Studies On Antimicrobial Resistance Of Escherichia Coli Isolated From Guangdong Province And The Molecular Epidemiology Of Integrons/gene Cassettes

The development and spread of resistant organisms is a major global problem and many countries have become increasingly concerned about the potential dangers to public health. Because of this, the use of antimicrobial agents in veterinary services is widely debated. In order to assess the risk of transfer of antimicrobial resistance from animals, standardized and continuous data must be collected from the veterinary, agricultural and medical departments. In this dissertation, an investigation of the use of antimicrobials in food animals and the susceptibility of bacteria in food animals and in food of animal origin was done. Besides, the epidemiology of integrons/gene cassettes in Escherichia coli isolates collected from pig farm was carried out.1. Antimicrobial susceptibility testing of 21 antimicrobial agents was performed on a total of 1524 commensal Escherichia coli isolates collected from pigs industry (522), poultry industry (220) ,dairy cattle farm (200), pig slaughterhouse (120) ,retail meats (148), and healthy student volunteers (85) in Guangdong province between Nov.2001 to Aug. 2002. Antibiograms and their interpretation were made using the disk diffusion method following the NCCLS(1999) standards.Among the 21 antimicrobial agents tested, resistance of E.coli isolated from all sources was the most frequent for sulfonamides, tetracyclines, and penicillins. Isolates from swine farm and poultry farm presented significantly more resistant than those from dairy cattle farm. These results were correlative with the use of antimicrobial agents in livestock production. The resistance of E.coli isolated from young animals (including porket, chickling, and calf) were higher than those from adults animals, from infected animals were higher than those from healthy animals, and from medicating animals were higher than those from non-medicating animals.These may be due to younger animals were at a greater risk of contracting disease, and had a higher frequent prophylactic medication.The susceptibility of E.coli isolates collected from farmers and environment was consistent with those of collected from animals, and the same multi-resistant patterns of E.coli isolates from different sources were identified. It suggested that the spread of resistant bacteria among animals, environment and humans occurred on the farm. The susceptibility of E.coli strains isolated from pig carcasses consistent with those of pre-slaughtered pig, the susceptibility of E.coli isolates collected from retail beef,pork and chicken were corresponded to those of market-age cattle, pig and poultry,and a large proportion of resistant E.coli strains were isolated from cooked food. The results suggested that the resistant bacteria have access to enter human body through contaminated food.2. One-hundred and five (54.7%) strains of Escherichia coli which carry class I integrons were identified by PCR with primers designed to hybridize to integrase genes from 192 E.coli isolates collected from one pig farm. To further characterize the integrons of E.coli isolates, using primers specific for the 5 ' -CS and 3 ' -CS, touch-down PCR was used to amplify and sequence the gene cassettes associated with the identified class I integrons, and six different sized amplicons were identified. The DNA sequence of each amplicon was compared with published gene cassettes from genbank and confirmed the presence of the aminoglycoside adenyltransferase (aadA), dihydrofolate reductase (dhfr), beta-lactamase (bla), streptothricin acetyl-transferase (sat), and erythromycin esterase (ereA) genes, which confers resistance to strepto-mycin/spectinomycin, trimethoprim, β -lactams, streptothricin, and erythromycin, respectively.Six different class I integrons were prevalent in pig farm, which carried different genecassette (type and number). The most prevalent class I integron represented 36.6%, which carried dhfr and aadA2 two types of gene cassettes. One E.coli strain could carry one or two integrons, and almost all the integrons were located on the plasmids. Isolates collected from one pig material or from the same pigpen material had the similar integron profiles, but isolates with the same resistant patterns not always carried the same integrons. All isolates carried class I integrons were multiresistant, especially resistant to Sulfonamides (100%), streptomycin (90%), and spectinomycin (75%).Class I integrons carried dhfr and aadA cassettes confered resistance to trimethoprim and streptomycin represented 78% and 100% of cassettes detected, respectively. It was likely that selection for cassettes carrying dhfr genes and aadA genes had occurred in pig farm because trimethoprim was used for antibacterial growth promoter, and the extensive and long-term use of streptomycin, this could therefore account for their high prevalence, other cassettes identified in class I integrons were bla,sat,and ereA.The presence of these integrons and gene cassettes did not account for the total phenotypic resistance of all the isolates and did not exclude the presence of other mobile DNA elements in the resistant E.coli isolates.