Study On The Integrons And Multiresistance In Shigella Spp.

As an important pathogen of infectious diarrhea, Shigella spp. can cause bacillary dysentery, also known as shigellosis. Shigellosis is the third high incidence in infectious diseases and the emphasis of prevention work in infectious diseases in China. Antibiotics have been used to cure shigellosis clinically for a long time. Antimicrobial therapy has been effective not only in allaying the dysenteric syndrome of shigellosis, but also reducing the duration of illness and the fecal excretion of the bacterium which preventing the further transmission. However, with the wide use of antibiotics, resistant isolates of Shigella spp., especially multiresistant isolates with multi-drug resistance (MDR), occurred. In case of shigellosis caused by a multidrug-resistant isolate, the safe and effective treatment options is limited both in developing and developed country. The multiresistance of Shigella spp. will cause not only clinical failures in the treatment of shigellosis but also the public health crises with persisting and spreading worldwide as a multidrug resistant organism. Therefore, the MDR of Shigella spp. and its molecular mechanisms have been paid great attentions.Antibiotic resistance of Shigella spp. can be attained through intrinsic or acquired mechanisms. Intrinsic mechanisms are those such as gene mutations found on chromosome, for example, mutations within the drug's targets, DNA gyrase and topoisomerase, to get the resistance to quinolones, and those such as enzymatic mechanisms of drug modification, altered membrane permeability and enhanced efflux pump expression, to get MDR. Acquired mechanisms involve the extrachromosomal elements acquired from other bacteria in the environment. Although classically attributed to chromosomal mutations, resistance is most commonly associated with acquired mechanisms. There are different types of mobile DNA segments such as plasmids, transposons, and bacteriophages which can transfer resistance determinants among organisms within the same genus or between distant organisms as gram-positive and gram-negative bacteria and then contribute to multidrug resistance in bacteria. Recently, integrons have been observed to take part in the transfer of resistance genes. Integrons are genetic elements composed of an integrase, gene cassettes and the site-specific recombination site for gene cassettes. Integrons themselves are not mobile but integrase can excise and integrate the gene cassettes from and into the integron. Integrons can reside on transposons, conjugative plasmids, or the chromosome, facilitating the acquisition and dissemination of gene cassettes and so accounting for their wide distribution and their significant association with the multiresistance phenotype in Enterobacteriaceae. Many studies showed that integron containing isolates were more likely to be resistant to drugs and integron-gene cassettes were related to the multiresistance of Shigella spp.. Class 2 integron was found more frequently than class 1 integron in Shigella spp. in studies all over the world. The types of gene cassettes discovered in isolates of Shigella were not various relatively. Neither the classes of integron nor the types of integrated cassettes were correlated completely to the resistant phenotype of Shigella spp.. The resistant phenotype could only be partially explained by the presence of integrons. It's necessary to investigate the relationship among integron, gene cassettes and multiresistance, and to evaluate the status and contribution of integrons to the multiresistance of Shigella spp..In current study,83 isolates of S. flexneri and 7 isolates of S. sonnei were determined the susceptibility to antimicrobial agents and screened for the presence of integrons and gene cassettes. The clone and functional analysis of integrons and gene cassettes were conducted to investigate the contribution of integrons to the multiresistance of Shigella spp..Methods1 Susceptibility testNinety isolates of Shigella spp. were determined their susceptibility to eight antimicrobial agents, which were ampicillin (AMP), tetracycline (TET), trimethoprim-sulfamethoxazole (SXT), chloramphenicol (CHL), nalidixic acid (NAL), ciprofloxacin (CIP), gentamicin (GEN) and cefazolin (CFZ), by the disk diffusion method on Mueller-Hinton agar according to the recommendations of the Clinical and Laboratory Standards Institute. The minimal inhibitory concentration of TET, AMP, CHL, CIP, streptomycin (STR), trimethoprim (TMP) and sulfamethoxazole (SSS) were also conducted. Escherichia coli ATCC 25922 was used as a quality control strain.2 Screening for integrase and various region of integron by PCR and PCR-RFLPIntegrase of class 1, class 2 and class 3 were detected by PCR from the total DNA extracted by boiling method. Total DNA and plasmid DNA were extracted from all isolates respectively by Axyprep Bacterial Genomic DNA Miniprep Kit to detect the various regions of integrons. The primers hep58 and hep59 were designed to amplify the various region of typical class 1 integron, primers hep58 and ISR for that of atypical class 1 integron, and primers hep74 and hep51 for that of class 2 integron. The PCR products of various regions with similar length were examined by restriction fragment length polymorphism analysis with PvuⅠ, HindⅢor HinfⅠThe identical restriction profiles were regarded as the same array of gene cassettes.3 Southern blottingSouthern blotting analysis was applied according to the manufacturer's instructions of DIG DNA labeling and detection kit. After electrophoresis of the total DNA digested by PstⅠand BamHⅠ, or the plasmid DNA in 0.8% agar gel, the DNA transferred to Hybond N+nylon membrane and fixed in hot air drying oven, the digoxigenin-labelled DNA probes specific to intI1 and intI2 by random primering were used to identify the location of integrons.4 Conjugation testConjugation was carried out by using E. coli DH5a as a recipient in all the 90 isolates. The over night culture of purified donor and recipient cells were diluted 100-fold in 2mL of fresh LB broth. The growth continued at 37℃until OD600 reached about 0.6. Then 0.1 mL each of the donor and recipient cultures was mixed in 1 mL of fresh LB broth. After incubation at 37℃without shaking for 16-18h, the mixture was plated on MacConkey agar. The morphological differences between E. coli and Shigella on MacConkey were used to select the recipients. The change of the susceptibility to antimicrobial agents was measured by the disk diffusion method as described above.5 Sequence analysis and phylogenetic tree of integron and gene cassettesThe integrase and various region of typical class 1 integron, atypical class 1 integron and class 2 integron were amplified by PCR respectively. The PCR products of representative isolates were sequenced and then analyzed by using software BLAST on the website of the National Center for Biotechnology Information. Several sequences from different class, order or genus were selected among all sequences with 99%-100% identify. Alignment of multiple sequences was conducted by the software ClustalX. The construction of phylogenetic tree was carried out by the neighbor joining (NJ) method and maximum parsimony (MP) method respectively using software MEGA 4.1. Bootstrap test was selected to validate the phylogeny in each process.6 Cloning and functional analysis of integron and gene cassettesThe interested PCR products (including the gene cassettes of typical class 1 integron located in conjugatable plasmid named as pvl, the gene cassettes of class 2 integron located in genome named as pv2, and the whole gene of class 2 integron named as intI2pv2) of representative isolates were cloned into pucl8 or pMD-18 by the Takara TA cloning kit, and then transformed into E. coli DH5a. The minimal inhibitory concentration to antibiotics of positive clones DH5a with puc-pvl and puc-pv2 were determined respectively. Conjugation was carried by using E. coli DH5a and positive clone DH5a with puc-intI2pv2 as recipient respectively, and S. flexneri 05100 as donor. The resistances to antibiotics of conjugants were compared.7 Continuous cultivation without antibiotic pressure for the multiresistant isolates of Shigella spp.Ten isolates of S. flexneri with different resistant phenotypes and positive integrons were cultured continuously without any antibiotic pressure. Single clone of each isolate was inoculated in fresh LB broth at 37℃overnight. On day one, the suspension was diluted 100 times in fresh LB broth and cultured continuously at 37℃with 200rpm shaking for 3-4h till OD600 reaching about 0.08-0.12. The process was repeated three times a day. The same process was repeated for each isolate for 10 days. The isolate after every 5 inoculation was subjected to the suscepbility test and detection of integrons by PCR.Results1 Prevalence of resistance and integrons in Shigella spp.Ninety Shigella strains (83 S. flexneri and 7 S. sonnei) showed high resistant rate to TET, NAL, AMP, SXT and CHL at 93.3%,92.2%,91.1%,82.2%and 75.6% respectively. Multidrug resistance was detected in 86 isolates (95.6%). Among all the Shigella spp., the most frequent resistant profiles were AMP-TET-SXT-CHL-NAL (31/90,34.4%). PCR analysis revealed that 79 (87.8%) isolates carried integron of class 1 only (3.3%), class 2 only (10.0%) and both (74.4%). No intI3 was found.89.5%(77/86) isolates of multidrug resistant Shigella spp. carried integron. The prevalence of intI2 was significantly different between multidrug resistant isolates and non-multidrug resistant isolates (p<0.05).2 Characteristics of integron and gene cassettes in Shigella spp.Four gene cassette arrays were found. The sequences were submitted to the GenBank database and the accession numbers are FJ895301, FJ895302, GQ214137 and EF634237 respectively. There were overall 56 isolates of multiresistant S. flexneri carrying three integrons simultaneously. They are the gene cassettes of classic class 1 integron dfrA17-aadA5 or dfrA12-orfF-adA2 located in conjugatable plasmid, the gene cassettes of atypical class 1 integron blaoxa-30-aadA1 located in genome and the gene cassettes of class 2 integron dfrA1-sat1-aadA1 located in genome.The prevalence of gene cassettes of atypical class 1 integron blaoxa-30-aadA1 in isolates resistant to AMP-TET-CHL association was significant higher that in isolates without resistance to AMP-TET-CHL association (p<0.05).3 Sequence analysis and phylogenetic tree of integron and gene cassettesExcept for integrase gene of class 1 integron intI1 and gene cassettes blaoxa-30-aadA1 of atypical class 1 integron, the sequences of following fragments, gene cassettes of class 1 integron dfrA17-aadA5, dfrA12-orf-aadA2, integrase gene of class 2 integron intI2, and gene cassettes of class 2 integron dfrA1-sat1-aadA1, were all found having genetic differentiation.4 Cloning and functional analysis of integron and gene cassettesThe positive clone of DH5a with puc-pv1 (dfrA17-aadA5) increased the MIC to STR from 0.5μg/ml to 4μg/ml, the MIC to TMP from 2μg/ml to 64μg/ml. The positive clone of DH5a with puc-pv2 (dfrA1-sat1-aadA1) increased the MIC to STR from 0.5μg/ml to 32μg/ml, the MIC to TMP from 2μg/ml to 64μg/ml.No resistance was transferred by conjugation from the plasmid of S. flexneri 05100 carrying gene cassettes of class 1 integron dfrA17-aadA5 to the recipient DH5a. However, the resistance to SXT and AMP was transferred by conjugation from the plasmid of S. flexneri 06208 carrying gene cassettes of class 1 integron dfrA12-orf-aadA2 to the recipient DH5a. With S. flexneri 05100 as the same donor, no changes of MIC to antibiotics were detected after conjugation with the positive clone of DH5a with puc-pv2 (dfrA1-sat1-aadA1) and DH5a as recipient respectively.5 The results of continuous cultivation without antibiotic pressure for the multiresistant isolates of Shigella spp.After 30 times of inoculation without antibiotic pressure, the isolate of S. flexneri 05015,05105,06208 lost the single resistance to CHL, SXT and CIP respectively without any change of the integrons'status. The isolate of S. flexneri 06218 lost all the resistance (to TXT, SXT and AMP) as well as the only integron, class 2 integron and gene cassettes dfrA1-sat1-aadA1, located in genomic DNA.Conclusions1. The isolates of Shigella spp. are usually resistant to common antibiotics and most of them are multidrug resistant (95.6%). The positive of intI2 is related to the multiresistance of Shigella spp. The positive of gene cassettes blaoxa-30-aadA1 of atypical class 1 integron is related to the associated resistance to AMP-TET-CHL.2. It's the first report of coexistence of three integons and gene cassettes, the classic class 1 integron located in conjugatable plasmid, the atypical class 1 integron located in genome and the class 2 integron located in genome.3. The genetic differentiations occur among sequences of integrase and gene cassettes from different class, order or genus.4. The gene cassettes of integron in Shigella spp. lead to the specific resistance and the level of resistance was different in different gene cassette.5. The plasmid carrying integron in Shigella spp. can transfer the resistance unrelated to the integron.6. The class 2 integron and resistant gene cassettes may take part in the assemble or excise of multiple resistant genetic structure by participating the activity of other mobile DNA segments, accordingly having indirect association with the multiresistance of Shigella spp..