Study on Resistant Mechanism of Klebsiella Pneumoniae and Pseudomonas spp.
|Keywords||Klebsiella pneumoniae Pseudomonas spp. integron metallo-β-lacatmases AmpCβ- lacatmases|
1. ObjectiveKlebsiella pneumoniae and Pseudomonas spp. have emerged as importantnosocomial bacterial pathogens, and have became major pathogenic bacteriaof respiratory tract infection. Their resistance to antibiotics increasedin recent years. The objective of this study was to study the resistanceand its mechanism of Klebsiella pneumoniae and Pseudomonas Spp. isolatedin Shanotou, China. And this will contribute to a better control of thestrain’s prevalence and to a better selection of appreciate antibioticsin this area.2. Methods2.1 Seventy-four nonrepetitive clinical isolates of Klebsiellapneumoniae producing extended-spectrum beta-lactamases （ESBLs） wereisolated. Antibiotic susceptibility testing was performed by E-test.PCR were performed to amplified integrase and the variable region ofclass 1 integron with specific primers. PCR product of the variableregion was further identified by sequencing.2.2 Clinical isolates of Pseudomonas aeruginosa resistant to imipenemwere collected. DNA was extracted by boiling and PCR was performedwith specific primers for VIM, IMP and SPM metallo -β-lactamases, oprD2 and PSEβ-lactamase. 2.3 Eight pairs of Pseudomonas aeruginosa isolates before and aftertreatment with antibiotics were isolated from the same patient.Random amplified polymorphic DNA was performed for genetyping.Cefoxitin induced test and cefoxitin three-dimensional test wereperformed to detect the production of AmpCβ-lactamase.2.4 A Fluorescent pseudomonads clinical isolate highly resistant toextend-spectrum cephalosporins was isolated. Minimal inhibitoryconcentration to multiple antibiotics were detected by micro-dilution broth method. Alkaline lysis method was used to extractplasmid DNA. PCR was performed with primers for class 1 integrase,VIM, IMP and SPM metallo-β-lactamase.3. Resu Its3.1 Most of the Klebsiella pneumoniae isolates were multi-resistant.Sixty-nine isolates were IntI1 gene positive but no IntI2 and IntI3genes were found. Thirteen different gene cassettes and elevendifferent cassettes combinations were detected. Dfr and aadA cassetteswere predominant and cassette combination dfrAl2, orfF and aadA2 wasmost frequently found. No gene cassettes enconding ESBLs were found.3.2 Cefoperazone/tazobactam showed better activity in vitro to imipenem-resistant Pseudomonas aeruginosa isolates than other antibiotics. Noisolates produced metallo-β-lactamase. Six isolates produced PSEβ-lactamase. Most of the isolates failed to produce an amplicon withprimer for oprD2.3.3 Before treatment with antibiotic, most of the isolates werehyperinducible strains producing AmpCβ-lactamase and after treatment,minimal inhibitory concentration to ceftazidime increased and all theisolates became hyperproducing AmpCβ-lactamase strains.3.4 The isolate were highly resistant toβ-lactam antibiotics includingcarbapenems but sensitive to ciprofloxacin. Metallo-β-lactamase encoding gene was found to be integrated as a gene cassette within aclass 1 integron on a plasmid about 20kb. DNA sequencing showed thatpart of the sequence had i00％amino acid identity with IMP-8metallo-β-lactamase.4. Conclusions4.1 Integrons were prevalent and played an important role in multidrugresistance in ESBL-producing Klebsiella pneumoniae. The production ofESBLs and integrons will threaten the usefulness of antibiotic.4.2 Cefoperazone/tazobactam can be used in the infection caused byimipenem-resistant Pseudomonas aeruginosa. The major mechanism ofPseudomonas aeruginosa resistant to imipenem is the loss of OprD2expression.4.3β-lactam antibiotics that can induced the production of AmpCβ-lactamase can not be used for infection caused by Pseudomonasaeruginosa strains producing hyperinducible AmpCβ-lactamase.4.4 IMP metallo-β-lactamase encoding gene was located on class 1integron and this will facilitate the spread of resistance gene.