A Pilot Study On Inter-relationship Between Integron And Bacterial Antibiotic Resistance

1. DISTRIBUTION AND ASSOCIATION OF INTEGRON IN ESBL-PRODUCING AND NON-PRODUCING BACTERIABackgroundHorizontal dissemination of integrons has now been known to be the main cause of rapid resistance transmission. Integrons are assembly platforms that incorporate exogenous open reading frames (ORFs) by site-specific recombination and convert them to functional genes by ensuring their correct expression. ESBL genes and other drug resistant genes frequently remain together in the same plasmid, leading to the formation of highly resistant bacteria posing a greater challenge to the treatment of infectious diseases. Till date, researches about ESBL genes are quite common but those about integrons in ESBLs carrying bacteria are relatively less.ObjectiveThis research is about the analysis of integrons in the ESBLs clinical isolates and non-ESBLs isolates of Escherichia coli and Klebsiella pneumoniae detected in the third Xiangya Hospital of Central South to University. Our aim is to study the presence and distribution of these genetic elements in relation to the drug resistance brought by them and to analyze the different kinds of effects brought by the presence of integrons in ESBLs producing and non-producing bacteria and thus to investigate the changes in the degree of resistance of the ESBLs genes in the presence of integrons.MethodsA total of172strains of ESBL producing and non-producing Escherichia coli and Klebsiella pneumoniae that were isolated in the Third Xiangya Hospital of Central South University from June2009to December2009were collected. PCR was used to detect the presence of integrons. The variable region of integron was sequenced and also analysed by RFLP study using Alu I digestion.ResultAmong ESBL producing strains, the finding of class Ⅰ integron was69%and among ESBL non-producing strains, the finding was only40.2%, the difference having statistical significance, P value<0.05. Out of total17antibiotics studied, ESBL-producers having integron were significantly resistant to12kinds of antibiotics compared to ESBL-producers without integron. But in case of ESBL-non-producers, strains having integron were significantly resistant to only8kinds of antibiotics compared to strains without integron. Similarly, study of the relationship of integron and ESBL from another angle showed that, among integron positive strains, ESBL-producers were significantly resistant to9different antibiotics compared to ESBL-non-producers. But in case of integron negative strains, ESBL-producers were significantly resistant to only8antibiotics compared to ESBL-non-producers.84%of integron positive strains showed the presence of variable region, whereas remaining15.9failed to do so. Though co-existence of ESBL and integron was profound, no ESBL gene was found in the variable region of integron. Integrons of multiple gene cassettes were found, the gene cassette of dfrA17and aadA5together, were most numerous (41.4%).36.2%of bacterial strains carried multiple integrons.ConclusionESBL genes and class Ⅰ integron not only co-exist but their co-existence also increases the resistance efficiency of bacteria leading them to be multi-drug resistant. 2. INTEGRON INTEGRASE EXPRESSION IN NON-LETHAL ANTIBIOTIC SELECTIVE PRESSURE.BackgroundUnder stressful conditions, bacteria are known to induce responses that increase genetic variation which can bestow a selective damage. The responses include the SOS response, the general stress response, the heat-shock response, and the stringent response, all of which impact the regulation of error-prone polymerases. Under different selective pressures, once the bacterial antibiotic resistance genes are produced, these resistance genes are transferred to different bacterial strains via the site-specific recombination mechanism of the integron, thus leading to the development of resistance in these new strains. This is one way sub-inhibitory concentration of antibiotics can trigger antibiotic resistance. The other way is through induction of other genetic defence mechanism like SOS response, The General Stress response etc, in which bacteria respond to stressful conditions by changing their pattern of gene expression so that the stress is relieved.ObjectiveIn this research, we intend to sort out what effect sub-inhibitory concentration of antibiotics can have on the expression of integron integrase, a gene that is known to play a pivotal role in horizontal transfer of antibiotic resistance genes among bacteria. MethodsTwo strains of Eschericahia coli (E9and E22) that had a sequence of dfrA17-aadA5genes in their integron variable region, were selected for the study of integrase expression under the influence of antibiotic pressure. We used three antibiotics to give stress to our desired strains of bacteria. Those antibiotics were Trimethoprim (a chemotherapical agent), Streptomycin (an aminoglycoside) and Levofloxacin (a synthetic fluoroquinolone). The MIC of these antibiotics for each of the strains were detected. The bacteria were grown continuously for four months in LB broth containing sub-inhibitory concentration of these three antibiotics and also in antibiotic-free LB broth. Integron integrase expression was estimated in an interval of every one month from each of these four different growth conditions. The comparison of integrase expression was done with the original untreated strain. In order to provide proper nutrition, the media with proper antibiotic concentration was changed every48hours. The level of gene expression was quantitatively determined by using TranstartTM Green qPCR SuperMix UDG system. Relative expression of integron integtrase was determined by using Δ Δ Ct method, using16S rRNA as internal control.ResultMaximum increase of integrase expression was seen in sub-inhibitory concentration of Levofloxacin for both the bacterial strains. For E9strain, the expression of integrase went up to an level of77.70fold in a period of four months. For E22strain, the expression went up to a level of18.25in this time period. The expression of integrase under Levofloxacin stress was greater than that in untreated bacteria in every lot of comparison. Stress under Trimethoprim showed no increase in integrase expression in either of the strains when Trimethoprim treated strains were compared with the original untreated strains. Under Streptomycin stress, expression of integrase was seen to increase in both E9and E22strains to some extent, though not as much as that in Levofloxacin stress. In E9the maximum expression went upto13.73fold, while in E22it went up to3.7fold. The expression of integrase in antibiotic-free growth condition showed an irregular pattern of expression level for both E9and E22strains. Maximum expression for E9was4.2fold while for E22, it was1.9fold.ConclusionA prolonged non-lethal stress of antibiotic selective pressure can provoke integron integrase induction, the degree of provocation, however, can vary among different antibiotics and different strains.