Detection of virulence and ß-lactamase encoding genes in Enterobacter aerogenes and Enterobacter cloacae clinical isolates from Brazil

ABSTRACT Enterobacter cloacae and E. aerogenes have been increasingly reported as important opportunistic pathogens. In this study, a high prevalence of multi-drug resistant isolates from Brazil, harboring several β-lactamase encoding genes was found. Several virulence genes were observed in E. aerogenes, contrasting with the E. cloacae isolates which presented none.

High prevalence of plasmid-mediated quinolone resistance determinants in Enterobacter cloacae isolated from hospitals of the Qazvin, Alborz, and Tehran provinces, Iran

Abstract: INTRODUCTION: Plasmid-mediated quinolone resistance (PMQR) is a growing clinical concern worldwide. The main aims of this study were to detect qnr-encoding genes and to evaluate the clonal relatedness of qnr-positive Enterobacter cloacae isolates. METHODS: A total of 116 E. cloacae isolates that were not susceptible to quinolone were obtained from seven hospitals in Tehran, five hospitals in Qazvin, and two hospitals in Karaj (Iran). Bacterial identification was performed using standard laboratory methods and API 20E strips. Quinolone resistance was determined using the Kirby-Bauer disk diffusion method according to the Clinical Laboratory Standards Institute (CLSI) guidelines. PCR and sequencing were employed to detect qnrA, qnrB, and qnrS genes, and clonal relatedness was assessed using the enterobacterial repetitive intergenic consensus (ERIC)-PCR method. RESULTS: In total, 45 (38.8%) and 71 (61.2%) of isolates showed high- and low-level quinolone resistance, respectively, and qnr-encoding genes were detected in 70 (60.3%) of them. qnrB1 [45 (38.8%) isolates] was the most commonly detected gene, followed by qnrS1 [28 (24.1%) isolates] and qnrB4 [18 (15.5%) isolates] either alone or in combination with other genes. The results of the ERIC-PCR revealed that 53 (75.7%) qnr-positive isolates were genetically unrelated. CONCLUSIONS: This study describes, for the first time, the high prevalence of the qnrB1, qnrS1, and qnrB4 genes among E. cloacae isolates in Iran. The detection of qnr genes emphasizes the need for establishing tactful policies associated with infection control measures in hospital settings in Iran.

KPC and VIM producing Enterobacter cloacae strain from a Hospital in Northeastern Venezuela / Enterobacter cloacae productor de KPC y VIM en un Hospital del Noreste de Venezuela

An 83-year-old male patient is admitted to the central hospital in Cumaná, Venezuela with severe urinary infection, history of hospitalizations and prolonged antimicrobial treatments. A strain of Enterobacter cloacae was isolated showing resistance to multiple types of antibiotics (only sensitive to gentamicin), with phenotype of serine- and metallo-carbapenemases. Both, blaVIM-2 and blaKPC genes were detected in the isolate. This is the first report of an Enterobacteriaceae species producing both KPC carbapenemase and VIM metallo carbapenemase in Venezuela. This finding has a great clinical and epidemiological impact in the region, because of the feasibility of transferring these genes, through mobile elements to other strains of Enterobacter and to other infection-causing species of bacteria.

En un paciente masculino de 83 años, que ingresó al Hospital de Cumaná, Venezuela, con diagnóstico de infección urinaria severa, antecedentes de hospitalización y diferentes tratamientos antimicrobianos durante largos periodos de tiempo, se aisló una cepa de Enterobacter cloacae, la cual evidenció resistencia a múltiples tipos de antibióticos (solo sensible a gentamicina) y con fenotipo de carbapenemasas de tipo serina y metalobetalactamasa. Los genes blaVIM-2 y blaKPC fueron detectados en esta cepa. Este representa el primer reporte de una especie de Enterobacteriaceae productora simultánea de carbapenemasa KPC y metalobetalactamasa VIM en Venezuela. Esto tiene un gran impacto clínico y epidemiológico en la región por la posibilidad de transferencia de estos genes a otras cepas de Enterobacter u otras especies bacterianas causantes de infecciones, por medio de elementos móviles.

Molecular epidemiology of CTX-M producing Enterobacteriaceae isolated from bloodstream infections in Rio de Janeiro, Brazil: emergence of CTX-M-15

OBJECTIVE: The present studywas designed to evaluate the molecular epidemiology of CTX-M producing Klebsiella pneumoniae, Enterobacter cloacae and Escherichia coli isolated from bloodstream infections at tertiary care hospitals in the State of Rio de Janeiro, Brazil. MATERIAL AND METHODS: A total of 231 nonduplicate Enterobacteriaceae were isolated from five Brazilian hospitals between September 2007 and September 2008. The antimicrobial susceptibility testing was performed by disk diffusion method according to the Clinical Laboratory Standard Institute. Isolates showing resistance to third-generation cephalosporins were screened for ESBL activity by the double-disk synergy test. The presence of blaCTX-M , blaCTX-M-15 and blaKPC genes was determined by Polymerase Chain Reaction (PCR) amplification andDNA sequencing. The molecular typing of CTX-M producing isolateswas performed by pulsed-field gel electrophoresis (PFGE). RESULTS AND DISCUSSION: Ninety-three isolates were screened as ESBL positive and 85 (91%) were found to carry CTX-M-type, as follows: K. pneumoniae 59 (49%), E. cloacae 15 (42%), and E. coli 11 (15%). Ten isolates resistant for carbapenems in K. pneumoniae were blaKPC-2 gene positive. Among CTX-M type isolates, CTX-M-15 was predominant in more than 50% of isolates for K. pneumoniae, E. coli, and E. cloacae. PFGE analysis of CTX-M producing isolates showed the predominance of CTX-M-15 in 10 of 24 pulsotypes in K. pneumoniae, 6 of 13 in E. cloacae and 3 of 6 in E. coli. CTX-M-15 was also predominant among KPC producing isolates. In conclusion, this study showed that CTX-M-15 was circulating in Rio de Janeiro state in 2007-2008. This data reinforce the need for continuing surveillance because this scenario may have changed over the years.

Aminoglycoside Susceptibility Profiles of Enterobacter cloacae Isolates Harboring the aac(6')-Ib Gene / 대한진단검사의학회지

The aminoglycoside 6'-N-acetyltransferases of type Ib (aac(6')-Ib) gene confers resistance to amikacin, tobramycin, kanamycin, and netilmicin but not gentamicin. However, some isolates harboring this gene show reduced susceptibility to amikacin. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommends a revision of the phenotypic description for isolates harboring the aac(6')-Ib gene. In this study, we determined the aminoglycoside susceptibility profiles of 58 AAC(6')-Ib-producing Enterobacter cloacae isolates. On the basis of the CLSI and EUCAST breakpoints, a large proportion (84.5% and 55.2%, respectively) of these 58 isolates were found to be susceptible to amikacin. However, among the isolates that were shown to be anikacin-susceptible according to the CLSI and EUCAST breakpoints, only 30.6% and 18.8% isolates, respectively, could be considered to have intermediate resistance on the basis of the EUCAST expert rules. Further studies should be conducted to determine the aminoglycoside susceptibility profiles of aac(6')-Ib-harboring isolates from various geographic regions and to monitor the therapeutic efficacy of amikacin in infections caused by these isolates.

Resistencia a Cefepime en aislamientos de Enterobacter cloacae provenientes de hospitales de Bogotá, Colombia / Resistance to Cefepime in Enterobacter cloacae isolates from hospitals in Bogotá, Colombia

OBJETIVO: Detectar la presencia de genes codificantes de beta-lactamasas que pueden conferir resistencia al cefepime en aislamientos de Enterobacter cloacae, para los cuales este antibiótico se considera una opción terapéutica importante. MATERIALES Y MÉTODOS: Se analizaron 28 aislamientos provenientes de 4 hospitales de Bogotá, recolectados durante el año 2003. Se determinó fenotípicamente la producción de enzimas tipo cefalosporinasa y beta-lactamasas de espectro extendido. La presencia de genes bla codificantes para beta-lactamasas se detectó mediante amplificación por reacción en cadena de la polimerasa. Se evaluó por conjugación la posible transferencia de los genes bla que codifican para cefotaximasas. RESULTADOS: Las pruebas microbiológicas mostraron que un 57 por ciento de los aislamientos eran productores de beta-lactamasas de espectro extendido. En 82 por ciento de los aislamientos se detectaron, genes blaTEM, blaSHV y blaCTX-M. Siete de los 9 aislamientos que portaban genes blaCTX-M del grupo 1 estuvieron en el rango de intermedios o resistentes a cefepime. Estos aislamientos produjeron transconjugantes resistentes a cefotaxima. CONCLUSION: Se encontró relación entre la resistencia a cefepime y la presencia de cefotaximasas en E. cloacae. Los genes blaCTX-M estuvieron presentes en 32 por ciento de los aislamientos, indicando una diseminación importante en estos hospitales. La facilidad de transferencia de estos genes entre especies y géneros de enterobacterias es una razón importante para detectarlos y controlar su proliferación en el medio hospitalario. Estos resultados sugieren proceder con cautela en el uso de cefepime como alternativa terapéutica en las infecciones causadas por E. cloacae ante la posible presencia de cefotaximasas en estos aislamientos.

Caracterización fenotípica y genotípica de la resistencia enzimática a las cefalosporinas de tercera generación en Enterobacter spp. / Phenotypic and genotypic characterization of resistance to third-generation cephalosporins in Enterobacter spp.

Enterobacter spp. es un patógeno intrahospitalario que presenta múltiples mecanismos de resistencia a los antibióticos b-lactámicos. Se caracterizaron fenotípica y genotípicamente las diferentes b-lactamasas presentes en 27 aislamientos consecutivos e ininterrumpidos de Enterobacter spp. (25 Enterobacter cloacae y 2 Enterobacter aerogenes). También se evaluó la habilidad de diferentes métodos fenotípicos para detectar b-lactamasas de espectro extendido (BLEE) en estos microorganismos. En 15/27 aislamientos (63%) se observó resistencia a las cefalosporinas de tercera generación. En 12 de los aislamientos resistentes se detectó un alto nivel de producción de cefalosporinasa cromosómica, siendo 6 de ellos también productores de PER-2. Dicha resistencia en los 3 aislamientos restantes se debió exclusivamente a la presencia de BLEE, PER-2 en 2 de ellos y CTX-M-2 en un caso. Sólo CTX-M-2 se detectó con todas las cefalosporinas probadas en los ensayos de sinergia, utilizando el método de difusión, mientras que cefepima mejoró la detección de PER-2 en 7/8 aislamientos productores de esta BLEE, 4/8 utilizando la prueba de doble disco y 7/8 comparando discos de cefepima con y sin el agregado de ácido clavulánico. El método de dilución empleado solo detectó 1/9 BLEE al comparar las cefalosporinas con y sin el agregado de inhibidor.

Enterobacter spp. are becoming increasingly frequent nosocomial pathogens with multiple resistance mechanism to b-lactam antibiotics. We carried out the phenotypic and genotypic characterization of beta-lactamases in 27 Enterobacter spp. (25 Enterobacter cloacae y 2 Enterobacter aerogenes), as well as the ability of different extended spectrum b-lactamase (ESBL) screening methods. Resistance to third generation cephalosporins was observed in 15/27 (63%) isolates. Twelve resistant isolates produced high level chromosomal encoded AmpC b-lactamase; 6 of them were also producers of PER-2. Resistance to third generation cephalosporins in the remaining 3 isolates was due to the presence of ESBLs, PER-2 in 2 cases, and CTX-M-2 in the other. Only CTX-M-2 production was detected with all tested cephalosporins using difusion synergy tests, while cefepime improved ESBLs detection in 7/8 PER-2 producers, 4/8 in the inhibitor aproximation test and 7/8 with double disk test using cefepime containing disk with and without clavulanic acid. Dilution method, including cephalosporins with and without the inhibitor detected 1/9 ESBLs producers.

Molecular epidemiology of a nosocomial outbreak due to Enterobacter cloacae and Enterobacter agglomerans in Campinas, São Paulo, Brazil

A total of 73 isolates (57 Enterobacter cloacae and 16 Enterobacter agglomerans), recovered during an outbreak of bacteremia in the Campinas area, São Paulo, Brazil, were studied. Of these isolates, 61 were from parenteral nutrition solutions, 9 from blood cultures, 2 from a sealed bottle of parenteral nutrition solution, and one was of unknown origin. Of the 57 E. cloacae isolates, 54 were biotype 26, two were biotype 66 and one was non-typable. Of 39 E. cloacae isolates submitted to ribotyping, 87.2 percent showed the same banding pattern after cleavage with EcoRI and BamHI. No important differences were observed in the antimicrobial susceptibility patterns among E. cloacae isolates exhibiting the same biotype, serotype and ribotype. All E. agglomerans isolates, irrespective of their origin, showed same patterns when cleaved with EcoRI and BamHI. The results of this investigation suggest an intrinsic contamination of parenteral nutrition solutions and incriminate these products as a vehicle of infection in this outbreak

Phenotyping and genotyping methods applied to investigate the relatedness of Brazilian isolates of Enterobacter cloacae

In order to evaluate the resolving power of several typing methods to identify relatedness among Brazilian strains of Enterobacter cloacae, we selected twenty isolates from different patients on three wards of a University Hospital (Orthopedics, Nephrology, and Hematology). Traditional phenotyping methods applied to isolates included biotyping, antibiotic sensitivity, phage-typing, and O-serotyping. Plasmid profile analysis, ribotyping, and macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) were used as genotyping methods. Sero- and phage-typing were not useful since the majority of isolates could not be subtyped by these methods. Biotyping, antibiogram and plasmid profile permitted us to classify the samples into different groups depending on the method used, and consequently were not reliable. Ribotyping and PFGE were significantly correlated with the clinical epidemiological analysis. PFGE did not type strains containing nonspecific DNase. Ribotyping was the most discriminative method for typing Brazilian isolates of E. cloacae