Insight Into Molecular Mechanism Of Multiresistance Of Coexistence Of Hyperproduced AmpC Enzyme And ESBLs In Enterobacter Cloaca

To understand the resistance phenotypes and resistance patterns of clinical isolates producing ESBLs and hyperproduction of AmpC-type beta-lactamase in Enterobacter cloacae to most clinically useful antibacterial agents,and to research the mechanism of resistance to non-β-lactam antibiotics.[Methods](1)The MICs of 21 antimicrobial agents were determined by the twofold agar dilution method in accordance with the CLSI guideline.(2)To identify the mutations in the gyrA and parC genes corresponding to the QRDR of the E.coli gyrA and parC gene by PCR amplification and sequence analysis.(3)QnrA,qnrB,qnrS and aac(6')-Ib-cr genes were screened by PCR amplification with specific primers and sequence analysis in 25 ESBLs-producing Enterobacter cloacae.(4)Class 1,2 and 3 integrons were detected by PCR amplification and sequencing of the class 1,2 and 3 integrase specific int-1 gene,int-2 gene and int-3 gene.The sizes of the variable regions of integrons were determined by CS-PCR,using primers for the 5-CS and 3- CS regions,DNA sequencing was used to identify the genetic content of the integron-variable regions.(5)Screening for the aminoglycoside-modifying enzymes(AMEs)and 16S rRNA methylases genes was carried out by PCR amplification and DNA sequencing in 14 clinical isolates.[Results](1)Carbapenems shows the most activity against all tested strains (susceptibility,100%),All of strains were resistant to Ampicillin-sulbactam,Cefoxitin,third-generation cephalosporins and aztreonam,All of strains were not susceptible to Piperacillin-tazobactam,Cefoperazone-sulbactam and Cefepime,resistance rates were 50%,85.71%and 85.71%respectively. Aminoglycosides and fluoroquinolones susceptibility varied from10.25%to 25%. 85.71%of the isolates were multidrug-resistant and coresistant to 3-5 antimicrobial groups.(2)78.58%of strains had double amino acid at Ser-83→Phe and Asp-87→Ala in GyrA equivalent to Ser-83 and Asp-87 in the E.coli GyrA and ParC,7.14%of strains had single amino acid change at Ser-83→Tyr in GyrA,71.43%strains han a single amino acid change at Ser-80→Ile in ParC,71.4%strains had amino acid change in GyrA and ParC,12 fluoroquinolone- resistant strains harboured one to three amino acid change,2 fluoroquinolone-susceptible strains no amino acid change.(3)The qnrA and qnrB2 gene were detected in 4%and 16%of the 25 ESBL-producing isolate respectively,No isolates carried qnrS and aac(6')-Ib-cr gene.The qnrB2-1ike genes showing 86%identity with qnrB2(GenBank accession no.EU131184),showed six amino acid changes.All of qnr positive isolates carried blaSHV-12 and had no mutation in gyrA and parC genes.80%of qnr positive isolates was susceptible to Ciprofloxacin,Gatifloxacin,Ofloxacin and Levofloxacin,20%of qnr positive isolates showed decreased fluoroquinolone susceptibility.(4)The positive rates of AMEs genes of aac(6')-Ⅰ,aac(3)-Ⅱ,ant(3")-Ⅰand ant (2")-Ⅰwere 85.71%,21.43%,78.57%and 14.29%respectively,but the AMEs genes of aac(3)-Ⅰ,aac(6')-Ⅱand aph(3')-Ⅵwere not found.11 isolates(78.57%) were positive for the armA gene,All isolates were negative for rmtB and rmtC genes. All armA-positive isolates exhibited high-level aminoglycoside resistance(MICs＞256 mg/L)to gentamicin,tobramycin,amikacin and netilmicin,90.90%isolates produced CTX-M-3 type ESBLs,9.1%produced SHV-12 type ESBLs.(5)85.71%isolates carried class 1 integrons,whereas no Class 2 integrons and Class 3 integrons were detected.Class 1 integrons with various insert sizes were found in 78.57%(11/14)of the isolates.The predominant gene cassettes were aadA and dfrA conferring resistance to aminoglycoside and trimethoprim.1 strain had an amplicon 2121bp and was revealed to contain two gene cassettes of dfrA12-aadA2 conferring resistance to trimethoprim and to streptomycin/spectinomycin,dfrA12 gene cassette and dfrA12-aadA2 gene cassette arrays is the first found in E.cloacae;An amplicon of 1865bp by detected in 1strains carried two gene cassettes dfrA17-aadA5;An amplicon of 1776bp in 1 strains carried two gene cassettes dfrA16-aadA2;An amplicon of 1236bp in 1strains carried one gene cassettes aadA4,aadA4 gene cassette is the first found in E.cloacae;Amplicons with sizes of 1194 bp were found in six strains harboured one gene cassette aadA2;An amplicon of 962bp in 1 strains contain aadB gene cassette which encodes aminoglycoside 2"-adenyltransferase conferring resistance to gentamycin and kanamycin.[Conclusion](1)The majority of isolates produced ESBLs and hyperproduction of AmpC enzyme in Enterobacter cloacae were multi-drug-resistant,carbapenems had the most activity,all of the strains were not susceptible toβ-lactam-β-lactamase-inhibitor combinations,third-and fourth-generation cephalosporins,only 10.25%to 25%of the isolates were susceptible to aminoglycosides and fluoroquinolones.(2)The predominant mechanism of quinolones resistance was amino acid change at the Ser-83 and Asp-87 codons,GyrA subunit of DNA gyrase is a primary target of quinolones,only a single amino acid change at Ser-83 in GyrA is sufficient to generate resistance to quinolones.(3)The prevalence of qnr among ESBL-producing E.cloacae was 20%,qnrB2 was the most prevalent,whereas no qnrS were detected.All qnr-positive isolates are associated with SHV-12- type ESBLs,all qnr-positive isolates were susceptible or showed decreased susceptibility to quinolones.(4)The synthesis of aac(6')-I,aac(3)-Ⅱ,ant(3')-Ⅰ,ant(2')-Ⅰ)and armA 16S rRNA methylase were the predominant mechanism of resistance to aminoglycoside,all armA16S rRNA methylases-positive isolates confer high-level resistance to all clinically important aminoglycosides,All 16S rRNA methylase-bearing strains was associated with CTX-M-3and SHV-12 type ESBLs.(5)Class 1 integrons seem to play a major role in horizontal uptake and dissemination of the antibiotic resistance gene cassettes in clinical Enterobacter cloaca.The aadA(aadA2,aadA4 and aadA5)and dfrA(dfrA12,drfl6 and dfrA17)gene cassette was the most frequently found resistance gene in the variable region of integrons,resistant gene cassettes might horizontal disseminate through the integron between different bacteria species under antibiotic selective pressure.