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The Analysis of the Drug Resistance and Integrons of Aeromonas Hydrophila Clinical Isolated Strains

Author: XiaoGenHui
Tutor: WangPing
School: Jiangxi Agricultural University,
Course: Preventive Veterinary Medicine
Keywords: Aeromonas hydrophila aerolysin gene multi-drug resistant integron resistance gene cassette
CLC: S941
Type: Master's thesis
Year: 2012
Downloads: 61
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Abstract


Aeromonas Hydrophila is not only an important aquatic organisms pathogen which causes great economic losses to the aquaculture industry, but also is an important zoonotic pathogen that can be passed through water-borne, foodborne dissemination and wound infection,which caused illnesses such as pneumonia, septicemia and food poisoning. So the public health significance caused by this pathogen has been being concerned at home and abroad. In recent years, scholars have illustrated the existence of Integrons-gene Cassettes System which exist in gram positive and gram negative bacteria.With the help of integrase, The bacteria can be resistant to drug or multi-drugs when the captured drug resistance gene expressed,which depend on the promoter of integrons’ upstream.The plasmids and transposons can be used as the carrier which can lead to the spread of resistance genes between inner-species and inter-species in bacterium,resulting in accelerating the spread of drug resistance.Therefore, this experiment will study the distribution, structure and function of Integrons-gene Cassettes System, discuss the relevancy of the phenotypes of multiple drug-resistant and Integrons-gene Cassettes System in bacteria. Utilizing bioinformatics to analysis the sequence and structure of gene cassette in variable region, which helps to understand integrase sites involving in the ways of gene cassettes’ capture and excision,these will be available for the dynamics research of Integrons which mediate the level spread of resistance genes in Aeromonas Hydrophila.18strains are isolated from the tissue samples of diseased Chinese soft-shelle and finless eel,which feeded in scale aquacultural grounds in jiangxi province in2008-2011,the isolates are identificated as Aeromonas Hydrophila by the bacteria’s colonies and morphologic observation, cultivating characteristics, biochemical identification, virulence factors and PCR detection.In order to understand the drug-resistant phenotype and feature of Aeromonas hydrophila Jiangxi isolates. This study detectes the sensitivity of isolates to21kinds of commonly used antibiotics by the K-B disk diffusion method.10isolates of Chinese soft-shelled turtle are mutiple resistant to13~20antibiotics, The average of multiple drug resistance rate is76.7%.6/8isolates of finless eel are resistant to the16~21antibiotics, one isolate is resistant to5antibiotics,and another isolate is resistant to13antibiotics The average of multiple drug resistance rate of turtle and finless eel isolates are75.6%,。The results show that18isolates from different sources are serious resistant to multi-drugs,It was small that the diversity of multiple drug resistance rate between the isolates from different source. we analysis the durg resistance spectrum of strains such as ciprofloxacin, levofloxacin, doxycycline and Ttetracycline through the explaination table and scatter plot by the software WHONET5.4. The isolate which is highly resistant to tetracycline is serious cross-resistant to doxycycline ciprofloxacin, levofloxacin. The integrons play an important role in the molecule mechanism of drug resistance in Aeromonas hydrophila, this study will detect the integron/gene cassette system of isolates and analyse the integrons’structural characteristion. Designing the primers of Class Ⅰ, Class Ⅱ integrase gene, the variable region in integron and qacE△1-sull gene of3’conserved domain by the software premer premier5.0to detect the18strains by PCR and purify and sequence the positive products The results showed that there are six soft-shelled turtle isolates carring the Class Ⅰ integron,16isolates carring the class Ⅱ integrons, two finless eel isolates carring neither class Ⅰ or class Ⅱ integron. The sequences homology of Class Ⅰ and Ⅱ integrase genes between the isolates and other bacteria are more than99.6%and99.4%. while the sequences homology between Class Ⅰ and Ⅱ integrase gene is only22.1%, which confirm the integrons belong to different types.In order to understand integrase sites involving in the ways of gene cassettes’ capture and excision. Utilizing bioinformatics to analysis the amplifications of the integrons’ variable region. There are five different gene cassettes with the different fragment length from1452bp (of InI-A),1530bp (of InI-B),1238bp (of InI-C),1840bp (of InI-D) to2478bp (of InI-E). Comparing related sequence and analysising their functions by the softwares. we find that both InI-A and InI-B carrying the acc-(6’)-Ib-cr and arr-3gene cassette which mediates the resistance of aminoglycosides, quinolones and rifampicin to the Aeromonas hydrophila respectively. The InI-C carries GGDEF family proteins or diguanylate cyclase, DGC which can catalytic GTP and synthesis C-di-GMP.The InI-D carrying aadA2and,dfrA12which mediates resistance of aminoglycosides, Trimethoprim respectively, it contains an unknown proteina named orfF. The InI-E carries the cmIA6and aadA2gene cassette which mediates the resistance of chloramphenicol, and aminoglycosi-des respectively,The integron variable region of Inl-A has three recombination sites:aatC (59bp), aatll and aatI2, the core sequence of the latter two are highly similar,only a lack of a base T after the core bit point "CCCTAAA" in attI2.Furthermore the sequence homology of aatI1aatI2and attC between the Klebsiella pneumoniae bacteria (GenBank accession EU543272) and InI-A is more than99%. The resistance gene acc-(6’)-Ib-cr is integrated between the specific points of aatI1and aatC (59bp).and the arr-3gene is integrated between the specific points of aatC (59bp) and aatI2.

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