Genotype Detection Of Extended-spectrum β-lactamases Of Pathogens Isolated From Chicken

The detection rate of E. coli. producing extended-spectrum P-lactamases (ESBLs) are relatively high and the proportion is rising year by year. Most ESBLs-producing strains posseses multi-drug resistance to Penicillins, cephalosporin of three generations, Quinolones, sulfonamides, amino-glycoside and so on. ESBLs is mutagenesis by the individual amino acids of commonβ-lactamases TEM-1, TEM-2 and SHV-1. ESBLs encoded by plasmid easily epidemic outbreak to epidemic transmission in the same species and even between different species of bacteria. At present,the genotypes of ESBLs are discovered about 400 varieties according to the difference of genotype and enzyme substrate encod of plasmid, the genotype of ESBLs are divided into five classes:TEM,SHV-type,CTX-M-type,OXA-type and the other type. There are numerous hypotype of each genotype. The genotypes and genetic subtypes is different according to the bacteria and antibiotic selection pressure suffered. TEM-type and CTX-M-type is the most genotype in Escherichia coli. ESBLs of the rare types such as PER type, VEB type, GES type, BES type have also been on the rise. Applied hetero-antibacterials in various countries and regions, prevail the different genotype of ESBLs. The spread of ESBL s, as well as their clinical multi-drug resistance caused great difficulty to clinical antibiotic treatment. Timely and accurate detection of ESBLs has an important significance to prevent epidemic, infection control in hospitals and clinical treatment.From May,2007 to December,2008, we detected ESBLS by CLSI commended methods to 35 E. coli.from chicken We detected MIC by duplation dilution and applied K-B disk diffusion method in susceptibility testing of general all purpose antibacterials and their combination, make detection of the rudiment grouping of drug resistance gene to E. coli from chicken by molecular biology methods.The results of test showed there were 22 strains producing ESBLs among 35 strains Chicken E. coli. The resistant rate of ESBLs-producing strains to 15 antibiotics is from 0 to 100%, which is higher than that of non-ESBLs-producing strains. The susceptibl rate of Carbapenems e.g. Imipenem and Amikacin to producing and non-producing ESBLs is 100%. Yet The resistant rate of ESBLs-producing strains to Cephalosporins is from 80% to 95%, The resistant rate of non-ESBLs-producing strains to Cephalosporins is23.1%. There is significant difference. ESBLs-Producing strains represent a wide range of multiple resistances and cross resistance. Whenβ-lactam antibiotics were combined with P-lactamase inhibitors or other types of antibiotics the antibacterial activity of their ESBLs-producing strains enhanced22 ESBLs-producing clinical strains could amplification 861 bp destination-segment of Specificity by PCR with TEM-type-primer,SHV-type-primer; 905bp destination-segment of specificity by PCR with CTX-M-type-primer, and 831bp destination-segment of specificity by PCR with OXA-1-type-primer. Four pairs of primer sequences below:TEM-type:Ups-primer:5'-GGGGATGAGTATTCAACATTTCC-3',Down-primer:5'-GGGCAGTTACCAATGCTTAATCA-3';SHV-type:Up-primer:5'-GGTTATGCGTTATATTCGCCTGTG-3',Down-primer:5'-TTAGCGTTGCCAGTGCTCGATCA-3';CTX-type:Up-primer:5'-GGGCTGAGATGGTGACAAAGAG-3',Down-primer:5'-CGTGCGAGTTCGATTTATTCAAC-3';OXA-type:Up-primer:5'-TGAAGGGTTGGGCGATTT-3',Down-primer:5'-TTAGCGTTGCCAGTGCTCGATCA-3'.18 strains of 22 E. coli plasmid the fragment TEM-phore, However, the subtypes of TEM genes is TEM-1, classified as commonβ-lactamase. SHV-type primers did not amplification,9 strains found to carry fragment CTX,6 strains E.coli were also found to fragment TEM-phore and CTX-phore,6 strains found to fragment OXA-phore,5 strains were also found to fragment TEM-1-phore and OXA-1-phore, PCR products were sequenced after cloning, the results of sequence analysis showed that the sequence homology of various strains producing TEM-type enzyme is 99.2% to 100%, there are two mutations compared to EU979560 in Gene Bank. Yet it is still broad-spectrum TEM-1 type enzyme because it didn't cause mutations of amino acid sites.The sequence homology of various strains producing CTX-type enzyme is from 99.2% to 100%, the genotypes of strains ESBLs-producing are CTX-M-14,CTX-M-65 and CTX-M-24. The sequence homology of the submitted sequence 1148066 and published on Gne Bank sequence EU213262 (CTX-M-65) and EF418608 (CTX-M-65) is 100%, so the ESBLs produced by the strains isolated in this test is CTX-M-65-type. The sequence homology of the submitted sequence 1148088,1148090 and 1148092 and published on Gne Bank sequence DQ304479 (CTX-M-14) is 100%, so the ESBLs produced by the strains isolated in this test is CTX-M-14-type. The sequence homology of the submitted sequence 1148075 and published on Gne Bank sequence EU418917 (CTX-M-24) is 100%, so the ESBLs produced by the strains isolated in this test is CTX-M-24-type.