临床分离产AmpCβ┐内酰胺酶阴沟肠杆菌的耐药性分析

              作者：凌保东 余娴 谢勇恩 雷军  

【摘要】  　　目的 研究我院临床分离阴沟肠杆菌的产AmpC酶耐药情况及ampC基因型。 方法 收集临床分离的耐药阴沟肠杆菌15株;检测产AmpC酶和超广谱β-内酰胺酶（ESBLs）的菌株;测定MIC值;PCR扩增检测ampC基因及序列测定。 结果 15株菌中8株菌（53.3%）产AmpC酶，3株菌（20.0%）产ESBLs。产AmpC酶的菌株除对亚胺培南全敏感外，对其它抗菌药不同程度耐药。ampC基因与阴沟肠杆菌ECLC074的ampC基因高度同源。 结论 产AmpC酶是阴沟肠杆菌对β-内酰胺类抗生素耐药的主要机制之一。阴沟肠杆菌ECLC074ampC基因是我院主要的阴沟肠杆菌ampC基因型。产AmpC酶的阴沟肠杆菌常呈多重耐药，亚胺培南是此类菌所致感染的最有效药物。

【关键词】  阴沟肠杆菌 头孢菌素酶 ampC基因

　　A variety ofβ-lactamases have been classified into classes A，B，C，and D according to their amino acid homologies［1］ .AmpCβ-lactamases，mostly conferring resistance to manyβ-lactam antibiotics（cephamycins and broad-spectrum cephalosporins），are included in class Cβ-lactamases.Chromosomally encoded AmpCβ-lacta-mases are present in Enterobacter spp.，Shigella spp.，Serratia marcescens，Citrobacter freundii，Morganella morganii，Providencia spp.，Pseudomonas aeruginosa，and Escherichia coli［2］ .Enterobacter cloacae are becom-ing increasingly important as nosocomial pathogens［3］ .In view of the spreading risk of AmpC resistance deter-minants among enterobacterial isolates，it is necessary to elucidate the AmpC resistance mechanism.This study was conducted to determine the prevalence and genotypes of AmpCβ-lactamases among clinical isolates of Enterobacter cloacae.Materials and methods  Clinical strains15randomly chosen piperacillin-resistant E.cloacae were isolated from February2003to January2004in the Affiliated Hospital of North Sichuan Medical College.

    Detecting for production of ESBLs and AmpCβ-lactamases:Crudeβ-lactamase preparations were obtained by sonication and the qualitation detection ofβ-lactamase was performed by nitrocefin.The production of ESBLs was tested according to NCCLS Screening and Confirmatory test.AmpCβ-lactamase was examined by three dimension test ［4］ .

    Antibacterial susceptibility:MICs of antibacterials to AmpCβ-lactamase producers were determined by two-fold agar dilution method.E.coli ATCC25922and K.pneumoniae ATCC700603were used as reference strains.PCR of ampC genes，the genomic DNA of AmpCβ-lactamase producers，was prepared with the genomic DNA mini preparation kit（Sangon，Shanghai，China）and used as the template DNA in PCR analysis.The PCR primers were AmpCF（5′-GACTCGCTATTACGGAAG-3′）and AmpCR（5′-TTACTGTAGCGC-CTCGAG-3′）.AmpCF and AmpCR are consensus sequences of many AmpCs of E.cloacae（such as AY125471，AY536040，AY302261，AF411149and etc.）.PCR was performed in50μl reaction mixtures including0.5U Taq DNA polymerase（TaKaRa，Dalian，China）.The PCR mixture was submitted to a denatu-ration step（3min at94℃）and then30cycles of amplification（1min at94℃，1min at55℃，1min at72℃）and5min at72℃for the last step.The PCR products were analyzed by electrophoresis in a1.2%agarose gel.TA cloning of ampC gene:PCR products were extracted by DNA gel extraction kit（V-gene，Hangzhou，China）and ligated into the pMD18-T Vector（TaKaRa，Dalian，China）which is carrying the ampicillin resis-tance gene.The ligation mixture was inserted into CaCl 2 competent E.coli JM109cells by transformation.Transformants were cultivated overnight on LB plates containing ampicillin，IPTG，and X-Gal.White colonies were incubated overnight in LB broth.Plasmids were extracted from transformed E.coli JM109cells and veri-fied by EcoRI and HindⅢdigesting.

    DNA sequence analysis:DNA of plasmids extracted from transformed E.coli JM109cells was sequenced by Genecore Biotechnology Ltd.The nucleotide sequences and deduced amino acid sequences of AmpCβ-lacta-mases were analyzed with BLAST in GenBank.

    Results

    β-lactamases produced by E.cloacae:All strains producedβ-lactamases.8（53.3%）isolates produced AmpCβ-lactamases and other3isolates（20.0%）produced ESBLs.

    Antibacterial susceptibility:The results of MICs of8AmpCβ-lactamase producers are summarized in Tab.1.All isolates were resistant to cephalosporins，cefoxitin and aztreonam，while they were susceptible to imipenem.In addition，they had the different resistant rates against the other antibacterials.

    Sequence analysis of ampC genes:The PCR products of8AmpCβ-lactamase producers were1146bp（Fig.1）.Compared with E.cloacae ECLC074ampC gene（AY536040），the ampC genes of5strains had100%homologies，and those of the remaining3strains（Ecl3-51，Ecl3-94，Ecl3-107）had99%homologies（Tab.2）.Compared with E.cloacae ECLC074AmpC amino acid sequence（Fig.2），the deduced amino acid sequences of6strains had100%homologies and those of the remaining2isolates had one amino acid residue mutation，Ecl3-94L-169→P，Ecl3107A-254→V，respectively（Tab.2）.   Tab.1     Antibacterial susceptibility of8E.cloacae isolates producing AmpCβ-lactamase

   AmpCβ-lactamase is an inducible enzyme produced by E.cloacae and many of the other Gram-negative bacilli.AmpC expression is under the control of a regulatory gene system including ampD，ampG，ampE and ampR which are involved in the induction of AmpCβ-lactamase［5］ .Recently ESBLs produced by E.cloacae have also been reported in Europe    ［6，7］ and United States［8］ .In this study，AmpCβ-lactamases were produced in8isolates（53.3%）and ESBLs were producted in other3isolates（20.0%），which emphasize that in E.cloacae isolates，the resistance to extended-spectrum cephalosporins is related to both hyperproduction of AmpCβ-lactamase and ESBLs.The Enterobacter cloacae ECLC074ampC gene and the ampC genes of8strains were highly homologous，which indicates E.cloacae ECLC074ampC gene is the main gene style of AmpCβ-lactamase-producing strains of E.cloacae in the hospital.AmpCβ-lactamases of2strains had one amino acid residue mutation，however the activity binding site of serineβ-lactamase SXXK and characteristic regions YXN，KTG  ［9］ and Thr70 ［10］ didn′t mutate.So we plan to explore the effect of these substitutions in the AmpCβ-lactamases of E.cloacae strains on their substrate specificities later on.

    Result of antibacterial susceptibility testing showed that AmpCβ-lactamase producers were multi-drug resistant.Sulbactam and tazobactam can′t inhibit the activity of AmpCβ-lactamases，so the combination ofβ-lactamase inhibitor andβ-lactam does not work in treatment of infections caused by AmpCβ-lactamase produc-ers.The fourth generation cephalosporins carrying amino thiazole side chain have a low affinity for AmpCβ-lactamases，and can penetrate the cell membrane of bacteria more rapidly，so they are presumed to be the preferred choice for infections caused by AmpCβ-lactamase producers.However，in this study only one isolate with hyperproduction of AmpCβ-lactamase was susceptible to cefepime.Imipenem is extremely stable toβ-lactamases，so imipenem is the most effective antibiotic for treating AmpCβ-lactamase-producers.In addition，this study showed that amikacin was also an effective agent which was similar to the results of some other reports   ［11，12］ .

    To conclude，we should pay more attention to the prevalence of Enterobacter cloacae producing AmpCβ-lactamases in hospital.In the one hand，the most effective antibacterials should be used according to the result of antibacterial susceptibility test.In the other hand，the reasonable measures should be taken against the prevalence of AmpCβ-lactamase-producing strains.

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