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ObjectiveTo investigate the identification methods of all the genotypes of Acinetobacterspeciese, including Acinetobacter calcoaceticus-Acinetobacter baumannii complex(ACB); to analyze the clinical characteristics of inpatients with infectious diseasescaused by non-baumannii Acinetobacter (NbA), the susceptibility pattern of thoseisolates; to detect metallo-beta-lactamase and integrase genes, to explore the role inmediating β-lactam antibiotics resistance, and finally to illucidate the moleculermechanisms involved and thus to provide reasonable guidance and reference forclinical antibiotic therapy.Methods1. All40NbA strains and20ACB complex strains were collected non-repetitivelywith a duration of more than1year (from February2012to April2014) from TheFirst Affiliated Hospital of Bengbu Medical College, The First Affiliated Hospital ofAnhui Medical University. Bacterial identification was carried out using GNI card ofVITEK-2compact. Additionally, for more accurate discrimintation between ACBcomplex species, all the20ACB complex strains were further subject to polymerasechain reaction (PCR) amplification to detect the A. baumannii-specific expression ofthe class D carbapenemase gene OXA-51,16S rRNA gene and ACB complex gyrBgenes, which specifically expressed in each species.2. The antimicrobial susceptibility patterns of all NbA strains were tested by diskdiffusion test (K-B method), all the clinical date of the isolates were collected and therelated clinical infection risk factors and its antimicrobial susceptibility patterns wereanalyzed. 3. Antibiotic-resistant genes which lead to β-lactam antibiotics resistance andspeading were amplified by PCR, such as: Metallo-beta-lactamase genes (SIM-1,VIM-2, IMP-4) and typeⅠ, typeⅡ and typeⅢ integrase genes.Results1. Totally all60strains bacteria were identified as Acb20strains by VITEK2compact,29strains were identified as A. lwoffii,9strains as A. junii and2strains as A.haemolyticus.16S rRNA gene amplification and DNA sequencing were performed inall20ACB complex strains, as16Acinetobacter baumannii strains,3A.calcoaceticus strains and one uncultured Acinetobacter strain. The ACB complexstrains were further undergone gyrB gene screening by PCR, of them3A.calcoaceticus strains and one13TU strain were detected in ACB complex, togetherwith16Acinetobacter baumannii strains. Additionally, OXA-51carbapenemase genewas positive in80%(16/20) strains, which was considered as Acinetobacterbaumannii specific house-keeping gene.2. All44strains NbA (29A. lwoffii strains,9A. junii strains,2A. haemolyticus strains,3A. calcoaceticus strains and1Acinetobacter genomic species13TU strain), ofwhich61.4%were A.lwoffii, were potentially gathered in Cardiology Department,Respiratory Department and ICU wards. Respiratory tract infection was the mostlyfound infectious diseases of our study (account for65.9%).3. Of all the NbA44isolates,3(6.8%) isolates (2A. lwoffii strains,1A. haemolyticusstrains) were multi-resistant, including all β-lactam antibiotics,17strains (38.6%)were susceptible to all antimicrobial agents except β-lactam antibiotics, while the rest24isolates were fully susceptible.4.9of the44(20.5%) NbA isolates carried SIM-1gene, including2A. lwoffii strains(resistant to all antimicrobial agents),5A. lwoffii strains and2A. junii strains(resistant only to β-lactam antibiotics);4strains (9.1%) carried VIM-2gene, including1A. lwoffii and1A. haemolyticus strain (resistant to all antimicrobial agents), and1A.lwoffii strain,1A. junii strain(resistant only to β-lactam antibiotics); while IMP-4gene was not detected in all NbA strains. 5. In all44NbA strains,5(11.4%) harbored Class1integrase, of which3weremultiresistant bacteria strains, and2strains β-lactam antibiotics resistant bacteria (1A. lwoffii isolate and1A. junii isolate);6(13.6%) isolates harbored Class2integrase,all of which3were resistant only to β-lactam antibiotics (3A. lwoffii strains and1A.haemolyticus strain and2A. junii); No class3integrase were found in all the NbAisolates.Conclusions1. Species identification of all Acinetobacter genospecies is a great chanllege globally.Commercially available species identification methods such as VITEK2have limiteddiscriminative efficiency, which necessitate the combination of additional genotypingmethods. OXA-51,16S rRNA and gyrB genes detaction by PCR are among the mostpromising and economic methods mostly used. Our finding showed that thecombination of species specific genes detection and VITEK2can provide moreaccurate genospecies identification in ACB complex. As showed in our study, gyrBgene is better than16S rRNA in identifying closely phylogenetic related species.2. All of the NbA strains were isolated non-repetitively from the specemens frominpatients of Cardiology Department, Respiratory Department and ICU wards. themajority of NbA were from the respiratory tracts. A. lwoffii is the most frequentisolates of all NbA, which deserves more clinical attention.3. NbA strains in our study were generally of low antibiotics resistance whencomparing with Acinetobacter baumannii clinical isolates. It is notbable that severalmultiresistant strains have been found, the importance of NbA is probablyunderestimated.4. beta-lactams-resistant genes as blaSIM-1and blaVIM-2had been found in9β-lactams resistant isolates (A. lwoffii、A. junii、A. haemolyticus?) and4β-lactamssusceptible strains (A. lwoffii、A. junii、A. haemolyticus?) in our study, suggesting theinvolvements of metallo-beta-lactamase and some additional mechanisms β-lactamsresistance, till now we have rarely found the report of A. lwoffii, A. junii, A. haemolyticus habouring blaSIM-1, blaVIM-2gene in China and and it is the firstreport in our province. Integrase genes of class1and2intergrons were found in NbAstrains, with the higher positive rate of class2integron. All strains habouring class1and2integrases were resistant to β-lactams, speculating that the class1and2intergrons play a role in antibiotic resistance, which deserves further investigation.
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