Investigation And Mechanism Of Drug Resistance In H. Parasuis

Haemophilus parasuis is the causative agent of Glasser’s disease in swine, charac-terized by fibrinous polyserositis, meningitis and polyarthritis, and it also caused great economic losses in the global pig industry.15H. parasuis serovars have been recognized based on immunodiffusion of heat-stable polysaccharide antigens, while some isolates are still non-type serovars. H. parasuis has strong host specificity. H. parasuis could easily infect the pigs ranging from2weeks to4months and can cause severe diseases to the pigs, especially for the early-weaned piglets. The prevention and control of H. parasuis disease can be achieved through vaccine immunization and antibiotics therapy. The vac-cines usually confer protection against challenge with homologous serovar, but the results of the cross-protection are variable. So antibiotics were used as the main prevention strategy. However abuse of antibiotics would encourage an increase in resistance to the antibiotics, decreasing the efficiency of them. In recent years, the drug resistance cases in H. parasuis have been reported in many countries. So research the resistance mechanism and investigating the drug resistance in H. parasuis is very important for us to control this disease. Firstly, this study investigated the epidemiology about the drug resistance, isola-tion and identification of endogenous plasmids in clinical isolates of H. parasuis. Sec-ondly, the mutation sites that penicillin binding proteins were analyzed to identify the mechanism of the resistance to ampicillin and the endogenous plasmids in clinical isolates. At the same time, a whole-genome DNA microarray was used to profile the transcription-al response of H. parasuis SH0165induced tilmicosin at sub-inhibitory and inhibitory concentrations.The principal results were presented as followed:1. Investigating epidemiology about the drug resistance in H. parasuis, isolation and identification of endogenous plasmidsOur study investigated the epidemiology about the drug resistance in H. parasuis to ten kinds of common antibiotics, and the endogenous plasmids were also isolated and identified based on the92strains isolated in2009. The results revealed that20strains (21.74%) were resistance to penicillin,22strains (23.91%) were resistance to ampicillin,22strains were resistance to lincomycin and14strains (15.22%) were resistance to amoxicillin, while83strains (90.22%) were sensitive to cefotaxime and chloramphenicol. The detected22resistance genes of the strains were aadB gene, tetB gene and sulll gene which included20strains,11strains and8strains, respectively. At the same time, six en- dogenous plasmids of H. parasuis were identified, and the genomes of five of them were sequenced. Beside the plasmid FZJ5839is a recessive plasmid, the other plasmids were the resistant plasmids. Plasmid pHPS1019encoded the tetracycline resistance gene tetB and the replication protein rep. Plasmid FHN1020and FZG1012encoded the streptomy-cin resistance gene strA and the sulfasulfonamide resistance gene sulll, the movement proteins MOB family, but they are the different sources of resistance plasmids. Plasmid FJS5863carried β-lactam resistance gene blaROB-1and aminoglycoside modification bi-functional enzyme genes aacA-aphD, encoded the mobile family proteins and the replica-tion protein rep. The recessive plasmid FZJ5839is one of the biggest plasmids, which had been sequenced (11,812bp), encoded the movement protein mobA_mobL, recombinase rec, replication protein rep and splitting protein par A. Antimicrobial agents sensitivity profiling revealed that strains were resistant to the streptomycin, sulfadimidine, gentami-cin, kanamycin and ampicillin, beside of the tetracycline. The detected resistance genes of the endogenous plasmids were presents the resistant genes blaTEM, dhfrV, strA and sulll. Finally, the resistance plasmids of H. parasuis were analyzed by the phylogenetic tree, we found that have high similarity with the resistance plasmids in Pasteurella and the re-sistance plasmids in Haemophilus.2. Analysis of genes encoding PBPs in clinical isolates of H. parasuisBased on the genomic sequence of H. parasuis SH0165, we summarized the PBPs types, including six classes of PBPs, which has four HMM PBPs [two A classes (mrcA and pbp1B) and two B classes (ftsl and ftsI-2)], two LMM PBPs (dacA and dacB), one monofunctional transglycosylase mtgA and one peptidoglycan carboxy-terminal protease prc. We investigated the H. parasuis isolates (including22strains isolated in2009and34strains isolated in2010) resistance to β-lactam antibiotics (penicillin, ampicillin, oxacillin, ceftiofur, amoxicillin, clavulanic acid) and found that the resistance strains to ampicillin and oxacillin have reached95%. At the same time, two endogenous plasmids of H. para-suis were isolated in2012. PBPs genes of twenty strains (each of ten strains in2009and in2012) that resistance to ampicillin or carried endogenous plasmid were sequenced. Then we compared them with the PBPs gene sequence of the H. parasuis SH065and the standard H. parasuis serotype5, which had been found that the gene was highly con-served among the strains. Analysis of genes encoding PBPs through the relationship of the evolutionary tree, we predicted that the PBPs genes mutation sites of these strain and located the mutation sites on the three dimensional structure of the PBPs. We predicted that the mutation sites of the transpeptidase in PBPs have related to the (3-lactam antibiot-ics of the resistance, the mutation sites of the transglycosylase in PBPs have related to the transfer of the endogenous plasmid in the bacteria. The mrcA gene had inserted seven amino acids (ATENTTD) in the mutation sites638of the FJS5863plasmid of09FA17strain. It has7amino acids insertion and11mutation sites in the downstream of the mrcA gene and causes the changes in the3D structure of the mrcA protein. We speculated that these variations were related to the plasmid FJS5863carried the resistance gene blaвов-1. The dacB gene of the eight strains had199amino acids insertion in the downstream of the stop codon, that this difference was related to the endogenous plasmids. Others, the dacA gene of the five strains had changed the amino acid located in the sites of358to encode the termination codon.3. Transcription profile of H. parasuis SH0165response to tilmicosinBased on the genomic sequence of H. parasuis SH0165,2052probes were designed and synthesied in situ microarray chip. To gain a more detailed understanding of the mo-lecular mechanisms underlying H. parasuis response to tilmicosin treatment, microarray technology was applied to analyze the variation in gene expression of isolated H. parasuis SH0165treated in vitro with sub-inhibitory (0.25μg/mL) and inhibitory (8μg/mL) con-centrations. Tilmicosin treatment induced differential expression of405genes (p≤0.05, Fc≥1.5), the encoded products of which are mainly involved in the heat shock response, protein synthesis, and intracellular transportation. The sub-inhibitory and inhibitory con-centrations of tilmicosin induced distinctive gene expression profiles of shared and unique changes, respectively. These changes included302genes mainly involved in protein ex-port and the PTS system to sustain cell growth, and198genes mainly related to RNA polymerase, recombination, and repair to inhibit cell growth. In silico analysis of func-tions related to the differentially expressed genes suggested that adaptation of H. parasuis SH0165to tilmicosin involves modulation of protein synthesis and membrane transport. Collectively, the genes comprising each transcriptional profile of H. parasuis response to tilmicosin provide novel insights into the physiological functions of this economically significant bacterium and may represent targets for future molecular therapeutic strate-gies.