Extended-Spectrum Beta-lactamase and Metallo-beta-lactamase Production among Escherichia coli and Klebsiella pneumoniae Strains from Urine of Pregnant Women in Afikpo, Ebonyi State, Nigeria

Aim: The aim of this study was to determine the occurrence of Extended-spectrum beta-lactamase (ESBL) and Metallo-beta-lactamase (MBL) among Escherichia coli and Klebsiella pneumoniae strains from pregnant women attending Mater Misericordia Hospital Afikpo, Ebonyi state, Nigeria. Study Design: This is a laboratory based prospective study carried out on pregnant women suspected of having urinary tract infection and was requested to undergo diagnosis at microbiology laboratory of the hospital. Place and Duration of Study: The study was conducted in the Department of Science Laboratory Technology, Akanu Ibiam Federal Polytechnic, Unwana, Afikpo, Ebonyi State, Nigeria from October, 2022 to January, 2023. Methodology: Clean-catch midstream urine samples were collected from 206 pregnant women suspected of having urinary tract infection and were requested to undergo medical diagnosis at microbiology laboratory of the hospital. The urine samples were processed following standard microbiological procedure. Antimicrobial susceptibility testing was determined using the disc diffusion method, while ESBL phenotypes were determine by the Double-Disc Synergy Test (DDST). Disc potentiation test was performed to check for MBL production. Results: Out of the 206 urine samples processed, 24 (11.7 %) E. coli and 12 (5.8 %) K. pneumoniae were isolated. The antimicrobial susceptibility of the isolates recorded a 100 % resistance with Amoxicillin/Clavulanic acid and Cotrimoxazole. The Gram-negative isolates showed a high sensitivity of 100 % to Netilmicin, Meropenem and Ofloxacin. Overall, 35 (97.2 %) multidrug resistance (MDR) was observed of the bacteria isolates. A total of 9 (37.5 %) E. coli and 4 (33.3 %) K. pneumoniae was found positive for ESBL production whereas, 5 (20.8 %) E. coli and 2 (16.7 %) K. pneumoniae were MBL positive. Conclusion: The level of drug resistance in this study underscores the need for regular surveillance for effective management of urinary tract infection in pregnancy.

Phenotypic detection of metallo-beta-lactamase production in clinical isolates of Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital

Introduction: Carbapenem resistance due to metallo-beta-lactamase (MBL)-producing bacteria is an emerging threat worldwide. This study aimed to detect the MBL production in clinical isolates of E. coli and Klebsiella pneumoniae species in our hospital setting and to evaluate the efficiency of two phenotypic methods for the detection of MBL production. Materials and Methods: The present study was carried out in the Department of Microbiology, MGM Medical College and Hospital, Aurangabad, Maharashtra, for a period of 2 years from April 2018 to March 2020. From a total of 12,324 various clinical specimens, 393 isolates of E. coli and Klebsiella pneumoniae species were tested for MBL production. MBL was detected by two different phenotypic methods, i.e., combined disc test and E-test. Results: Out of 393 isolates, 130 (33.07%) isolates were resistant to imipenem on screening of which 71 (18.06%) were Klebsiella pneumoniae and 59 (15.01%) were E. coli. About 43.66% Klebsiella pneumoniae isolates and 40.67% E. coli isolates were MBL-positive by the combined disc test. Using the E-test, MBL production was found to be 46.47% and 45.76% in Klebsiella pneumoniae and E. coli, respectively. Conclusion: Routine screening of MBL-producing organisms should be performed in diagnostic laboratories to control the spread of resistance and for the proper management of antibiotic therapy. E-test is better than the combined disc test for the detection of MBL-producing gram-negative bacilli.

Three strains ST1642 Escherichia coli producing NDM-5 carbapenemase from neonatal department / 临床检验杂志

Objective@#To analyze the molecular epidemiological characteristics of Escherichia coli producing NDM-5 carbapenemase in the neonatal department of our hospital. @*Methods@#Three carbapenem-resistant Escherichia coli strains（E1, E2 and E3） isolated from neonatal ward of our hospital from August to September of 2017 were collected. Vitek 2 Compact system combined with K-B disk method was used for drug sensitivity test. The resistance genes were detected by PCR amplification. Plasmid replicon typing was detected by PCR. Plasmid conjugation tests were performed to explore the conjugating transfer of plasmids in the three strains. The homology of the three strains was analyzed by multiple locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE). @*Results@#Drug susceptibility test showed that the three bacteria were resistant to most β-lactam antibiotics except Aztreonam, and resistant to quinolones and SMZ-TMP, but sensitive to aminoglycosides drugs. PCR and sequencing results indicated that the three strains carried bla SHV gene and extended-spectrum beta-lactamase gene (bla SHV , bla TEM and bla CTX-M ). The plasmid replicon type was IncX3. Transfer test of E1 strain was successful. MLST results indicated that all the three strains were ST1642 type. MLST and PFGE results indicated that the bands of the three bacteria were identical. @*Conclusion@#Both NDM-5 carbapenemase and extended-spectrum beta-lactamase were detectable in the three strains of carbapenem-resistant bacteria from neonatal department. MLST and PFGE results suggested that the three strains were from the same clonal source.

Duplex PCR for rapid detection of the drug-resistant genes KPC and NDM / 医学研究生学报

ObjectiveThe nucleic acid technology for detecting drug-resistant genes has become one of the powerful tools for monitoring and controlling the spreading of drug-resistant bacteria. This study was to establish a method for rapid detection of the drug-resistant genes KPC and NDM and provide some guidance in clinical drug use and monitoring the prevalence of drug-resistant bacteria in the hospital.MethodsAccording to the conserved regions of Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM), we designed the primers of duplex PCR, optimized the amplification system and established a method for simultaneous detection of the drug-resistant genes KPC and NDM. Then, we analyzed the sensitivity and specificity of the method and applied it to the detection of Pseudomonas aeruginosa and Klebsiella pneumoniae.ResultsThe sequences of KPC and NDM exhibited a 100% consistency with those of the original ones. Target fragments of the desired size of 151 bp were detected in the KPC-2 positive standard and Klebsiella pneumoniae ATCC BAA 1705 standard strains, and those of the desired size of 261 bp were observed in the NDM-2 positive standard strain and NDM-positive pneumococcal bacteria, neither with non-specific amplification. Sequencing of the PCR products showed a 100% consistency between the sequences of the products and those of the drug-resistant genes KPC-2 and NDM-1. The detectable limits of KPC and NDM for duplex PCR were 7×102 and 5×102 copies per reaction respectively. Drug-resistant genes were detected in 12 (92.3%) of the 13 carbapenems-resistant strains, including 10 KPC-positive (83.3%) and 2 NDM positive ones (16.7%), but neither KPC nor NDM in the other 10 carbapenems-sensitive strains. In the 13 strains of Pseudomonas aeruginosa, KPC was detected in 2 (33.3%) of the 6 carbapenems-resistant ones, but neither KPC nor NDM in the other 7.ConclusionThe duplex PCR method can be used for rapid and effective detection of the drug-resistance genes KPC and NDM, with the advantages of high sensitivity and specificity, and is therefore of great significance for guiding clinical drug use and monitoring the spreading of carbapenems-resistant bacteria in the hospital.

Prevalence and antimicrobial susceptibility of non-fermenting Gram-negative bacilli isolated from clinical samples at a tertiary care hospital

Abstract INTRODUCTION: We compared the prevalence and antimicrobial susceptibility of non-fermenting gram-negative bacilli (NFGNB) isolated from clinical samples at a Brazilian tertiary care hospital in 2008 and 2013. METHODS: Collected data included patient's name, age, sex, inpatient unit, laboratory record number, type of biological material, culture test result, and antimicrobial susceptibility of isolated strains. RESULTS: Out of 19,112 culture tests analyzed, 926 (4.8%) were positive for NFGNB. Among these, 45.2% were metallo-beta-lactamase (MBL) producing strains. CONCLUSION: Between 2008 and 2013, the number of MBL-producing NFGNB isolates increased by 21.5%, which was accompanied by a consequent reduction in susceptibility to antimicrobials.

Identification of São Paulo metallo-beta-lactamase-1-producing Pseudomonas aeruginosa in the Central-West region of Brazil: a case study

Abstract: Metallo-beta-lactamase production is an important mechanism for carbapenem resistance of Pseudomonas aeruginosa , which represents an emerging public health challenge. We report the case of a patient admitted to an intensive care unit, with sepsis caused by multidrug-resistant São Paulo Metallo-beta-lactamase-1-producing P. aeruginosa . This is the first case of infection by this pathogenic strain in the State of Mato Grosso do Sul, Brazil. Thus, infection control measures are required for preventing future spread and outbreaks.

Multidrug resistance mediated by co‑carriage of extended‑spectrum beta‑lactamases, AmpC and New Delhi metallo‑beta‑lactamase‑1 genes among carbapenem‑resistant Enterobacteriaceae at five Indian medical centres.

In this study, we evaluated the coexistence of extended‑spectrum beta‑lactamases (ESBL), AmpC and New Delhi metallo‑beta‑lactamase‑1 (NDM‑1) genes among carbapenem‑resistant Enterobacteriaceae (CRE) recovered prospectively from patients at multiple sites. The study included 285 CRE strains from 2782 Gram‑negative Bacilli collected from multiple centres during 2007–2010, of which 87 were characterised. Standard and reference laboratory methods were used for resistance determination. Detection of blaNDM‑1, blaAmpC, blaTEM, blaSHV and blaCTX‑M was done by polymerase chain reaction. High levels of antimicrobial resistance observed among study isolates. Co‑carriage of ESBLs, AmpC and NDM‑1 was 26.3%. Nosocomial origin among the co‑carriage isolates was 64.3%, with 9.2% associated mortality.

Endemic Indian clones of Klebsiella pneumoniae‑harbouring New Delhi metallo‑beta‑lactamase‑1 on a hybrid plasmid replicon type: A case of changing New Delhi metallo‑beta‑lactamase plasmid landscapes in India.

Purpose: blaNDM genes are MBL genes that confer resistance to carbapenems. Globally, they are associated with diverse clones and plasmids. In this study, we characterised three isolates of Klebsiella pneumoniae‑harbouring blaNDM1 from patients undergoing chronic haemodialysis and renal transplantation. Materials and Methods: 3 blaNDM1‑producing K. pneumoniae were isolated from end‑stage renal disease patients undergoing haemodialysis and renal transplantation from a nephrology unit. All the three isolates were screened for clinically relevant resistant genes. Plasmid replicon content was analysed by polymerase chain reaction based replicon typing. Conjugation assays were done using azide‑resistant Escherichia coli J53 as the recipient strain. Multilocus sequence typing and variable number tandem repeat typing were done to find the clonality. Replicon sequence based typing was attempted to find the diversity of replicon‑associated sequences in IncHI3 plasmids. Results: All the 3 blaNDM positive isolates possessed the New Delhi metallo‑beta‑lactamase‑1 (NDM‑1) allele with an IncHI3 plasmid which was not transferable in one isolate. The isolates were found to be sequence type 14 (ST14; 2 nos) and ST38 both of which were previously reported to be the NDM‑producing K. pneumoniae STs prevalent in India. Replicon sequence analysis revealed limited sequence diversity within the repHI3 and repFIB locus. Conclusion: To the best of our knowledge, this is the first report of IncHI3, a newly assigned enterobacterial plasmid incompatibility group from India. This could either be a case of importation or a widely circulating NDM plasmid type in India.

Prevalence of metallo beta lactamase in clinical isolates of pseudomonas aeruginosa in a tertiary care hospital.

Background: Increasing Carbapenem resistance in Pseudomonas aeruginosa is an emerging threat and a matter of particular concern. Aim: Our study was conducted to find out the prevalence of Metallo beta lactamase producing Pseudomonas aeruginosa and compare various phenotypic MBL detection methods. Methods: A prospective study was conducted on 86 non duplicate Pseudomonas aeruginosa strains isolated from clinical specimens for a period of 2 year from April 2011 to march 2013. Total 86 isolates of Pseudomonas aeruginosa were included in the study. All clinical samples were processed according to standard microbiological method. The MIC for Imipenem and Meropenem were determined by broth dilution method. As per CLSI any isolate having an MIC of >8μg/ml was considered resistant.42 isolates were both or either resistant to IMP and MRP. These 42 isolates were tested for MBL production by (a) IMP EDTA E test (17 MBL positive isolate detected), (b) IMP & IMP EDTA disc diffusion test (17 MBL positive isolate detected), (c) IMP & EDTA double disc synergy test (14 MBL positive isolate detected). Results: 48.84% isolates were resistant to Carbapenem and 19.76% isolates were found to be MBL producer. Colistin showed 100% susceptibility in all the MBL positive isolates. Conclusions: Among the 3 test done IMP & IMP EDTA test is easy to perform, cost effective and as sensitive as E test. Our results strongly suggest that for the MBL isolates should be detected on routine basis and the antibiotic prescribed accordingly.

Isolation and Identification of L·adecarboxylata Producing IMP-1 Metallo-beta-lactamase / 现代检验医学杂志

Objective To investigate the phenotype and genotype in a carbapenem resistant L.adecarboxylata strain.Methods Microbial identification and drug susceptibility test was done with VITEK II Compact.Carbapenemases genes were detec-ted by PCR methods.Results The strain of carbapenem resistant L.adecarboxylata produced IMP-1 carbapenemase.Con-clusion L.adecarboxylata was found only rarely in the clinical isolates.This was the first Isolate of L.adecarboxylata pro-ducing IMP-1 metallo-beta-lactamase.

Epidemiology and Characteristics of Metallo-beta-Lactamase-Producing Pseudomonas aeruginosa / 감염과화학요법

Metallo-beta-lactamase-producing Pseudomonas aeruginosa (MPPA) is an important nosocomial pathogen that shows resistance to all beta-lactam antibiotics except monobactams. There are various types of metallo-beta-lactamases (MBLs) in carbapenem-resistant P. aeruginosa including Imipenemase (IMP), Verona integron-encoded metallo-beta-lactamase (VIM), Sao Paulo metallo-beta-lactamase (SPM), Germany imipenemase (GIM), New Delhi metallo-beta-lactamase (NDM), Florence imipenemase (FIM). Each MBL gene is located on specific genetic elements including integrons, transposons, plasmids, or on the chromosome, in which they carry genes encoding determinants of resistance to carbapenems and other antibiotics, conferring multidrug resistance to P. aeruginosa. In addition, these genetic elements are transferable to other Gram-negative species, increasing the antimicrobial resistance rate and complicating the treatment of infected patients. Therefore, it is essential to understand the epidemiology, resistance mechanism, and molecular characteristics of MPPA for infection control and prevention of a possible global health crisis. Here, we highlight the characteristics of MPPA.

Emergence of Acinetobacter pittii Harboring New Delhi Metallo-beta-Lactamase Genes in Daejeon, Korea

Carbapenemase production has been reported worldwide in gram-negative bacteria, including Acinetobacter species. We detected carbapenemase-producing Acinetobacter pittii in clinical isolates in Daejeon, Korea. Twenty-one ertapenem-resistant A. pittii isolates screened with a disk diffusion method were characterized by using the Epsilon test, four multiplex PCR assays, and a multilocus sequence typing (MLST) scheme. A total of 21 A. pittii isolates harbored the metallo-beta-lactamase (MBL) gene bla(IMP-1) or bla(NDM-1). Nineteen isolates containing bla(IMP-1) were resistant to imipenem and meropenem, but two isolates harboring bla(NDM-1) were susceptible to them. The sequence types (STs) of the two New Delhi MBL (NDM-1)-producing A. pittii isolates were ST70 and ST207, which differed from the STs (ST63, ST119, ST396, and a novel ST) of the IMP-1-producing A. pittii. This is the first report on NDM-1-producing A. pittii isolates in Korea. Our results emphasize that the study of NDM-1-producing gram-negative bacteria should involve carbapenem-susceptible as well as carbapenem-resistant isolates.

Further Modification of the Modified Hodge Test for Detecting Metallo-beta-Lactamase-Producing Carbapenem-Resistant Enterobacteriaceae

BACKGROUND: The modified Hodge test (MHT) was designed to detect carbapenemase-producing Enterobacteriaceae (CPE). This study evaluated variables to improve the performance of MHT. METHODS: Carbapenem-resistant Enterobacteriaceae isolated from November 2010 to March 2013 at the Asan Medical Center, were evaluated, including 33 metallo-beta-lactamase (MBL) producers and 103 non-CPEs. MHT was performed by using two carbapenem disks (ertapenem and meropenem; Becton Dickinson, USA), three media (Mueller-Hinton agar (MHA), MacConkey agar (MAC), and zinc-enriched MHA), and two inoculums (0.5-McFarland [McF] suspension and a 10-fold dilution of it.) PCR was performed to detect beta-lactamase genes of the MBL, AmpC, and CTX-M types. RESULTS: The sensitivity of MHT for detecting New Delhi metallo-beta-lactamase (NDM) producers was highest using ertapenem and 0.5-McF, 52.0% on MHA and 68.0% on MAC, respectively. NDM-producing Klebsiella pneumoniae (NDMKP) were detected with higher sensitivity on MAC (78.6%) vs. MHA (28.6%) (P=0.016), but VIM-producing Enterobacter, Citrobacter, and Serratia were detected with higher sensitivity on MHA (78.5%) vs. MAC (14.3%) (P=0.004). MBL producers were consistently identified with lower sensitivity using meropenem vs. ertapenem, 39.4% vs. 60.6% (P=0.0156), respectively. The effects of zinc and inoculum size were insignificant. Enterobacter aerogenes producing unspecified AmpC frequently demonstrated false positives, 66.7% with ertapenem and 22.2% with meropenem. CONCLUSIONS: The MHT should be adjusted for the local distribution of species and the carbapenemase type of MBL producers. MAC and ertapenem are preferable for assessing NDMKP, but MHA is better for VIM. Laboratory physicians should be aware of the limited sensitivity of MHT and its relatively high false-positive rate.

Increasing Carbapenem-Resistant Gram-Negative Bacilli and Decreasing Metallo-beta-Lactamase Producers over Eight Years from Korea

The trends and types of carbapenemase-producing Gram-negative bacilli were analyzed from clinical specimens collected between 2005 and 2012 at a Korean teaching hospital. The proportions of carbapenem-resistant Acinetobacter spp. increased markedly to 66%. Metallo-beta-lactamase producers significantly decreased and the majority shifted from the bla(VIM-2) type to the bla(IMP-1) type.

Characteristics of Metallo-beta-Lactamase-Producing Pseudomonas aeruginosa in Korea / 감염과화학요법

BACKGROUND: The aim of this study was to investigate the molecular epidemiological characteristics of metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa clinical isolates in Korea. MATERIALS AND METHODS: Three hundred and twenty nine P. aeruginosa clinical isolates were collected from 23 general hospitals in Korea from March to June 2014. Species were identified by matrix-assited laser desorption/ionization-time of flight and 16S rRNA sequencing. Antimicrobial susceptibility was determined by disk diffusion methods. Further, minimum inhibitory concentrations of carbapenems were determined by Etest. Polymerase chain reaction and sequencing were performed to identify genes encoding MBLs. Multi-locus sequence typing and pulsed-field gel electrophoresis were performed to determine epidemiological characteristics of MBL-producing P. aeruginosa isolates. RESULTS: Of the 329 isolates, 229 (69.6%) were susceptible to the carbapenems tested, including imipenem and meropenem; while 100 (30.4%) were non-susceptible to more than one of the carbapenems. Genes encoding imipenemase-6 (IMP-6) and Verona imipenemase-2 (VIM-2) MBLs were identified in 21 (6.4%) isolates (n = 17 and 4, respectively). All MBL-producing isolates showed multi-drug resistant phenotype, and a majority (n = 19) of the isolates were identified as sequence type 235 (ST235). The remaining isolates (n = 2) were identified as ST309 and ST463. CONCLUSION: P. aeruginosa ST235 might play an important role in dissemination of MBL genes in Korea.

A possible alternative to the error prone modified Hodge test to correctly identify the carbapenemase producing Gram-negative bacteria.

Context: The modified Hodge test (MHT) is widely used as a screening test for the detection of carbapenemases in Gram‑negative bacteria. This test has several pitfalls in terms of validity and interpretation. Also the test has a very low sensitivity in detecting the New Delhi metallo‑β‑lactamase (NDM). Considering the degree of dissemination of the NDM and the growing pandemic of carbapenem resistance, a more accurate alternative test is needed at the earliest. Aims: The study intends to compare the performance of the MHT with the commercially available Neo‑Sensitabs ‑ Carbapenemases/ Metallo‑β‑Lactamase (MBL) Confirmative Identification pack to find out whether the latter could be an efficient alternative to the former. Settings and Design: A total of 105 isolates of Klebsiella pneumoniae resistant to imipenem and meropenem, collected prospectively over a period of 2 years were included in the study. Subjects and Methods: The study isolates were tested with the MHT, the Neo‑Sensitabs ‑ Carbapenemases/MBL Confirmative Identification pack and polymerase chain reaction (PCR) for detecting the blaNDM‑1 gene. Results: Among the 105 isolates, the MHT identified 100 isolates as carbapenemase producers. In the five isolates negative for the MHT, four were found to produce MBLs by the Neo‑Sensitabs. The Neo‑Sensitabs did not have any false negatives when compared against the PCR. Conclusions: The MHT can give false negative results, which lead to failure in detecting the carbapenemase producers. Also considering the other pitfalls of the MHT, the Neo‑Sensitabs ‑ Carbapenemases/MBL Confirmative Identification pack could be a more efficient alternative for detection of carbapenemase production in Gram‑negative bacteria.

Incidence of blaNDM-1 gene in Escherichia coli isolates at a tertiary care referral hospital in Northeast India.

Purpose: Increasing reports on New Delhi metallo-β-lactamase-1 (NDM-1) producing Escherichia coli constitute a serious threat to global health since it is found to be highly resistant to most of the currently available antibiotics including carbapenems. This study has been performed to find out the incidence blaNDM-1 in E. coli isolates recovered from the various clinical samples at a tertiary care referral hospital in Northeast India. Materials and Methods: A total of 270 non-duplicated E. coli isolates were recovered from the various clinical samples at a tertiary care referral hospital in Northeast India. All isolates with reduced susceptibility to meropenem or ertapenem (diameter of zones of inhibition, ≤21 mm) were further phenotypically confirmed for carbapenemase production by modified Hodge test. All screened isolates were also subjected to the polymerase chain reaction detection of blaNDM-1 gene and additional bla genes coding for transmission electron microscopy, SHV, CTX-M, and AmpC. Results: Out of 270 E. coli isolates, 14 were screened for carbapenemase production on the basis of their reduced susceptibility to meropenem or ertapenem. All screened isolates were found to be positive for blaNDM-1 . Each of the blaNDM-1 possessing isolate was also positive for two or more additional bla genes, such as blaTEM , blaCTX-M and blaAmpC . Phylogenetic analysis showed very less variation in blaNDM-1 gene with respect to blaNDM-1 possessing E. coli isolates from other parts of India and abroad. Conclusions: Our findings highlight the incidence of blaNDM-1 in E. coli isolates with a reduced susceptibility to meropenem or ertapenem.

Loss of blaVIM-2 and blaIMP-1 during the Storage of Gram-Negative Bacilli, Antimicrobial Susceptibility of the Gene-Lost Strain, and Location of the Gene in the Cell / 대한임상미생물학회지

BACKGROUND: Gram-negative bacilli can be stored in cystine tryptic agar (CTA) at room temperature for over 1 year, but we experienced a loss of imipenem resistance among VIM-2-producing isolates. The aims of this study were to determine the frequency of loss of IMP-1 and VIM-2 genes during storage in CTA at room temperature and to document any change in the MIC of antimicrobial agents and the location of the gene. METHODS: Bacteria were isolated from clinical specimens at Severance Hospital collected from 1995-2000. Modified Hodge and double disk synergy tests were performed for screening of MBL-production isolates, and blaIMP-1 and blaVIM-2 were detected by PCR. Loss of resistance was tested in CTA at room temperature. PFGE and hybridization using a blaVIM-2 probe were carried out to determine the location of the VIM-2 gene. RESULTS: When VIM-2- and IMP-1-producing strains of eight P. aeruginosa and two Acinetobacter spp. were stored in CTA at room temperature, some isolates lost imipenem resistance after 3 days and 90% lost resistance after 15 weeks. Loss of resistance genes resulted in a decrease of the MIC of imipenem from 32-128 mug/mL to 0.5-8 mug/mL for P. aeruginosa, and from 32 mug/mL to 0.25-4 mug/mL for Acinetobacter spp. Hybridization of I-CeuI and S1-digested and PFGE suggested that VIM-2 genes are located on approximately 50-100 kb or 400 kb plasmids. CONCLUSION: Isolates may lose resistance genes when stored in CTA at room temperature. Therefore, it is necessary for MBL-production tests including the Modified Hodge test and double disk synergy test and detection of MBL genes.

Metalo beta lactamasas: el fin de los beta lactámicos? / Metallo beta lactamases: the end of beta lactams?

Uno de los grupos de antibióticos más importantes, es el grupo de los beta-lactámicos. Para ejercer su acción antimicrobiana los beta-lactámicos requieren penetrar la pared celular y atacar las Proteínas Ligadoras de Penicilinas (Penicilin Binding Proteins: PBP). El mecanismo más utilizado por los bacilos Gram negativos para adquirir resistencia a beta-lactámicos, es la inactivación de las drogas por las enzimas beta-lactamasas. La producción de beta-lactamasas tipo carbapenemasas es un mecanismo de resistencia de gran importancia. Estas enzimas, codificadas por genes que en su mayoría están localizados en elementos genéticos tales como los integrones o insertados en elementos móviles como transposones y plásmidos, se han extendido rápidamente entre los agentes patógenos de importancia clínica, como Enterobacterias, P. aeruginosa y A. baumanii. Las metalo-beta-lactamasas (MbetaL) pertenecientes al grupo B de Ambler y el grupo 3 de Bush, poseen cuatro características principales: (i) Poseen actividad contra los carbapenemes, (ii) No hidrolizan los monobáctamicos como el aztreonam, (iii) Son inhibidas por quelantes como el EDTA o el Mercapto acetato de Sodio; y (iv) Requieren cationes +2 divalentes, generalmente Zn como cofactor para su actividad catalítica. La aproximación de un disco conteniendo un agente quelante a uno que contiene un carbapeneme podría resultar una herramienta útil para la detección de MbetaLs. El efecto sinérgico entre los carbapenemes [imipenem (IPM) y/o meropenem (MEN)] y el agente quelante sería indicativo de la presencia de MbetaL. La aparición de metalo-beta-lactamasas como una amenaza sustancial a la salud debe impulsar a las autoridades de salud para formular un plan de contención, para su implementación a nivel nacional. Este plan debe asegurar la detección temprana de casos, la vigilancia permanente de brotes y una estrategia en entornos con presencia espor dica o ausencia completa de productores metalo-beta-lactamasa.

One of the most important groups of antibiotics, is the group of beta-lactams. To exert its antimicrobial action the beta-lactam require penetrate and attack the cell wall Penicillin Binding Proteins (PBP). The mechanism used by Gram negative bacilli to acquire resistance tobeta-lactams, is drug inactivation by beta-lactamase enzymes. The production of beta-lactamase carbapenemases is a resistance mechanism of great importance. These enzymes encoded by genes that are located mostly in genetic elements such as integrons or inserted in mobile elements such as transposons and plasmids, have spread rapidly among clinically important pathogens such as Enterobacteriaceae, P. aeruginosa and A. baumannii. The metallo-beta-lactamases (MbetaL) in group B and group 3 Ambler Bush, have four main characteristics: (i) possess activity against carbapenems, (ii) not hydrolyze monobactams like aztreonam, (iii) are inhibited by chelating agents such as EDTA or sodium acetate Mercapto and (iv) require divalent cations, usually Zn+2 cofactor for its catalytic activity. The approximation of a disc containing a chelating agent containing one carbapeneme could be a useful tool for detecting MbetaLs. The synergistic effect between carbapenems [imipenem (IPM) and/or meropenem (MEN)] and the chelating agent would be indicative of the presence of MbetaL. The emergence of metallo-beta-lactamase as a substantial threat to health should prompt health officials to develop a plan of containment, for implementation at the national level. This plan should ensure early case detection, continuous surveillance of outbreaks and strategy in environments with sporadic presence or complete absence of metallo-beta-lactamase.

Detection and characterization of metallo-β-lactamases in Pseudomonas aeruginosa by phenotypic and molecular methods from clinical samples in a tertiary care hospital / Detección y caracterización de la metallo-betalactamasa en la Pseudomonas aeruginosa mediante métodos fenotípicos y moleculares a partir de muestras clínicas en un hospital de atención terciaria

AIMS: The aim of this study was to detect and characterize the presence of metallo-β-lactamase (MBL) production in multidrug resistant (MDR) P aeruginosa collected from clinical samples in a tertiary care hospital. METHODS AND MATERIALS: A total of 67 non-repetitive isolates of MDR P aeruginosa recovered from various clinical specimens were screened for MBL production by IPM/MEM-EDTA combined disc test. Polymerase chain reaction was performed on all isolates using blaIMP and blaVIM consensus primers to characterize them genotypically. RESULTS: Among 67 P aeruginosa isolates, 62.7% (42/67) and 70.1% (47/67) were resistant to imipenem and meropenem respectively and 47 (70.1%) were found to be MBL producers. Among this 47 MBL-producing isolates, 41 (61.1%) strains carried the blaVIM gene and 2 (3%) strains carried the blaIMP gene. Three strains were phenotypically negative but positive genotypically for blaVIM gene. One strain was resistant to both imipenem and meropenem but did not show phenotypic positivity. CONCLUSION: This study confirms the dissemination of blaVIM genes among MDR Pseudomonas aeruginosa and hence it is indispensible to identify and aptly control the threat of horizontal and vertical transfer.

OBJETIVO: El objetivo de este estudio es descubrir y caracterizar la presencia de producción de metallo-betalactamasa (MBL) en P aeruginosa resistente a los multifármacos (RMF), recogida de muestras clínicas de un hospital de atención terciaria. MÉTODO: Un total de 67 aislados no repetitivos de P aeruginosa RMF obtenidos de varios specímenes clínicos, fueron tamizados en busca de producción de MBL, mediante una prueba de disco combinado IPM/MEM-EDTA. Se efectuó una reacción en cadena de la polimerasa sobre todos los aislados, usando iniciadores de consenso blaIMP y blaVIM para la caracterización genotípica. RESULTADOS: Entre los aislados de P aeruginosa, 62.7% (42/67) y 70.1% (47/67) fueron resistentes al Imipenem y al Meropenem respectivamente, mientras que se halló que 47 (70.1%) eran productores de MBL. De los 47 aislados productores de MBL, 41 (61.1%) cepas eran portadoras del gen blaVIM en tanto que 2 (3%) cepas eran portadoras del gen blaIMP. Tres cepas fueron fenotípicamente negativas, pero genotípicamente positivas con respecto al gen blaVIM. Una cepa fue resistente tanto al Imipenem como al Meropenem, pero no mostró positividad fenotípicamente. CONCLUSIÓN: El presente estudio confirma la diseminación de los genes blaVIM entre las Pseudomonas aeruginosa RMF. Es importante identificar así como controlar adecuadamente la amenaza de la transferencia horizontal y vertical.