Compreensão de mecanismos fenotípicos e genotípicos relacionados à produção de β-lactamases do tipo AmpC em Enterobacteriaceae

Abstract

The production of AmpC-type β-lactamase in Enterobacteriaceae is reported worldwide and has great clinical and epidemiological importance due to the therapeutic restriction, detection difficulty, dissemination of plasmid AmpC and mutation of the natural gene causing multidrug resistance in the case of ESAC producers (Extended Spectrum AmpC). In this study, AmpC production was carried out considering two groups of enterobacteria, suspected of producing the enzyme: 18 strains from human clinical specimens (FFUP - Portugal) and 20 from bovine mastitic milk (UFRRJ - Brazil), respectively. After this, a total of 238 Escherichia coli from brazilian dairy farms were used to detect AmpC according to previously developed phenotypic and biochemical methodologies, and by PCR (ACC, AmpC universal, ampR, CIT, CMY, DHA, EBC , MOX and FOX). Sequencing of the promoter and the entire ampC gene was performed on 12 strains to evaluate mutations related to AmpC overproduction and ESAC production. All isolates of human origin were phenotypically expressed as AmpC producers and were confirmed in 72.2% by the inhibition test with boronic acid (AB) and pI 9 in 44.5%. The PCR technique revealed: AmpC universal in 83.4%, CIT in 66.7%, CMY in 16.7% and DHA in 55.6%. Although 61.1% of these isolates suspected plasmidial ampC only 38.9% presented the mobile element. Complete inhibition by cloxacillin could not be observed in either donor or recipient. Of the 20 animal enterobacteria isolates, only one Proteus mirabilis demonstrated phenotypic characteristics of AmpC producer whose gene transfer was confirmed by conjugation. Possibly, the other isolates lost the resistance plasmid during transport to the FFUP, which would justify the initial suspicion of enzyme production. Among the 238 E. coli, 11.76% presented decreased susceptibility to cefoxitin, characteristic of pAmpC or super producers of AmpC or ESAC. There was no confirmation of AmpC production by inhibition with cloxacillin and AB nor by MIC determination. The absence of pAmpC was confirmed by PCR and also by conjugation. Two isolates suspected of overproducing the enzyme and 4 ESAC suspects were analyzed for the promoter region and entire gene. Three isolates (2 suspects) were found to have mutations at the promoter region (-1, +58) confirming the overproduction of AmpC. Entire gene sequencing demonstrated mutations responsible for amino acid variations in the Ω loop regions (191, 209) and R2 (298, 300, 304). All the analyzed strains presented variations in the regions responsible for the greater flexibility of the enzyme, propitiating the hydrolysis of other β-lactams, such as cefepime. Thus, the study allowed an important survey of epidemiological data on resistance genes in circulating enterobacteria in the studied portuguese hospital and it is the first report of mutations suggesting overproduction of AmpC and ESAC detection in E. coli in brazilian cattle dairy samples