Initial Study Of The Phenotypes Of Ampc β-lactamase And Related Genes Of Ampc And Ampd In Enterobacter Cloacae

Enterobacter cloacae is the common pathogenicbacteria to cause the infection of respiratory tract, urogenital tract and wound. In the past twenty years, with the extensive use of the third and fourth generation cephalosporin and immunosuppressant also various kinds aggressive operations, the infection of multidrug resistant has been much concerned in gram-negative bacilli, among Enterobacter cloacae and Enterobacter aerogenes are now the most important organism in the nosocomial infection. Significant amount of research had shown that the main mechanisms of drug resistance are high-producing AmpC B-lactamase in Enterobacter cloacae. A quickly and precisely method that can detect the AmpC B-lactamase high-producing bacteria is in favour of clinic doctors using antibiotics and administration for the nosocomial infection.Objective:1. Detect the different phenotypes of AmpC B-lactamase in Enterobacter cloacae with the multi-substrates synergy-antagonize test.2. Initial study of Enterobacter cloacae carrying ampC and ampD genes and the relationship between the amino acid substitution sites of AmpD and the phenotypes of AmpC B-lactamases.Methods:1. Clinical isolates of Enterobacter cloacae were collected from Jan,2006 to Jun, 2009 in the respiratory microbiological laboratory of the Second Hospital, Tianjin Medical University and were reappraised with classic biochemistry conformation.The different phenotypes of AmpC B-lactamases were detected with MSSAT.2. The ampC and ampD gene fragments were detected by PCR amplification.The different phenotypes of the product of ampD were selected for DNA sequencing, sequences were aligned with Enterobacter cloacae 14 of Kopp U's report (accession number in GenBank:Z14003.1) and the relationship between the mutants sites of ampD and the phenotypes of AmpC lactamases was analysed. Results:1. Totally we have reappraised 31 clinical isolates of Enterobacter cloacae from Jan,2006 to Jun,2009.18 strains were separated from respiratory wards,7 strains from the outpatients section and 6 strains from intensive care unit wards.The different AmpC lactamases phenotypes of Enterobacter cloacae were detected by multi-substrates synergy-antagonize test and 2 strains (2/31,6.5%,1 from sputum specimen and 1 from midstream specimen of urine) were identified as non-AmpC B-lactamases producing,15 strains (15/31,48.4%,12 from sputum specimen, 2 from midstream specimen of urine and 1 from nasopharyngeal swab specimen) were identified as high producing AmpC B-lactamases,9 strains (9/31,29.0%, 9 from sputum specimen) as partially derepressed phenotype,5 strains (5/31, 16.1%,4 from sputum specimen and 1 from midstream specimen of urine) as fully derepressed phenotype.2. All clinical isolates were found to possess of ampC and ampD by PCR and agarose gel electrophoresis.Sequence analysis revealed that all 3 different phenotypes isolates have possessed of some gene mutations of ampD. The corresponding amino acid substitution sites of high inducing phenotype were Asp-59â†'Asn, Ala-71â†'Arg, Leu-72â†'Val, Asp-75â†'His, Gln-103â†'His, Thr-122â†'Ser, Glu-131â†'Gln, Lys-132â†'Gln, Val-136â†'Ile, Gln-138â†'His, Arg-143â†'Leu, Glu-160â†'Ala, His-175â†'Arg, Thr-179â†'Ala, Thr-180â†'Ala and Thr-187â†'Asn. The mutant sites of partially derepressed phenotype were Ala-71â†'Arg, Leu-72â†'Val, Asp-75â†'His, Gln-103â†'His, Ala-128â†'Ser, Glu-131â†'Gln, Lys-132â†'Gln, Val-134â†'Ala, Gln-138â†'Arg, Arg-143â†'Leu, Glu-160â†'Ala, Ser-172â†'Pro, His-175â†'Arg and Thr-180â†'Ala.The mutant sites of fully derepressed phenotype were Ala-71â†'Arg, Leu-72â†'Val, Asp-75â†'His, Ala-158-â†'Val and Ile-186â†'Thr.Conclusion:1. The sources of 31 clinical isolates of Enterobacter cloacae are mainly sputum specimen and mainly from respiratory wards. This study have detected that the AmpCβ-lactamases are mainly high-inducing phenotype, partialy derepressed phenotype is in the second place and fully derepressed phenotype is relatively lower by multi-substrates synergy-antagonize test.2. The relevant size fragments of ampC and ampD were acquired by PCR. Sequence analysis of ampD gene shows that the amino acid substitution sites of 128,134,138,172 maybe result in partialy derepressed effects and substitution sites of 158,186 maybe result in lead to fully derepressed effects.