Detection Of AmpC Beta-lactamase In Isolates Of Enterobacteriaceae From Several Hospitals Of Anui Province And Analysis Of The Resistance Of AmpC-producing Isolates

ObjectiveTo detect of AmpC beta-lactamase in 106 clinical isolates of Enterobacteriaceae in several hospitals in Anhui province in 2005 and study the resistance of AmpC-producing isolates against 10 antibiotics.To investigate distribution of other drug resistance genes of the AmpC-producing isolates.Materials and MethodsIsolatesThe 106 clinical isolates of Enterobacteriaceae were collected from 23 hospitals of Anhui province in 2005.MethodsA total of 106 clinical isolates of Enterobacteriaceae were consecutively collected from different clinical significance specimens in 23 hospitals of Anhui province in September 2005 (from 1 september, 2005 to 30 september, 2005), and were identified by the standard methods in each cooperative hospitals in the entire month (from 1 september, 2005 to 30 september, 2005). All isolates were identified with the API identification System, and excluded the repeated isolates from the same patients. The AmpC-producing isolates were choosed by cefoxitin firstly, and identified by the three-dimensional test and multiple PCR, the susceptibility to Antibiotics of AmpC-producing isolates of Enterobacteriaceae were studied by agar dilution method. The conjugation experiment was performed in AmpC-producing isolates, according to multiple PCR results, two pairs of entire coding gene primers were designed, and performed in the consequence experiments and the sequence was determined by direct sequencing of PCR products, carried out by the dideoxy chain termination procedure of Sanger on an ABI377 automatic sequencer (Invotrigen Biocompany, Shanghai, China).Adopting PCR methods, universal primers of blaCTX-M, blaSHV, blaTEM and blaOXA were used for ESBLs+ isolates screening, and analyse the other genotypes of the AmpC-producing isolates.Results61 isolates were intermediate-susceptible resistant to cefoxitin were choosed by means of Kirby-Bauer (K-B), and 35 AmpC-producing isolates were confirmed by the three-dimensional test and multiple PCR, and accounted for 33.02% (35/106). From the size of PCR products (DHA-405bp, EBC-302bp, CIT-462bp), the genotypes of AmpC beta-lactamase can be concluded. Of 35 AmpC-producing isolates, resistance to ceftazidim and ceftriaxone was above 55%, resistance to cefoperazone-sulbactam and cefepime was 65.7% and 45.7%, respectively.35 AmpC-producing isolates were used in the conjugation experiment, and four transconjugants were captured. A total of DNA from four transconjugants was used in PCR by two pairs of entire coding gene primers. Then BLAST program was used to ascertain the genotypes of the sequencing of PCR products at Genbank. The amino acid sequence showed the gene of 3 isolates had 100% identity with that of DHA-1, and the gene of 1 isolates had 99% identity with that of ACT-1, the nucleotide sequence data was presented to GeneBank, and the accession number was acquired (DQ986958).The results of PCR with the universal primers of blaCTX-M, blaSHV, blaTEM and blaOXA showed 2 isolates carried blaCTX-M-1, 4 blaCTX-M-9, 1 blaSHV, 14 blaTEM, 1 blaOXA-1. And there existed above 3 drug resistance genes in 3 strains. ConclusionsAmpC-producing isolates in several hospitals of Anhui province have powerful resistace to antibiotics, not only to the third-generation cephalosporins, but also to the cephalosporins withβ-lactamase inhibitions. 4 isolates of plasmid-mediated AmpCβ-lactamase had been found in 106 clinical isolates, and a novel genotype had been found. The number of chromosomal-mediated AmpCβ-lactamase isolates accounted for above 50%, and there existed other genotypes in AmpC-producing isolates.