Molecule Mechanism Of Integrons Mediate Multidrug Resistance Among Clinical Isolates Of Acinetobacter Baumanii

[Backgroud ] Acinetobacter baumannii has emerged as an important nosocomial pathogen that is responsible for a variety of nosocomial infections, comprising bacteremia, urinary tract infection, secondary meningitis, surgical-site infection, and nosocomial and ventilator-associated pneumonia. In recent years, several outbreaks of nosocomial infections patients, especially in intensive-care-unit (ICU) and blood ward caused by carbapenem-resistant A. baumannii have been documented. Because of the multiple antibiotic resistance exhibited by A. baumannii, nosocomial infections caused by this organism are difficult to treat. It is signification to investigate the molecular epidemiology and phenotypic of A.baumannii colonization and infection and to study the antibiotic resistant mechanisms in these strains.[Objective] To Screen multidrug-resistant clinical isolates of acinetobacter baumanii and to investigate phenotypes of these isolates. To determine P-Lactamases of the isolates and its genotypes. To detect integrase gene and its type by PCR and DNA sequencing and determine drug resistant gene cassette in variable region of integron, analysis drug resistant gene arrangement of integron structure. To investigate the effect of integron on drug-resistant phenotypic. To explore initially molecule mechanism of carbopenems-resistant acinetobacter Baumanii.[ Methods 1(1) Standard disk diffusion susceptibility tests of PIP, CAZ, CTX, IMP, AMK, and CIP were used as initial screen tests for Non-repetitive 82 isolates of acinetobacter baumanii. The standard agar dilution susceptibility tests was carried out on Mueller Hinton agar according to CLSI guidelines to determine minimal inhibition concentration(MIC) of 24 antimicrobial agents against total acinetobacter baumanii. (2 ) β-Lactamases of the isolates with decreasedsusceptibilities to PIP> CAZ. CTX, IMP> AMK, CIP by three dimensional extract test. The bla5//(/. blareA/> blaC7x-A/> b\aOXA-23- b\aoxA-24- bla/M/>andblaWA/geneof P-Lactamases for these strains were amplified and sequenced. Random Amplified Polymorphic DNA (RAPD) typing was used to investigate the homology of the resistant isolates. (3)Specificity primers of class 1,2,3 integrase were designed to detect incidence of integrase gene and its type by PCR and DNA sequencing among 82 isolates of acinetobacter baumanii. (4) PCR were used to determine drug resistant gene cassette in variable region of integrons by primers Int-CSF^ Int-CS and DNA sequencing. The results of PCR and DNA sequencing were BLAST on GeneBank(http://www.ncbi.nlm.nih.gov/BLAST/), analysis drug resistant gene type and arrangement in integron by DNAstar, explore molecule mechanism of carbopenems-resistant acinetobacter baumanii.[ Results ] (1) 82 strains of acinetobacter baumanii were identified by biochemical and Vitek system. 68 strains of mutli-resistant acinetobacter baumanii were screened by Standard disk diffusion susceptibility tests , of these isolates, 62(75.6%) appeared to carbopenems-resistant. (2) AmpC positivity was found to be 95.5%.(65/68), Of AmpC positivity, carbapenemase and AmpC secreted from isolates simultaneously were observed 88.2%(60/68), AmpC secreted alone of five strains were detected, one isolate appeared ESBL positivity, No found metal P-lactamase. Result of PCR typing of P-lactamase show TEM-1 type of broad-spectrum P-lactamase for total 68 strains of mutli-resistant acinetobacter baumanii, SHV> CTX-M type of P-lactamase were not found. The result of genotype carbopenemase show OXA-23 subtype was found 96.8%(60/62) strains carbopenems-resistant acinetobacter baumanii excluding two strains , OXA-24 carbopenemase and IMP* VIM metal P-lactamase were not detected in these strains. Genotypic analysis of 68 strains of mutli-resistant acinetobacter baumanii by RAPD revealed 7 patterns named A to G, including 5,8,5,46,2,l,lstrain(s),respectively. (3) The integrase gene PCRresulted in a frenquency of 23.2%( 19/82), Class 1 integrons were detected in 27.9%( 19/68) of the mutli-resistant acinetobacter baumanii, whereas no strains contained a class 2 or 3 integron. The sequences of these PCR amplification products were 100% identical to previously published sequences of intll. Integron carriage was significantly associated with an increase in antibiotic resistance. (4) PCR amplification was performed with primers for the 5'-and 3'-conserved segments. Integrons with various insert sizes were found in 68.1%(13/19) of the mutli-resistant acinetobacter baumanii. The rang of inserted gene cassette sizes detected varied from 1.3 to 3 kb. A band of 2476 bp were found in 7strains and DNA secquencing revealed that the amplicon contained four gene cassettes: aacCl > orfl > orfx' and aadDAl on class 1 integron, (GeneBank AJ784787), aacCl encoding an AAC(3)-Ia aminoglycoside acetyltransferase confers resistant to gentamicin;Orfl and orfx' encoding for unknown products;and aadDAl encoding an AAD(3")-Ia aminoglycoside adenyltransferase confers resistance to spectinomycin and streptomycin. An amplicon of 2235 bp detected in 2 strains carried three gene cassettes: BlaPSE-l-orfx'-aadAl, BlaPSE-1 conferring resistance to beta-lactams and aadAl conferring resistance to spectimomycin and streptomycin. BlaPSE-1 gene cassette is the first found in acinetobacter baumanii in our country. Sequencing revealed that 1 strain has an amplicon 2307bp and contain gene cassettes of aacA4 * CatB8, aadAl, conferring resistance to amikacin and tobramycin, chloromycin, spectinomycin (GeneBank AY922989). An amplicon of 1848bp contain aadA2 orfF-dhfrXII, conferring restance to spectinomycin and streptomycin -. sulfanilamide respectively (GeneBank AY748453). Sequencing revealed that 1 strain has an amplicon 1304bp and contain gene cassettes of aar-3? aacA4 conferring restance to rifampin, amikacin and tobramycin( GeneBank DQ 174291). Sequencing revealed that 1 strain has 1664bp contain gene cassettes of aacA4-aadAl conferring restance to aminoglycoside antibiotics. Integrons with various insert sizes were not detected in 6 strains of integrase gene positive.[Conclusion] The present study reveals that detection rate of mutli-resistant acinetobacter baumanii has been raisen rapidly, susceptivity of carbapenems antibacterials descent obviously and drug resistance of other antibacterials of UK IV generation cephalosporins and enzyme inhibitor compound increased remarkably over 80%. Many type pMactamase including TEM-1 ^ AmpC, OXA-23 of carbapenemase could been detected. OXA-23 carbapenemase and AmpC secreted from isolate simultaneously is molecule mechanism of carbopenems-resistant acinetobacter baumanii. Integrons are widespread in clinical mutli-resistant acinetobacter baumanii isolates. Six drug gene cassettes including aacA4 * aadAl> aadDAl, aadA2, CatB8, aar-3* dhfrXIL BlaPSE-1 were found in integron conferring resistance to aminoglycosides> Trimethoprim, chloromycin, rifampin, P-lactams. Six different arrangement of drug resistance gene cassettes were found in integron and two new arrangement of drug resistance gene cassettes (BlaPSE-l-orfx'-aadAland aacA4-aadAl) were detected. Drug gene cassette could play an important role in causing the antibacterial resistance on aminoglycosides and Trimethoprim. This study also demonstrated that all of the integrons contained antibiotic-resistant gene cassettes and the presence of new cassettes and novel combinations of gene cassettes. This indicates that continued antibiotic selective pressure does not only favor the incorporation of gene cassettes in integrons but also select new resistance gene variants and different combinations of cassettes. Moreover, such selection pressure may also promote the association of integrons carrying more than one antibiotic-resistant gene cassette with other genetic elements containing resistance determinants.