Study On The Carbapenemases And Integrons Among Carbapenem-resistant Acinetobacter Baumannii

Objective The study was conducted to determine the drug resistance, prevalence of carbapenemases and integrons among the isolates of carbapenem-resistant Acinetobacter baumannii. To analyze the molecular epidemiological characteristics of nosocomial resistant isolates, control spread of resistant isolates in the hospital.Methods A total of 103 non-repetitive imipenem-resistant A. baumannii were isolated from January 2008 to March 2010 in the General Hospital of Tianjin Medical University. The antimicrobial susceptibility test was performed by using VITEK-2 compact automatic system. Isolates of imipenem-resistant A. baumannii were screened for carbapenemases by modified Hodge test, improved three-dimensional test, EDTA synergy test and 2-mercaptopropionic acid synergy test. Isolates were then subjected to the multiplex PCR targeting genes encoding for OXA type carbapenemases (blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, and blaOXA-58-like), metallo-β-lactamases (IMP-1 and VIM-2) and integrases (intll and intI2). PCR was conducted to detect the insertion sequence ISAbal. Isolates harboring classⅠor classⅡintegrase genes were further investigated by PCR and sequencing of the variable regions. Random amplified polymorphic DNA (RAPD) genotyping was performed to assess genetic relatedness.Results The multidrug resistance status of A. baumannii clinical isolates was severe. All the 103 isolates exhibited resistant to meropenem. Cefoperazone-sulbactam was the most active antimicrobial agent tested in our study (susceptible rate:35.9%), amikacin was the second active agent (susceptible rate:29.1%). The rates of susceptibility to the other drugs were lower than 21%. Among the 103 isolates, 75(72.8%) demonstrated positive in the modified Hodge test,80(77.7%) were positive in the improved three-dimensional test. No metallo-p-lactamase was found in the 2-mercaptopropionic acid synergy test. The multiplex-PCR indicated that all strains possessed a blaOXA-51-like gene (100%), intll, blaOXA-23-like, blaOXA-24-like was detected in 92(89.3%),89(86.4%),2(1.9%), respectively. The co-existence of blaOXA-51-like+blaOXA-23-like+intI1, blaOXA-51-like+blaOXA-23-like, blaOXA-51-like+intI1, blaOXA-51-like+blaOXA-24-like was detected in 84,5,8,2 isolates, respectively. Four' isolates were only found positive for blaOXA-51-like-The blaOXA-58-like, IMP-1, VIM-2 and intI2 genes were all negative. Sequencing of the entire blaOXA-51-like, blaOXA-23-like, and blaOXA-24-like genes indicated the presence of blaOXA-66, blaOXA-23, and blaOXA-72, respectively. OXA-23-like gene of 87 strains was just on the down-stream of insertion sequence ISAbal. No ISAbal was detected upstream of blaOXA-51-like-Eighty-nine (96.7%) of the intI1 positive strains owned the variable region. PCR amplification and sequencing revealed that 81 intergron-positive A. baumannii isolates contained a 2300 bp gene cassette array with aacA4, aadAl and catB8. A 3000 bp PCR product was obtained from 8 isolates. Sequencing confirmed the presence of gene cassettes aacC1, orfX, orfX, orfX'and aadAla. Four integron-positive isolates didn't contain gene cassettes. RAPD of 103 isolates showed 7 genotypes. The RAPD patterns were as followed:pattern A (45 isolates), pattern B (23 isolates), pattern C (16 isolates), pattern D (10 isolates), pattern E (7 isolates) and pattern F (1 isolate, producing OXA-72), pattern G (1 isolate, producing OXA-72).Conclusion These findings show that multi-drug resistance in A. baumannii is a common problem. The modified Hodge test and improved three-dimensional test could be used for screening carbapenemases in A. baumannii. It is necessary to carry out PCR assays designed for the detection of genes encoding carbapenemases. The presence of OXA-23 carbapenemase was correlated with A. baumannii resistant to carbapenem. ISAbal has close relationship with OXA-23 carbapenemases genes, which suggests that ISAbal may provid the promoter for OXA-23 gene.The classⅠintegrons confer resistance to aminoglycoside and chloramphenicol. The result of RAPD indicates that there were carbapenem-resistant isolates clone spread in the hospital.