Diversidad genética de cepas extraintestinales de Escherichia coli productoras de las betalactamasas TEM, SHV y CTX-M asociadas a la atención en salud / Genetic diversity of extraintestinal Escherichia coli strains producers of beta-lactamases TEM, SHV and CTX-M associated with healthcare

RESUMEN Introducción. En Venezuela existen pocos reportes que describan las bases genéticas del potencial patogénico y filogenético de las cepas de Escherichia coli provenientes de hospitales. Objetivo. Determinar la diversidad genética de cepas extraintestinales de E. coli productoras de las betalactamasas TEM, SHV y CTX-M asociadas a la atención de salud. Materiales y métodos. Se estudió una colección de 12 cepas extraintestinales de E. coli con sensibilidad disminuida a las cefalosporinas de amplio espectro. La sensibilidad antimicrobiana se determinó por concentración inhibitoria mínima. La detección de los grupos filogenéticos, de los factores de virulencia y de los genes que codifican la resistencia antimicrobiana se hizo mediante la técnica de reacción en cadena de la polimerasa y la relación clonal se estableció mediante reacción en cadena de la polimerasa de elementos palindrómicos extragénicos repetitivos (Repetitive Element Palindromic-PCR, rep-PCR). Resultados. Todas las cepas analizadas presentaron resistencia a las cefalosporinas, y resistencia conjunta a quinolonas y aminoglucósidos. La distribución filogenética evidenció que los grupos A y B1 fueron los más frecuentes, seguidos por D y B2; en este último, se detectaron todos los factores de virulencia evaluados, y el gen más frecuente fue el fimH. En todas las cepas analizadas, se encontró bla CTX-M, con predominio de las bla CTX-M-8, y en dos de estas cepas se evidenció la presencia simultánea de bla CTX-M-9, variantes bla CTX-M-65 y bla CTX-M-147. Conclusión. Las cepas estudiadas demostraron diversidad genética y albergaron diferentes genes de virulencia y betalactamasas de espectro extendido (BLEE) sin predominio de ningún filogrupo en particular. Este estudio constituye el primer reporte de la variante bla CTX-M-65 en Venezuela y de la variante bla CTX-M-147 en el mundo, en cepas no relacionadas genéticamente aisladas de hospitales, situación que merece atención y la racionalización del uso de los antimicrobianos.

ABSTRACT Introduction: There are few reports from Venezuela describing the genetic basis that sustains the pathogenic potential and phylogenetics of Escherichia coli extraintestinal strains isolated in health care units. Objective: To establish the genetic diversity of extraintestinal E. coli strains producers of beta-lactamases TEM, SHV and CTX-M associated with healthcare. Materials and methods: We studied a collection of 12 strains of extraintestinal E. coli with diminished sensitivity to broad-spectrum cephalosporins. Antimicrobial susceptibility was determined by minimum inhibitory concentration. We determined the phylogenetic groups, virulence factors and genes encoding antimicrobial resistance using PCR, and clonal characterization by repetitive element palindromic-PCR rep-PCR. Results: All strains showed resistance to cephalosporins and joint resistance to quinolones and aminoglycosides. The phylogenetic distribution showed that the A and B1 groups were the most frequent, followed by D and B2. We found all the virulence factors analyzed in the B2 group, and fimH gene was the most frequent among them. We found bla CTX-M in all strains,with a higher prevalence of bla CTX-M-8; two of these strains showed coproduction of bla CTX-M-9 and were genetically identified as bla CTX-M-65 and bla CTX-M-147 by sequencing. Conclusion: The strains under study showed genetic diversity, hosting a variety of virulence genes, as well as antimicrobial resistance with no particular phylogroup prevalence. This is the first report of bla CTX-M alleles in Venezuela and in the world associated to non-genetically related strains isolated in health care units, a situation that deserves attention, as well as the rationalization of antimicrobials use.

Combined Use of the Modified Hodge Test and Carbapenemase Inhibition Test for Detection of Carbapenemase-Producing Enterobacteriaceae and Metallo-beta-Lactamase-Producing Pseudomonas spp

BACKGROUND: We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-beta-lactamase (MBL)-producing Pseudomonas spp. METHODS: A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum beta-lactamase [GES]-5, 9 New Delhi metallo-beta-lactamase [NDM]-1, 5 Verona integron-encoded metallo-beta-lactamase [VIM]-2, 3 imipenem-hydrolyzing beta-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain. RESULTS: Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively. CONCLUSIONS: Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.

Preparation, crystallization and preliminary X-ray crystallographic analysis of OXA-23, a carbapenemase conferring widespread antibiotic resistance.

OXA-23, a class D carbapenemase that confers widespread antibiotic resistance hydrolyzes the β-lactam rings of β-lactam antibiotics, presenting an enormous challenge to infection control, particularly in the eradication of pathogenic bacteria such as Acinetobacter baumannii, one of six top-priority dangerous pathogens. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing carbapenemases. In this study, OXA-23 was purified and crystallized at 298 K and X-ray diffraction data from OXA-23 crystal were collected at 2.03 Å resolution using synchrotron radiation. The crystal of OXA-23 belonged to space group P41 with unit cell parameters a = 82.47, b = 82.47 and c = 172.01 Å. Analysis of the packing density showed that the asymmetric unit probably contained two molecules with a solvent content of 73.64%.

An update on newer beta-lactamases.

The resistance to beta-lactam antibiotics is an increasing problem worldwide and beta lactamases production is the most common mechanism of drug resistance. Both global and Indian figures showed a marked increase in the number of beta-lactamases producing organisms. These enzymes extended spectrum beta-lactamases (ESBLs) are numerous and continuous mutation has led to the development of enzymes having expanded substrate profile. To date, there are more than 130 TEM type and more than 50 sulphydryl variable (SHV) type beta-lactamases found in Gram negative bacilli. ESBL producing Enterobacteriaceae are, as a rule, resistant to all cephalosporins and extended spectrum penicillins including the monobactam, aztreonam, while resistance to trimethoprim - sulphamethaxazole and aminoglycosides is frequently co-transferred on the same plasmid. Many ESBL producing organisms also express Amp C beta-lactamases. Amp C- beta-lactamases are clinically significant, as these confer resistance to cephalosporins in the oxyimino group, 7 alpha-methoxy cephalosporins, and are poorly inhibited by clavulanic acid. Carbepenems are the drugs of choice for the treatment of infections caused by ESBL producing organisms but carbapenemases (MBLs) have emerged and have spread from Pseudomonas aeruginosa to Enterobacteriaceae. The routine clinical microbiology laboratories should employ simple methods to recognize these enzymes using various substrates and inhibitors. These organisms may lead to therapeutic dead ends. Presently, the therapy relies on beta-lactam/ beta-lactamases inhibitor combinations, carbepenems and piperacillin - tazobactam plus aminoglycoside combination. Proper infection control practices and barrier precautions are essential to contain the organisms producing beta-lactamases.