PCR Assay Based on the gyrB Gene for Rapid Identification of Acinetobacter baumannii-calcoaceticus Complex at Specie Level.

BACKGROUND: The genus Acinetobacter sp. comprises more than 50 species, and four are closely related and difficult to be distinguished by either phenotypic or genotypic methods: the Acinetobacter calcoaceticus-baumannii complex (ABC). The correct identification at species level is necessary mainly due to the epidemiological aspects. METHODS: We evaluated a multiplex PCR for gyrB gene to identify the species of the ABC using the sequencing of the ITS 16S-23S fragment as a gold standard. Isolates identified as Acinetobacter calcoaceticus-baumannii from three hospitals at southern Brazil in 2011 were included in this study. RESULTS: A total of 117 isolates were obtained and 106 (90.6%) were confirmed as A. baumannii, 6 (5.1%) as A. nosocomialis and 4 (3.4%) as A. pittii by PCR for gyrB gene. Only one isolate did not present a product of the PCR for the gyrB gene; this isolate was identified as Acinetobacter genospecie 10 by sequencing of ITS. We also noted that the non-A. baumannii isolates were recovered from respiratory tract (8/72.7%), blood (2/18.2%) and urine (1/9.1%), suggesting that these species can cause serious infection. CONCLUSION: These findings evidenced that the multiplex PCR of the gyrB is a feasible and simple method to identify isolates of the ABC at the species level.

Molecular characterization of Klebsiella pneumoniae carbapenemase-producing isolates in southern Brazil.

Carbapenem-resistant Enterobacteriaceae have been frequently reported worldwide. They represent a serious concern because of the limited therapeutic options. The aim of this study was to investigate the molecular epidemiology of 14 Klebsiella pneumoniae carbapenemase (KPC) producers among 345 clinical isolates of Enterobacteriaceae with reduced susceptibility to carbapenems recovered from 11 separate hospitals in southern Brazil. The blaKPC-2 gene was detected in 14 isolates (4 %): six Enterobacter cloacae, five K. pneumoniae and three Serratia marcescens. Most of these isolates exhibited high-level resistance against ß-lactams and ciprofloxacin, while the most active drugs were polymyxin B and amikacin. Genetic environment analysis, based on the classical Tn4401 structure, revealed six distinct platforms. Plasmids carrying blaKPC-2 were not typable and most were approximately 20 kb. Only KPC carbapenemases were found among the isolates studied, highlighting the local relevance of these enzymes in acquired resistance to carbapenems in Enterobacteriaceae. Our results contribute to the understanding of carbapenem resistance in Enterobacteriaceae and to the molecular characterization of KPC-2-producing isolates in Brazil.

Hetero- and adaptive resistance to polymyxin B in OXA-23-producing carbapenem-resistant Acinetobacter baumannii isolates.

BACKGROUND: Resistance rates to polymyxin B in surveillance studies have been very low despite its increasing use worldwide as the last resort therapy for multidrug-resistant Gram-negative bacilli. However, two other resistance phenotypes, hetero- and adaptive resistance, have been reported to polymyxin. We aimed to investigate the presence of polymyxin B hetero- and adaptive resistance and evaluate its stability in carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates. METHODS: CRAB isolates were recovered from hospitalized patients at three Brazilian hospitals. Hetero-resistance was determined by population analysis profile (PAP). Adaptive resistance was evaluated after serial daily passages of isolates in Luria-Bertani broth containing increasing polymyxin B concentrations. MICs of polymyxin B of colonies growing at the highest polymyxin B concentration were further determined after daily sub-cultured in antibiotic-free medium and after storage at -80°C, in some selected isolates. RESULTS: Eighty OXA-23-producing CRAB isolates were typed resulting in 15 distinct clones. Twenty-nine randomly selected isolates (at least one from each clone) were selected for hetero- resistance evaluation: 26 (90%) presented growth of subpopulations with higher polymyxin B MIC than the original one in PAP. No isolate has grown at polymyxin B concentrations higher than 2 mg/L. Polymyxin B MICs of subpopulations remained higher than the original population after daily passages on antibiotic-free medium but returned to the same or similar levels after storage. Twenty-two of the 29 isolates (at least one from each clone) were evaluated for adaptive resistance: 12 (55%) presented growth in plates containing 64 mg/L of polymyxin B. Polymyxin B MICs decreased after daily passages on antibiotic-free medium and returned to the same levels after storage. CONCLUSIONS: The presence of subpopulations with higher polymyxin B MIC was extremely common and high-level adaptive resistance was very frequent in CRAB isolates.

First report of carbapenem-resistant Acinetobacter nosocomialis isolates harboring ISAba1-blaOXA-23 genes in Latin America.

In recent years, different resistance genes have been found in Acinetobacter spp., especially in the species A. baumannii. We describe two isolates of carbapenem-resistant A. nosocomialis harboring ISAba1-blaOXA-23 and blaOXA-51 found in patients from the city of Porto Alegre, southern Brazil. To the best of the authors' knowledge, this is the first report of carbapenem-resistant A. nosocomialis in Latin America.