RESUMEN
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.
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Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Infecciones por Enterobacteriaceae/epidemiología , Plásmidos/genética , beta-Lactamasas/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Carbapenémicos/farmacología , Farmacorresistencia Bacteriana/genética , Enterobacter cloacae/efectos de los fármacos , Enterobacter cloacae/genética , Enterobacter cloacae/aislamiento & purificación , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Humanos , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Mianmar/epidemiología , Secuenciación Completa del GenomaRESUMEN
The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has been increasing at medical institutions in Japan without even noticing. Recently, we performed a point prevalence survey for CRE carriage at a medical facility in northern Osaka that demonstrated an unexpectedly high prevalence of blaIMP-6-positive CRE, particularly at long-term care hospitals (LTCH). To identify the risk factors for CRE carriage, we collected clinical data of patients at a representative LTCH. Of 140 patients who were included in this study, 27 (19.3%) were colonized with metallo-beta-lactamase (IMP-6) producers. Pulsed-field gel electrophoresis of the IMP-6 producing Enterobacteriaceae suggested a non-clonal transmission of Escherichia coli, while a clonal spread was shown for Klebsiella pneumoniae. Risk factors for CRE colonization were a longer stay at the hospital stay and a lower independence state, as measured by Norton scales. We propose that a paradigm shift in infection control, inciting a coordinated regional effort to involve LTCHs, should be discussed in the aging society of Japan.
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Enterobacteriaceae Resistentes a los Carbapenémicos/metabolismo , Infecciones por Enterobacteriaceae/epidemiología , Heces/microbiología , Inosina Monofosfato/metabolismo , Anciano de 80 o más Años , Proteínas Bacterianas/metabolismo , Carbapenémicos/farmacología , Portador Sano/epidemiología , Portador Sano/microbiología , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Infecciones por Enterobacteriaceae/microbiología , Femenino , Estudios de Seguimiento , Hospitales , Humanos , Japón , Cuidados a Largo Plazo/métodos , Masculino , Prevalencia , Factores de Riesgo , beta-Lactamasas/metabolismoRESUMEN
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.
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Proteínas Bacterianas/genética , Heces/microbiología , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , beta-Lactamasas/genética , Carbapenémicos/farmacología , HumanosRESUMEN
BACKGROUND: Identification of carbapenemase-producing Enterobacteriaceae (CPE) in faecal specimens is challenging. This fact is particularly critical because low-level carbapenem-resistant organisms such as IMP-producing CPE are most prevalent in Japan. We developed a modified selective medium more suitable for IMP-type CPE. METHODS: Fifteen reference CPE strains producing different types of ß-lactamases were used to evaluate the commercially available CHROMagar KPC and chromID CARBA as well as the newly prepared MC-ECC medium (CHROMagar ECC supplemented with meropenem, cloxacillin, and ZnSO4) and M-ECC medium (CHROMagar ECC supplemented with meropenem and ZnSO4). A total of 1035 clinical samples were then examined to detect CPE using chromID CARBA and M-ECC medium. RESULTS: All tested strains producing NDM-, KPC-, and OXA-48-carbapenemases were successfully cultured in the media employed. Although most of the IMP-positive strains did not grow in CHROMagar KPC, chromID CARBA, or MC-ECC, all tested strains grew on M-ECC. When faecal samples were applied to the media, M-ECC medium allowed the best growth of IMP-type CPE with a significantly higher sensitivity (99.3%) than that of chromID CARBA (13.9%). CONCLUSIONS: M-ECC medium was determined as the most favourable selective medium for the detection of IMP-type CPE as well as other types of CPE.
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Proteínas Bacterianas , Técnicas de Tipificación Bacteriana/métodos , Medios de Cultivo , Infecciones por Enterobacteriaceae , Enterobacteriaceae , Inosina Monofosfato/metabolismo , beta-Lactamasas , Enterobacteriaceae/enzimología , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/metabolismo , Infecciones por Enterobacteriaceae/diagnóstico , Infecciones por Enterobacteriaceae/microbiología , Heces/microbiología , HumanosRESUMEN
The protective effect of blueberry (BB) on the clastogenic effects of MNNG and DMBA was evaluated with the induced micronucleus (MN) frequency as a biomarker, both in vitro and in vivo. Human hepatoma HepG2 cells, which contain most of the metabolic activating enzymes was used for the in vitro test. MN frequencies were determined in binucleated cells generated by blocking cytokinesis by use of cytochalasin-B. The MN frequency in vivo was determined in polychromatic erythrocytes (PCEs) from the bone marrow of treated mice. BB by itself was not toxic both in vivo and in vitro. There was no evidence of a potential physico-chemical interaction between BB and the test carcinogens in vitro. Pre-treatment with BB reduced the MN frequency induced by MNNG. But, simultaneous treatment and post-treatment with BB did not affect the frequency of MNNG-induced MN. BB did not affect the frequency of DMBA-induced MN in vitro under any test condition. Under in vivo conditions, BB reduced the frequencies of MNNG- and DMBA-induced MN in PCEs, but in the case of the protective effect of BB against DMBA a dramatic reduction in the percentage of PCEs was observed, suggesting increased cytotoxicity.
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9,10-Dimetil-1,2-benzantraceno/toxicidad , Arándanos Azules (Planta) , Metilnitronitrosoguanidina/toxicidad , Mutágenos/toxicidad , Línea Celular Tumoral , Humanos , Técnicas In Vitro , Pruebas de MicronúcleosRESUMEN
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.
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Enterobacteriaceae Resistentes a los Carbapenémicos , Infecciones por Enterobacteriaceae , Humanos , Klebsiella pneumoniae/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Tailandia/epidemiología , Filogenia , Infecciones por Enterobacteriaceae/epidemiología , beta-Lactamasas/genética , Proteínas Bacterianas/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
Early detection of coronavirus disease 2019 (COVID-19) is critical for both initiating appropriate treatment and preventing the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent. A simple and rapid diagnostic test that can be performed without any expensive equipment would be valuable for clinicians working in a low-resource setting. Here, we report a point-of-care detection technique for COVID-19 that combines the power of isothermal amplification (reverse transcription helicase-dependent amplification, RT-HDA) and dipstick technologies. The limit of detection of this diagnostic test is six copies of SARS-CoV-2 µl-1 in clinical specimens. Of the 22 clinical specimens tested, RT-HDA-coupled dipstick correctly identified all positive and negative specimens. The RT-HDA can be performed over a heating block and the results can be interpreted visually with the dipstick technology without any specialized equipment. Furthermore, the RT-HDA-coupled dipstick could be performed in a short turnaround time of ~2 h. Thus, the RT-HDA-coupled dipstick could serve as a point-of-care diagnostic test for COVID-19 in a low-resource environment.
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COVID-19 , COVID-19/diagnóstico , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral , Transcripción Reversa , SARS-CoV-2/genética , Sensibilidad y EspecificidadRESUMEN
The timely diagnosis of active tuberculosis disease (TB) is crucial to interrupt the transmission and combat the spread of Mycobacterium tuberculosis (Mtb), the causative agent for TB. Here, we demonstrate the development of a specimen-direct rapid diagnostic method for TB which consists of an isothermal amplification device, Tiny Isothermal Nucleic acid quantification sYstem (TINY), coupled with helicase-dependent amplification (HDA). HDA, an isothermal amplification technique is established over TINY using pUCIDT-AMP vector carrying IS6110, the target DNA sequence for Mtb. The limit of detection of this technique for detecting the IS6110 within a threshold time of 50 min is 2.5 × 105 copies of IS6110. HDA in TINY for TB detection was evaluated using three IS6110-positive Mtb strains - H37Rv, CDC 1551, and Erdman wild-type and one IS6110-negative Mycobacterium avium. For spiked oral swabs, HDA in TINY detects IS6110 without any non-specificity in relatively short turnaround time (<1.5 h), highlighting its potential utility as a specimen-direct point-of-care diagnostic for TB. TINY does not require an uninterrupted power supply and its lightweight and small footprint offers portability and easier operation in clinical settings with poor infrastructure. Overall, HDA in TINY could serve as an efficient rapid, and portable platform for the qualitative detection of TB at the point-of-care.
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Introduction. Among the causative agents of bloodstream infections (BSIs), methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococcus (VRE) are the key causative pathogens. Their rapid detection directly from Gram-positive cocci-positive blood culture specimens will promote timely treatment and help to implement effective infection control measures.Aim. We aim to develop a PCR-dipstick technique for the rapid detection of MRSA and VRE directly from positive blood culture specimens.Methodology. PCR-dipstick is a PCR-based multiplex detection technique where DNA-DNA hybridization is employed, and the results are interpreted with the naked eye. It was designed to target three drug resistance genes: mecA in MRSA and vanA/vanB in VRE from positive blood culture specimens. A total of 120 clinical isolates were used to evaluate the sensitivity and specificity of PCR-dipstick. Then, PCR-dipstick was examined for MRSA and VRE detection directly from positive blood cultures.Results. PCR-dipstick showed 100â% sensitivity and specificity in detecting mecA, vanA and vanB genes directly from bacterial colonies in comparison with multiplex PCR for genomic DNA followed by agarose gel electrophoresis. Further, it could differentially detect multiple resistant genes in pooled bacterial colonies (n=10). Ultimately, PCR-dipstick could detect MRSA and VRE in positive blood cultures in ~3 h.Conclusion. The results of the current study substantiate that PCR-dipstick can be used as an efficient detection system for MRSA and VRE directly from Gram-positive cocci-positive blood cultures. Its affordability and rapidity indicate that PCR-dipstick can be an effective tool for controlling nosocomial pathogens.
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Proteínas Bacterianas/genética , Infecciones por Bacterias Grampositivas/microbiología , Cocos Grampositivos/genética , Reacción en Cadena de la Polimerasa Multiplex/métodos , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Sangre/microbiología , Cultivo de Sangre , Farmacorresistencia Bacteriana , Infecciones por Bacterias Grampositivas/sangre , Infecciones por Bacterias Grampositivas/diagnóstico , Cocos Grampositivos/clasificación , Cocos Grampositivos/efectos de los fármacos , Cocos Grampositivos/aislamiento & purificación , Humanos , Reacción en Cadena de la Polimerasa Multiplex/instrumentaciónRESUMEN
Antimicrobial resistance (AMR) is a fundamental global concern analogous to climate change threatening both public health and global development progress. Infections caused by antimicrobial-resistant pathogens pose serious threats to healthcare and human capital. If the increasing rate of AMR is left uncontrolled, it is estimated that it will lead to 10 million deaths annually by 2050. This global epidemic of AMR necessitates radical interdisciplinary solutions to better detect antimicrobial susceptibility and manage infections. Rapid diagnostics that can identify antimicrobial-resistant pathogens to assist clinicians and health workers in initiating appropriate treatment are critical for antimicrobial stewardship. In this review, we summarize different technologies applied for the development of rapid diagnostics for AMR and antimicrobial susceptibility testing (AST). We briefly describe the single-cell technologies that were developed to hasten the AST of infectious pathogens. Then, the different types of genotypic and phenotypic techniques and the commercially available rapid diagnostics for AMR are discussed in detail. We conclude by addressing the potential of current rapid diagnostic systems being developed as point-of-care (POC) diagnostic tools and the challenges to adapt them at the POC level. Overall, this review provides an insight into the current status of rapid and POC diagnostic systems for AMR.
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Antibacterianos , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , Sistemas de Atención de PuntoRESUMEN
Shewanella sp., the progenitors of blaOXA-48 -like genes are increasingly reported with the possession of different blaOXA-48 -like variants. This study aims to characterize blaOXA-731 , a new variant of a blaOXA-48 -like gene identified in Shewanella sp. isolated from the aquatic environment in Myanmar. Phylogenetic analysis of the blaOXA-731 sequence with other blaOXA-48 -like variants showed that it has the highest nucleotide identity of 86.09% with blaOXA-48 . However, the active site motifs in OXA-731 were 100% identical to that in OXA-48. Whole-genome sequencing analysis showed that blaOXA-731 is not surrounded by any mobile genetic elements. The genetic context of blaOXA-731 was found as similar to other blaOXA-48 -like genes previously identified in Shewanella sp. S1 nuclease pulsed-field gel electrophoresis followed by Southern blotting confirmed the location of blaOXA-731 in the chromosome of the Shewanella genome. Cloning and expression studies showed that OXA-731 has ß-lactamase activity similar to OXA-48 and OXA-181, but it has no significant carbapenemase activity. Our results showed the significance of blaOXA-48 -like-carrying Shewanella sp. in the spreading of blaOXA-48 -like genes in the community.
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Proteínas Bacterianas/genética , Agua Potable/microbiología , Shewanella/enzimología , beta-Lactamasas/genética , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/enzimología , Cromosomas Bacterianos/genética , Pruebas de Sensibilidad Microbiana , Mianmar , Filogenia , Alineación de Secuencia , Shewanella/efectos de los fármacos , Shewanella/genética , Shewanella/aislamiento & purificación , beta-Lactamasas/metabolismoRESUMEN
BACKGROUND: Limited treatment options complicate management of infections with New Delhi metallo-ß-lactamase (NDM)-producing organisms. The efficacy of combination therapy with meropenem (MEM) and cefmetazole (CMZ) was assessed against NDM-producing Enterobacteriaceae. MATERIALS AND METHODS: Twelve Escherichia coli clinical isolates harbouring blaNDM-1 and a positive control E. coli BAA-2469 harbouring blaNDM-1 were studied. Minimum inhibitory concentrations (MICs) of MEM, ertapenem (ERT) and CMZ were determined by broth microdilution. Checkerboard and time-kill assays were performed to confirm the in vitro efficacy of the MEM/CMZ combination. Scanning electron microscopy, kinetic studies and whole-genome sequence analysis were used to determine the antimicrobial resistance mechanisms. RESULTS: MICs of MEM, ERT and CMZ in monotherapy ranged from 8 to 32, 16 to 128, and 32 to 512 µg/mL, respectively. In the checkerboard assay, MEM/ERT resulted in no synergy, whereas MEM/CMZ showed a synergistic effect in all the tested isolates. Furthermore, the MIC of MEM in combination decreased by 2- to 8-fold compared with that of MEM alone. The time-kill study revealed a bactericidal effect in 4 of 13 isolates at 24 h. Scanning electron microscopy showed spheroidisation of the bacterial cell in the MEM/CMZ combination; this was not observed in single antibiotic conditions. Kinetic studies indicated CMZ was a better antagonist for NDM-1 than ERT. Whole-genome sequence analysis did not reveal any explainable differences between isolates susceptible and those non-susceptible to combination therapy. CONCLUSION: In vitro studies showed the potential effectiveness of MEM/CMZ combination therapy against NDM-producing organisms.
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Antibacterianos/farmacología , Cefmetazol/farmacología , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Enterobacteriaceae/efectos de los fármacos , Meropenem/farmacología , Antibacterianos/uso terapéutico , Cefmetazol/uso terapéutico , Quimioterapia Combinada , Enterobacteriaceae/metabolismo , Infecciones por Enterobacteriaceae/microbiología , Ertapenem/farmacología , Humanos , Meropenem/uso terapéutico , beta-Lactamasas/biosíntesisRESUMEN
Purpose: Optimal treatment regimens are yet to be established for carbapenemase-producing Enterobacteriaceae (CPE). We assessed the in vitro efficacy of meropenem (MEM) and cefmetazole (CMZ) combination treatment against blaKPC-2-positive Enterobacteriaceae, in comparison with that of double-carbapenem therapy using ertapenem (ERT). Materials and Methods: We performed checkerboard assay for 10 blaKPC-2-positive clinical isolates and Klebsiella pneumoniae BAA-1705 (possessing blaKPC-2), with synergistic effect being defined by a fractional inhibitory concentration index of ≤0.5. Subsequently, we conducted time-kill assays using K. pneumoniae BAA-1705 with an initial inoculum of 104-107 colony forming unit (CFU)/mL. Bactericidal effect was defined as the reduction of initial bacterial count by ≥103 CFU/mL in 24 hr. Finally, we applied scanning electron microscopy to observe morphological changes induced by the combination of MEM and CMZ. Results: Checkerboard assays revealed a synergistic effect in 7 out of 11 blaKPC-2 -positive Enterobacteriaceae when the MEM and CMZ combination was used, and no effect when the MEM and ERT combination was used. The minimum inhibitory concentration of MEM decreased 4-8-fold when combined with CMZ. Time-kill assays with an initial inoculum of 5 × 105 CFU/mL revealed regrowth under the combination of MEM and ERT (0.25 × minimum inhibitory concentration [MIC] each), whereas the combination of 0.25 × MIC each of MEM and CMZ exhibited bactericidal effect. Scanning electron microscopy results demonstrated that the combination of 0.5 × MIC MEM and 0.5 × MIC CMZ facilitated bacterial cell lysis compared with each antibiotic alone. Conclusion: The combination therapy using MEM and CMZ potentially has bactericidal effect against KPC-producing Enterobacteriaceae.
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Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Cefmetazol/farmacología , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Meropenem/farmacología , beta-Lactamasas/metabolismo , Enterobacteriaceae Resistentes a los Carbapenémicos/metabolismo , Carbapenémicos/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Infecciones por Enterobacteriaceae/microbiología , Ertapenem/farmacología , Humanos , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/metabolismo , Pruebas de Sensibilidad Microbiana/métodosRESUMEN
The spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a serious threat to clinical practice and public health. These bacteria are present both in clinical settings and non-clinical environments. The presence of CPE in food stuffs has been reported, but sporadically so. Here, we screened for CPE in meat, seafood, and vegetable samples from local markets of Yangon, Myanmar. We obtained 27 CPE isolates from 93 food samples and identified 13 as Escherichia coli, six as Klebsiella pneumoniae, seven as Enterobacter cloacae complex, and one as Serratia marcescens. All except the E. cloacae complex harboured the carbapenemase genes blaNDM-1 or blaNDM-5, while all Enterobacter isolates carried the carbapenemase gene blaIMI-1. The blaIMI-1 gene was located in putative mobile elements EcloIMEX-2, -3, or -8. Using multi-locus sequence typing, E. coli, K. pneumoniae, and E. cloacae complex isolates were classified into 10, six, and five different sequence types, respectively. Our results demonstrate that diverse organisms with various carbapenemase genes are widespread in the market foods in Yangon, highlighting the need for promoting proper food hygiene and effective measures to prevent further dissemination.
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Proteínas Bacterianas/metabolismo , Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Microbiología de Alimentos , beta-Lactamasas/metabolismo , Animales , Proteínas Bacterianas/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Contaminación de Alimentos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Carne/microbiología , Mianmar , Verduras/microbiología , beta-Lactamasas/genéticaRESUMEN
Colistin is used as an alternative therapeutic for carbapenemase-producing Enterobacteriaceae (CPE) infections which are spreading at a very high rate due to the transfer of carbapenemase genes through mobile genetic elements. Due to the emergence of mcr-1, the plasmid-mediated colistin resistance gene, mcr-1-positive Enterobacteriaceae (MCRPEn) pose a high risk for the transfer of mcr-1-carrying plasmid to CPE, leading to a situation with no treatment alternatives for infections caused by Enterobacteriaceae possessing both mcr-1 and carbapenemase genes. Here, we report the application of PCR-dipstick-oriented surveillance strategy to control MCRPEn and CPE by conducting the PCR-dipstick technique for the detection of MCRPEn and CPE in a tertiary care hospital in Thailand and comparing its efficacy with conventional surveillance method. Our surveillance results showed a high MCRPEn (5.9%) and CPE (8.7%) carriage rate among the 219 rectal swab specimens examined. Three different CPE clones were determined by pulsed-field gel electrophoresis (PFGE) whereas only two MCRPEn isolates were found to be closely related as shown by single nucleotide polymorphism-based phylogenetic analysis. Whole genome sequencing (WGS) and plasmid analysis showed that MCRPEn carried mcr-1 in two plasmids types-IncX4 and IncI2 with ~99% identity to the previously reported mcr-1-carrying plasmids. The identification of both MCRPEn and CPE in the same specimen indicates the plausibility of plasmid-mediated transfer of mcr-1 genes leading to the emergence of colistin- and carbapenem-resistant Enterobacteriaceae. The rapidity (<2 h) and robust sensitivity (100%)/specificity (~99%) of PCR-dipstick show that this specimen-direct screening method could aid in implementing infection control measures at the earliest to control the dissemination of MCRPEn and CPE.
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The spread of carbapenemase-producing Enterobacteriaceae (CPE) is an increasing global public health concern. The development of simple and reliable methods for CPE detection is required in the clinical setting. This study aimed to establish a dual-wavelength measurement method using an ultraviolet-visible spectrophotometer to rapidly quantify imipenem hydrolysis in bacterial cell suspensions. The hydrolytic activities of 148 strains including various CPE strains (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter aerogenes containing the blaIMP, blaKPC, blaNDM, blaOXA, and blaVIM genes) were measured and analysed. A cut-off value was obtained for differentiation between CPE and non-CPE strains, and the method had high sensitivity (100%) and specificity (100%) within 60 min. Our system has potential clinical applications in detecting CPE.
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Enterobacteriaceae Resistentes a los Carbapenémicos/química , Enterobacteriaceae Resistentes a los Carbapenémicos/enzimología , Espectrofotometría Ultravioleta/métodos , Absorción Fisicoquímica , Antibacterianos/química , Proteínas Bacterianas/análisis , Infecciones por Enterobacteriaceae/diagnóstico , Hidrólisis , Imipenem/química , Meropenem/química , Curva ROC , Sensibilidad y Especificidad , Estadísticas no Paramétricas , Resistencia betalactámica , beta-Lactamasas/análisisRESUMEN
An NDM-5-producing Klebsiella pneumoniae ST147 strain was isolated from a Japanese patient who had not travelled abroad in at least 5 years. Whole-genome sequencing revealed a genomic rearrangement in an FII plasmid harbouring blaNDM-5 due to the replicative transposition of IS26. A hypothetical structure was proposed for its ancestral plasmid, and comparative genomic analysis of the plasmid suggested the dissemination of structurally similar plasmids harbouring blaNDM-5 in Asian and Middle Eastern countries.