 plasmids, and this type of plasmid was confirmed to transfer more effectively to recipient organisms at a relatively low temperature (25°C) compared to the IncFII plasmid. Moreover, various other plasmid types were identified harboring blaNDM-1, including IncFIB, IncFII, IncL/M, and IncA/C2, among clinical isolates of Klebsiella pneumoniae or Enterobacter cloacae complex. Overall, our results highlight three distinct patterns of the dissemination of blaNDM-harboring plasmids among CPE isolates in Myanmar, contributing to a better understanding of their molecular epidemiology and dissemination in a setting of endemicity.
Subject(s)
Carbapenem-Resistant Enterobacteriaceae/drug effects , Carbapenem-Resistant Enterobacteriaceae/genetics , Enterobacteriaceae Infections/epidemiology , Plasmids/genetics , beta-Lactamases/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Carbapenem-Resistant Enterobacteriaceae/isolation & purification , Carbapenems/pharmacology , Drug Resistance, Bacterial/genetics , Enterobacter cloacae/drug effects , Enterobacter cloacae/genetics , Enterobacter cloacae/isolation & purification , Escherichia coli/drug effects , Escherichia coli/genetics , Humans , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Myanmar/epidemiology , Whole Genome SequencingABSTRACT
We report here Klebsiella pneumoniae strains carrying chromosomal blaNDM-1 in Thailand. The genomes of these two isolates include a 160-kbp insertion containing blaNDM-1, which is almost identical to that in the IncHI1B-like plasmid. Further analysis indicated that IS5-mediated intermolecular transposition and Tn3 transposase-mediated homologous recombination resulted in the integration of blaNDM-1 into the chromosome from an IncHI1B-like plasmid. The spread of this type of carbapenem-resistant Enterobacteriaceae may threaten public health and warrants further monitoring.
Subject(s)
Chromosomes, Bacterial/chemistry , Genome, Bacterial , Klebsiella pneumoniae/genetics , Mutagenesis, Insertional , Plasmids/metabolism , beta-Lactamases/genetics , Anti-Bacterial Agents/therapeutic use , Carbapenems/therapeutic use , Chromosome Mapping , DNA Transposable Elements , Gene Expression , Homologous Recombination , Humans , Klebsiella Infections/drug therapy , Klebsiella Infections/epidemiology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/isolation & purification , Plasmids/chemistry , Thailand/epidemiology , Transposases/genetics , Transposases/metabolism , beta-Lactamases/metabolismABSTRACT
A PCR-dipstick chromatography technique was designed and evaluated for differential identification of blaNDM, blaKPC, blaIMP, and blaOXA-48 carbapenemase genes directly in stool specimens within 2 h. It is a DNA-DNA hybridization-based detection system where PCR products can be easily interpreted by visual observation without electrophoresis. The PCR-dipstick showed high sensitivity (93.3%) and specificity (99.1%) in directly detecting carbapenemase genes in stool specimens compared with multiplex PCR for genomic DNA of the isolates from those stool specimens.
Subject(s)
Bacterial Proteins/genetics , Feces/microbiology , Molecular Diagnostic Techniques/methods , Multiplex Polymerase Chain Reaction/methods , beta-Lactamases/genetics , Carbapenems/pharmacology , HumansABSTRACT
Clostridium botulinum HA is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and are made up mainly of ß-strands. Here, we demonstrate structural and functional reconstitution of whole HA and present the complete structure of HA of serotype B determined by x-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding.
Subject(s)
Botulinum Toxins/chemistry , Hemagglutinins/chemistry , Animals , Botulinum Toxins/genetics , Botulinum Toxins/toxicity , Botulinum Toxins, Type A , Clostridium botulinum type B/chemistry , Clostridium botulinum type B/genetics , Clostridium botulinum type B/pathogenicity , Crystallography, X-Ray , Hemagglutinins/genetics , Hemagglutinins/toxicity , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Foodborne and intestinal botulism are the most common forms of human botulism; both result from the absorption of botulinum neurotoxin (BoNT) from the digestive tract into the circulation. BoNT is a large protein toxin (approximately 150 kDa), but it is able to pass through the epithelial barrier in the digestive tract. Recent cellular and molecular biology studies have begun to unravel the mechanisms by which this large protein toxin crosses the intestinal epithelial barrier. This review provides an overview of current knowledge relating to the absorption of botulinum toxins (BoNT and BoNT complex) from the gastrointestinal tract, with particular emphasis on the interaction of these toxins with the intestinal epithelial barrier.
Subject(s)
Botulinum Toxins/metabolism , Intestinal Mucosa/metabolism , Neurotoxins/metabolism , Animals , Binding Sites , Botulinum Toxins/toxicity , Botulism/microbiology , Cadherins/metabolism , Clostridium botulinum/metabolism , Clostridium botulinum/pathogenicity , Hemagglutinins/metabolism , Humans , Intestinal Mucosa/microbiology , Intestines/microbiology , Multiprotein Complexes/metabolism , Neurotoxins/toxicity , Protein Binding , Proteolysis , TranscytosisABSTRACT
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.
Subject(s)
Carbapenems , Escherichia coli , Ralstonia , Humans , Aged , Carbapenems/pharmacology , Carbapenems/therapeutic use , Meropenem , Doripenem , Escherichia coli/genetics , Escherichia coli/metabolism , Japan , beta-Lactamases/genetics , beta-Lactamases/metabolism , Bacterial Proteins/genetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Imipenem , Microbial Sensitivity TestsABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.