Study On Drug Resistance Genes Of SHV Type Extended-spectrum β-lactamases(ESBLs) Produced By Gram-negative Bacteria

Objective To investigate the prevalence of SHV-producing gram-negative bacteria isolates collected from three affiliated hospitals of Central South University in Hunan provine.To indentify the subtype of SHV encoding gene and study the epidemiological aspect of SHV-producing isolates.Methods A total of 254 clinical isolates of multidrug resistant gram-negative bacteria were collected from three affiliated hospitals of Central South University in October 2004 to July 2005．The phenotype of ESBLs was screened by double-disk synergy test. The genotype of SHV enzyme was detected by multiplex PCR,amplifying the whole ORF(open reading frame)of SHV enzyme was conducted by specific primers and the PCR products were sequenced by automatic sequencer and the subtype of the gene was determinde by BLAST searching.The homology of SHV-producing strains were detected by RAPD(Random Amplified Polymorphic DNA)typing method.Results Of 254 clinical isolates of multidrug resistant gram-negative bacteria, 153 ESBLs-producing clinical strains were identified,including 142 Enterobacteriaceae isolates and 11 nonfermenter isolates. 104 of 142 ESBLs-producing Enterobacteriaceae isolates were confirmed to obtain positive results by multiplex PCR.85 isolates were found to produce blaTEM genes, 26 isolates produced blaSHV genes,and 10 isolates produced blaOXA-1 genes.6 of 11 nonfermenter isolates were confirmed to produce blaTEM genes by multiplex PCR. By SHV specific primers amplifying, 5 subtypes of SHV enzymes were detected: SHV-28, SHV-12,SHV-5,SHV-1 and SHV-11．SHV-producing strains are mainly composed of Klebsiella pneumoniae and Enterobacter cloacae. SHV-producing strains were detected by RAPD typing method, there were 6 RAPD types in 19 isolates of SHV-producing K.pneumoniae. 5 RAPD types in 5 isolates of SHV-producing E.cloacae .A high degree of diversity of RAPD patterns was observed among isolates of all species identified,we also found that several K.pneumoniae with the same RAPD pattern.Conclusions 1) This was the first detection of SHV-28,SHV-12, SHV-5 ESBLs and SHV-1,SHV-11β-lactamases in Hunan province. 2) SHV-type ESBLs were detected among a wide range of clinical bacteria,espically in Klebsiella pneumoniae and Enterobacter cloacae of Hunan province. 3) To some extend,clone spread has played a certain role in SHV enzymes dissemination. 4) SHV-type ESBLs had spread in Hunan provinve.It is of significance continuously monitoring SHV-type ESBLs strains and preventing their spread.