| Aminoglycosides have been widely used for treatment of a variety of bacterial infections, usually in combination withβ-lactam agents. More and more bacteria have been reported resistance to these drugs since their introduction into clinical use. Genes that are often located on conjugative plasmids include those encoding for extended-spectrumβ-lactamases (ESBLs) and determinants conferring resistance to aminoglycosides, and quinolones. Therefore, understanding the new mechanism of resistance, it can provide a reference for clinical medicine.1. The analysis of resistance phenotype and resistance genes to aminoglycoside among E.coliIn order to study the prevalent of aminoglycoside modifying enzymes and 16S rRNA methylases among avian Escherichia coli Strains from Shandong province, a total of 224 strains were tested by K-B (Kirby-Bauer) method to analyze the aminoglycoside susceptibility, by micro-dilution method to evaluate the MICs to gentamicin and amikacin and by PCR to examine the modifying enzyme genes and the 16S rRNA methylase genes which mediated high level resistance to aminoglycosides. The results indicated that the resistant incidence rates were exhibited to streptomycin (84.4%), gentamicin (57.1%), kanamycin (55.8%), neomycin (46.9%) and amikacin (40.2%); the present ratio of ant(3'')-Ia, aac(6')-Ib and aph(3')-IIa were 49.6%, 25.0% and 22.8% respectively. All of the three genes of 16S rRNA methylase were negative in low-level resistance, and RmtB was the high rate gene of 16S rRNA methylase with 53.1% positive rate among 49 strains of high level resistance to aminoglycosides. Seventy-five strains were detected with at least two genes and only one strain with four genes at the same time. The results revealed that the aminoglycoside modifying enzymes and 16S rRNA methylases were prevalent in avian Escherichia coli strains, and there were the highest coincidence between the resistance to aminoglycoside and the detection rate of the resistance genes.2. The characterization and detection of aminoglycosides-resistant plasmidE. coli XZDC was displayed a very high degree of resistance to many aminoglycosides, including amikacin (MIC, >1024μg/ml) and gentamicin (MIC, 512μg/ml), and the plasmid named pXZ was extracted by SDS alkaline lysis method. In order to investigate the detection rate of pXZ among the 224 E. coli isolates, three pairs of primers were designed according the sequence of pXZ and a multiplex PCR method was established. The sequencing result showed that the plasmid pXZ was a circular molecule of 76,635 base pair (bp) with a 51.65% guanine-plus-cytosine (G+C) content, and contains four resistance genes (rmtB, fosA3, blaTEM-1 and blaCTX-M-24).The annotated sequence of pXZ has been submitted to the GeneBank nucleotide sequence database accession number JF927996. Among the 224 E. coli isolates, 17 were found to contain similar plasmid as pXZ by multiplex PCR and restriction map analysis, and all the isolates showed high level resistance to aminoglycoside (MICs to gentamicin and amikacin≥512μg/ml).3. To definite the genotype of CTX-M-9 groupExtended-spectrum beta-lactamases (ESBLs) were divided into five groups: CTX-M-1 group,CTX-M-2 group,CTX-M-8 group,CTX-M-9 group and CTX-M-25 group according to the genotype differences between the every group. Different gene subtypes belongs to different groups, for example, the gene of blaCTX-M-24 was pertain to CTX-M-9 group. In order to detect the gene subtype of CTX-M-9 this contained in the pXZ-posotive isolates, PCR amplification and nucleotide sequencing were used to analyze the genotypes of ESBLs. The resulted showed that one isolate carried blaCTX-M-14 and others carried blaCTX-M-24.
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