Background:Emergence and spread of carbapenemase (blaOXA) genes in multidrug resistant Acinetobacter baumannii (MDR-AB) forming biofilm complicated treatment of the patients infected with this microorganism particularly in intensive care units (ICUs).Objectives:The current study aimed to determine the prevalence of molecular class-D OXA carbapenemase in biofilm and non-biofilm forming strains of MDR-AB.Materials and Methods:A total of 65 strains of MDR-AB were isolated from the patients hospitalized in the ICU of two hospitals in Kerman, Iran. The isolates were identified by conventional microbiological tests as well as API 20NE assay. Antibiotic susceptibility was carried out by disk diffusion method; minimum inhibitory concentration (MIC) of carbapenems was measured by E-test. The presence of blaOXA genes among the isolates were studied by duplex-polymerase chain reaction and application of appropriate primers. Biofilm formation was detected by microtiter plate method.Results:The isolates were highly resistant to ciprofloxacin, levofloxacin, piperacillin, nalidixic acid and third generation cephalosporins such as tigecycline (7%; n = 5) and colistin (13%; n = 8). Among the isolates, 77% (n = 50) exhibited high MIC (265μg/mL) for imipenem. Both the blaOXA-51 and blaOXA-23 like genes coexisted in all the isolates; while, blaOXA-24/40 like gene was only detected in 29 imipenem-resistant strains (P ≤ 0.05). The blaOXA-58 like gene was not detected among the isolated strains. Quantification of biofilm introduced 23 isolates (including blaOXA-24/40 strains) with efficient attachment to microtiter plate; while, those isolates without blaOXA-24/40, or imipenem-sensitive strains formed weak or no biofilm.Conclusions:Coexistence of the blaOXA-51, blaOXA-23 and blaOXA-24/40 like genes, along with formation of strong biofilm, in MDR-AB strains particularly with indiscriminate use of imipenem, complicated treatment of the patients infected with these bacteria in the hospitals understudy.

Acinetobacter baumannii is an important source of infections in intensive care units (ICUs) of our hospitals in Kerman, Iran and the most frequently isolated strains produce biofilm. There is a little information about role of iron (Fe) levels on acyl homoserine lactone (AHL) production and biofilm formation in this microorganism. In the present study, we investigated the influence of iron-III limitation on AHL, siderophore, catechol and virulence factors in the biofilm forming clinical strains of A. baumannii. A total of 65 non-duplicated multidrug resistance (MDR) strains of A. baumannii were isolated from patients in ICUs of 2 hospitals in Kerman, Iran. Antibiotic susceptibility, siderophore and other iron chelators, hemolysis, cell twitching motility, capsule, gelatinase and DNase were studied. Presence of quorum sensing, LuxI and LuxR genes was detected by multiplex-PCR. AHL activity quantified by colorimetric method and the functional groups were determined by Fourier Transform Infra-Red Spectroscopy (FT-IR). Biofilm formation was detected by microtiter plate technique. All of the isolates were resistant to third generation of cephalosporins, ciprofloxacin, levofloxacin, tetracycline, whereas, 78% and 81% were resistant to amikacin and carbapenems, respectively. The siderophore activity was highest at 20 mM Fe 3C (70%); however, it decreased to 45% as concentration of Fe 3C increased to 80 mM. Furthermore, screening of the isolates for LuxI and LuxR genes showed that presence of both genes required in the isolates with high AHL activity. FT-IR analysis indicated CDO bond of the lactone ring and primary amides. Significantly, a higher amount of AHL (70%) was detected in the presence of low concentration of iron-III (20 mM); as iron concentration increased to 80 mM, the AHL activity was reduced to 40% (P 0.05). All the isolates exhibited twitching motility and had a capsule. No any gelatinase or DNase activity was detected. Quantification of the biofilm formation introduced 23 isolates with efficient attachment to microplate wells and strong biofilm. We found that both the AHL production and biofilm formation were regulated by iron concentration in a dose dependent manner. These findings provide evidence that iron limitation plays an important regulatory role in AHL and siderophore production resulting in strong or weak biofilm, thereby helping the organism to persist in less available micronutrient environment.

Resistance-nodulation-division efflux system (RND) adeABC contributes to intrinsic resistance to various drug classes in Acinetobacter baumannii. Similarly, quorum sensing (QS) plays an important role in the biofilm formation and pathogenicity of this bacterium. The aims of this study were to evaluate the influence of iron limitation on the expression of efflux pump (adeABC) genes and QS (luxI, luxR) system by relative quantitative real-time polymerase chain reaction (qRT-PCR). In addition, DNA sequence and phylogenetic relatedness of biofilm-associated protein (Bap) gene was also investigated. Sixty-five multidrug-resistant isolates of A. baumannii were recovered from ICU patients of three hospitals in Kerman, Iran. The isolates were highly resistant to at least 11 antibiotics (MIC ≥64 μg/mL); however, 87% and 89% were susceptible to colistin and tigecycline, respectively (MIC 0.05 μg/mL) (p ≤ 0.05). We detected the presence of RND efflux pump, QS, and bap genes with the frequencies of 92% (adeA), 61.5% (adeB), 84.6% (adeC), 80% (luxI), 61% (luxR), and 66% (bap), respectively. qRT-PCR analysis showed that in some isolates, expression of both adeABC and luxI/R was increased more than fourfold in the presence of low iron (20 μm), suggesting the additional regulatory role of iron on both efflux pump and QS system. Alignment and phylogenetic analysis on the strong biofilm forming isolates confirmed that the fragments amplified were indeed part of bap gene and deduced sequence was similar to A. baumannii K9B410.

Infections and outbreaks caused by multidrug-resistant Acinetobacter baumannii (MDR-AB) are prevalent and have been reported worldwide over the past 20 or more years. Class 1 integron in MDR-AB plays an important role in the spread of antibiotic resistance in clinical settings. This study has been conducted to evaluate the detection of metallo-b-lactamase, characterization of class 1 integron and determination of clonal relatedness among A. baumannii hospital isolates. Sixty-five clinical isolates of MDR-AB were recovered from two Iranian hospital's intensive care units from February to August 2013. Integrase (intI1) and bla IMP genes were detected in 70.8 % (n=46/65) and 9.23 % (n=6/65) of the isolates using PCR assay, respectively. No other metallo-b-lactamase genes (bla VIM , bla SIM and bla NDM ) were detected. PCR sequencing of integron gene cassette revealed the following arrays: bla OXA10 -aacA4-bla IMP-55 -cmlA5 (as a novel array was designated In1243), aacC1 and aadA1. Analysis of bla IMP gene revealed a new allele designated as bla . Gene transfer experiment by conjugation showed the 36 kb conjugative plasmid harbouring In1243. The clonal assessment by repetitive extragenic palindromic PCR demonstrated a high-degree relatedness among the strains, but strains harbouring In1243 displayed a different repetitive extragenic palindromic PCR profile. In this study, we found that a novel class 1 integron (In1243) that encoded a new bla IMP allele resided on a transferable plasmid in non-clonal strains of MDR-AB.