Molecular Characterization Of Quinolones Resistance In Haemophilus Parasuis, And Mechanism Of Multi-drug Resistance In Streptococcus Suis

Bacteria drug resistance, as a phenotype of bacteria to resist the killing or inhibition of drug, is major responsible for impeding the prevention and therapy of bacterial diseases. Following the research and develop, drugs has been more and more extensively used. What’ more, abuse of drugs has been frequent. These conditions are beneficial to drug resistance. In recent years, the issue of drug resistance has become more and more serious, and is going to be higher resistant rate and more extensive resistance（multi-drug resistance（MDR））. It has been a serious human public health problem. Both Haemophilus parasuis（HPS） and Streptococcus suis（SS） are the important pathogenic bacteria of swine. Following the rapid development pig industry in recent years, the drug resistance of HPS and SS has been more and more serious. This paper focused on the mechanisms of drug resistance of HPS and SS.1. Molecular characterisation of resistance to quinolones in Haemophilus parasuis isolatedIn this study, 138 HPS isolated were choose as study object. Firstly, the E-test were used to detect the minimum inhibitory concentration（MIC） of 138 HPS isolated to enrofloxacin（EF） and levofloxacin（LF） respectively. According to the guidelines of the Clinical and Laboratory Standards Institute 2008（CLSI）, the resistance and susceptibility of all isolates were determined: The resistance rate of enrofloxacin was 60.1%. However, only 5.8% isolates were resistant to LF, all of which were also resistant to EF. Then, via sequence and analyse of DNA gyrase（Gyr A, Gyr B） and DNA topoisomerase IV（Par C, Par E）, 10 quinolone resistance related mutations were found. The mutation rate of Gyr A87 during all EF-resistant isolates was 100%. This result suggested that mutation of Gyr A87 should be crucial for the development of HPS to quinolones（QNs） resistance. Then the further analysis showed that there was synergy between 73Sâ†'R/I in par C and other point mutations with respect to QNs resistance. Furthermore, for the first time, the QNs resistance related mutation of Par E was found（Par E551 site mutation）. In order to prove the role of mutations of DNA gyrase and DNA topoisomerase IV for QNs resistance, in study, the site-directed mutagenesis was performed respectively: Gyr A（87Dâ†'N）, Par C（73Sâ†'R）, and Par E（551Tâ†'A）. The results showed that these 3 site mutations could significantly increase the MICs of HPS to EF and LF. And these data confirmed that the mutations of DNA gyrase and DNA topoisomerase IV do play an important role in QNs resistance. In addition, we also research the relationship between HPS QNs resistance and their hypothetical virulence factors. It was found that, compared with QNs-susceptive isolates, there were more hypothetical virulence factors（hhd A, fim B, and hsd R） in HPS QNs-resistant isolates. These data suggested that a positive relationship between QNs resistance and hypothetical virulence factors should occur in HPS isolates. This result was different from previous any related study, and enriched the body of knowledge concerning QNs resistance.2. The mechanism of multi-drug resistance of Streptococcus suis R61In our previous study, we found a ‘super drug-resistant’ SS isolate R61. Through the comparative genomics, the mechanism of MDR of R61 have been analyzed, but it was still incomplete. Therefore, in this study, we further researched the MDR mechanism of R61. Firstly, the R61 were cultured in different growth conditions（amoxicillin, chloramphenicol, cefotaxime, erythromycin, levofloxacin, tetracycline,no drugs, and adding 6 drugs at the same time）. When the strains grew at mid-log phase, the total bacterial proteins were extracted respectively. Then, via 2-D electrophoresis（2-DE）, we detected the differential expressed proteins by drug stimulation. A total of 46 differential expressed proteins（more than twice） were found, in which, up-regulated proteins were 32, and down-regulated proteins were 14. At the same time, these proteins were identified by mass spectroscopy（MS）. In order to further explain the MDR mechanism of R61, combining the function and characteristic of these differential expressed proteins, 13 proteins were choosed for the next research, in which, up-regulated proteins were 9, and down-regulated proteins were 4. The over-expression strains of these 13 proteins were constructed, and then were detected MICs. The data showed that the up-regulation of SSUR₀413（β-lactamase） and SSUR₂137（catabolite control protein A）, and the down-regulation of SSUR₁059（autolysin A） could significantly change the MICs of SS. Additional, the up-regulation of SSUR₀927（methyltransferase） could significantly increase the resistance of A7 to erythromycin. Taken together, our data proved that through up-regulating β-lactamase, increasing metabolism, and reducing degradation of autolysin, R61 could promote its own MDR.