Ampc ¦Â-lactamase In Beta-transition To The Sustained High-yield Type

Part 1Objectives:To reveal the influence of ampD specific mutations on the transformation of AmpCβ-lactamase from non-constitutive expression to constitutive high expression in Enterobacter cloacae.Methods:The chromosome-mediated constitutive high-productive strains of Enterobacter cloacae were selected with identification, drug sensitivity test, modified Cefoxitin three-dimensional test, and plasmid ampC gene amplification of bacteria. Chromosomal ampD was amplified by PCR and sequenced, and specific mutations were confirmed. Site-directed mutagenesis was used to mutate the wild-type Enterobacter cloacae for the above sites, and the ampD gene of clinical strains were complementated by wild-type ampD gene. The change of AmpCβ-lactamase types was detected by the modified Cefoxitin three-dimensional test and the AmpCβ-lactamase activity test.Results:For 121 clinical strains of Enterobacter cloacae,15 were chromosome-mediated constitutive high expression,8 had significant mutations of ampD and 7 strains by site-directed mutagenesis were established. Ecl MA (274 inserting A), Ecl MC (327 missing C) and Ecl MF (27 inserting G) changed from non-constitutive expression to constitutive high expression; but Ecl MB (371 inserting T), Ecl MD (515 missing C), Ecl ME (324 Câ†'A), Ecl MG (238Câ†'A 302Tâ†'A) did not change in AmpCβ-lactamase types. From the amino acid level, the amino acid changes of AmpD for the AmpCβ-lactamase type changes were:93-169 all changed (170/110 termination),109-132 all changed (133 termination),10-25 all changed (26 termination); while the AmpCβ-lactamase type did not change by the following amino acid changes:124-169 all changed (170 termination),172-187 all changed (187 no termination),108â†'termination,80Râ†'S,101Mâ†'K.Conclusions:The 3 significant ampD mutations of Enterobacter cloacae that lead to frame-shift changes of amino acid could converse AmpCβ-lactamase from non-constitutive expression to constitutive high expression. AmpD lactamases encoded by ampD gene decomposes the nien-peptide components including N-acetyl-muramyl tripeptide anhydride, and this decomposition is likely to rely on an hypothetical active region of AmpD lactamase. Our study revealed that the active region of ampD probably originated from 25 or later (the 9th amino acid of AmpD, or after), and terminated on or before 321 (the 107th amino acid of AmpD, or before). In the above active region, a single site mutation for a single amino acid change of AmpD may not impact the AmpCβ-lactamase type of Enterobacter cloacae. Of course, other mechanisms of ampD mutations affecting on the AmpCβ-lactamase were not excluded from the study. The above assumptions should be further studied.Part 2Objectives:To reveal the conversion rate of AmpC P-lactamase from inducible high expression to constitutive high expression in vivo, analyze the underlying diseases and the antimicrobial agents application of the patients, and play an initial role in the clinical control and reatment of AmpCβ-lactamase.Methods:The patients carrying gram-negative bacilli with inducible AmpCβ-lactamase were screened as research objects. The same patients'samples were collected and the strains were cultrued untill they changed to the AmpC P-lactamase of constitutive high expression. The bacterial species, the chronic diseases, the use of antimicrobial agents were recorded. Statistic analysis was performed using a statistical software package (Stata 8.0).Results:559 patients from two hospitals as study objects were enrolled in the study, 714 inducible AmpCβ-lactamase-producing gram-negative bacilli were isolated, including 527 strains of Pseudomonas aeruginosa,26 strains of Morganella Morganii,41 strains of Acinetobacter baumannii,25 strains of Serratia marcescens, 60 strains of Enterobacter cloacae,35 strains of Enterobacter aerogenes.33 strains of the above bacilli changed to constitutive high expression, the overall conversion rate was 4.6%. The conversion rates of bacilli were a variety:Pseudomonas aeruginosa 3.4%, Morganella Morganii 7.7%, Acinetobacter baumannii 4.9%, Serratia marcescens 0%, Enterobacter cloacae 13.3%, Enterobacter aerogenes 8.6%.There is a statistically significant difference of the conversion rate between the patients suffering from cerebral vascular disease and the patients without such disease (OR=4.9). When the underlying diseases and other conditions were equal, the number of days of cephalosporins use was negatively correlated with the conversion rate, while the days of carbapenem use was positively correlated with the conversion rate, but the days of application of other antibiotics had no obvious correlation with the conversion rate, the application ofβ-lactam antibiotics or non-β-lactam antibiotics did not produce significant differences in the results.Conclusions:Pseudomonas aeruginosa (73.8%) were the most common cause of nosocomial infections of inducible AmpC-producing bacteria; the conversion rate of Enterobacter cloacae (13.3%) was the highest. health care workers involved in the diagnosis and treatment of cerebral infarction or its complications with infections should pay more attention to emergence of the constitutive high expressed AmpCβ-lactamase. Under the same conditions, when the cephalosporins was necessary in the treatment of patients with nosocomial infection, the dosage ane the treatment should be enough to reduce the conversion rate; while large doses and a long time application of carbapenem was required, whether the AmpCβ-lactamase of the bacteria changed to the constitutive high expression should be payed attention and controled.