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.

Evaluation of heteroresistance to polymyxin B among carbapenem-susceptible and -resistant Pseudomonas aeruginosa.

One hundred and twenty-four Pseudomonas aeruginosa isolates were selected for antimicrobial susceptibility testing with anti-pseudomonal agents, MIC determination for polymyxin B and metallo-beta-lactamase detection (genes blaSPM, blaVIM-1, blaNDM-1 and blaIMP). According to the imipenem and/or meropenem susceptibility profile, a set of randomly selected isolates (12 isolates carbapenem-susceptible and 12 isolates carbapenem-resistant) were evaluated for heteroresistance to polymyxin B. Heteroresistance testing was performed by plating the isolates onto increasing concentrations of polymyxin B (from 0 to 8.0 mg l(-1)). The population analysis profile (PAP) was defined as the ratio of the number of colony-forming units on the plate with the highest concentration of polymyxin B at which bacterial growth occurred against the number of colony-forming units on the plate without antibiotic. Isolates presenting subpopulations that exhibited growth at polymyxin B concentrations ≥2 mg l(-1) were considered heteroresistant. Isolates containing subpopulations that grew at polymyxin B concentrations at least twice as high as the original MIC but <2 mg l(-1) were considered heterogeneous. Antimicrobial susceptibility testing results indicated a variable degree of susceptibility: high levels of resistance to gentamicin (30.6 %) and imipenem (29.0 %); low levels of resistance to aztreonam (1.6 %) and ciprofloxacin (4.8 %). All isolates were susceptible to polymyxin B: MIC50 and MIC90 were 1 mg l(-1) and 2 mg l(-1), respectively. Thirty-seven isolates (30 %) were carbapenem-resistant. Four isolates resistant to carbapenems were positive for blaIMP. There were no heteroresistant subpopulations in the carbapenem-susceptible group, but three isolates presented heterogeneous subpopulations. The PAP frequency ranged from 2.1×10(-4) to 6.9×10(-8). In the carbapenem-resistant group, one isolate was heteroresistant. Six isolates in this group presented heterogeneous subpopulations. In the resistant population, the PAP frequency ranged from 2.1×10(-7) to 2.6×10(-4). In this study, polymyxin B heteroresistance in P. aeruginosa was uncommon and occurred in only one carbapenem-resistant isolate, despite the fact that several isolates presented heterogeneous subpopulations with increased polymyxin B MICs.