Molecular Basis And Mechanism Of Drug Resistance In H. Parasuis

H. parasuis is a ubiquitous, opportunistic pathogen of swine and the causative agent of Glasser's disease, which was first described by K. Glasser in1910as a bacillus found in the serous exudates of pigs. It is an early colonizer of the upper respiratory tract of pigs. It invades the surrounding mucosa and is disseminated systemically via the bloodstream. Finally resulting in disease is manifested as systemic inflammation of serous surfaces of pigs.H. parasuis belong to the family of Pasteurellaceae, which currently includes at least10genera and more than50species of bacteria. Members of this family are polymorphic, G-cells ranging from coccobacilli to rod shaped, measuring<1.0μm in diameter. They are commonly isolated from vertebrates, especially mammals, birds, and their hosts. The genus Haemophilus are facultative anaerobes that are oxidase, catalase, and alkaline phosphatase positive. It includes fastidious organisms requiring complex media for growth. They have a strict requirement for X factor (protoporphyrin) and/or V factor (nicotinamide adenine dinucleotide) and grow optimally at35to37℃. H. parasuis forms smooth, gray colonies on chocolate agar or TSA growing to a diameter of approximately0.5mm. Some strains form capsules and tend to be coccobacillary whereas nonencapsulated strains tend to be filamentous in shape.The onset of acute Glasser's disease is usually seen within2days of the first signs of disease, such as coughing and dyspnoea, pyrexia (40to41℃), anorexia, serous nasal discharge. Clinical signs are dependent on the localization of the pathogen within the host. When pathogen located in the joints, it usually results in swelling, lameness, and lateral recumbent. Other signs include cyanosis of the extremities caused by the failure of peripheral circulation, reddened conjunctivae, subcutaneous edema of the ears and eyelids, and abortion. While pathogen located in the brain, meninges, or spinal cord causes central nervous system disturbances such as posterior paresis, tremors, and incoordination.Chronic disease in piglets can result in paleness, poor growth, and decreased feed efficiency. Chronic arthritis, intestinal obstruction, meningitis and peritonitis maybe observed in some cases. Survivors suffer chronic pericarditis and congestive cardiac failure which is frequently linked to sudden death. Acute disease may include arthritis, meningitis, pericarditis, polyserositis, pneumonia, and septicaemia. The mortality maybe reach50%if left untreated the acute disease and can have a potentially devastating economic impact on swine herds.Based on history, clinical signs and the presence of characteristic lesions can diagnose Glasser's disease. The characteristic lesions contain serofibrinous exudates in the peritoneal cavity or petechial renal hemorrhages, pericardial sac, fibrinopurulent exudates of the brain, or multifocal lesions of the lung, and so on. Isolation of H. parasuis is required for confirmation of Glasser's disease. At the same time, H. parasuis also needs to be differentiated from similar pathogens such as Streptococcus suis and Pasteurella spp.. Recently, a number of molecular methods have been developed for the identification of H. parasuis as an alternative to culture.Treatment with parenteral antibiotics may be effective after H. parasuis diagnosis is made. Reduction or elimination of stressful situations may also reduce outbreaks. In pig industry, transporting and handling of pigs is often necessary so that strategic administration of antimicrobials before, during, and after required transport has been used to reduce the occurrence of Glasser's and other stress-induced diseases. Limited cross-protection due to serovar heterogeneity and the existence of untypable isolates has meant that vaccine development is very difficult, although current understanding of the basis of immunity is rudimentary.The objective of this research is,(1) To detected the susceptibility of H. parasuis clinical isolated strain to nalidixic acid, enrofloxacin and ciprofloxacin, and identify the molecular mechnasim of H. parasuis resistant to quinolone antibacterial.(2) Isolated plasmid from clinical strain and sequenced, annotate the genes in the plasmid and compared with plasmid isolated from other Pasteurellaceae family bacteria.(3) The transcriptional profile of H. parasuis to subinhibitory concentration of florfenicol was investigated using the H. parasuis microarray developed by Agilent Corporation.1. Molecular mechanism of H. parasuis resistant to quinoloneThis thesis examined molecular changes in the quinolone-resistance determining regions (QRDR) of the topoisomerase genes, gyrA and parC of H. parasuis and their contribution to quinolone resistance. H. parasuis isolates and standard serotype strain were confirmed by16S rRNA PCR and then investigated for quinolone (nalidixic acid, ciprofloxacin and enrofloxacin) susceptibility and mutation in QRDR by PCR and DNA sequence. Additionally, plasmid-mediated quinolone resistance (PMQR) determinants were determined by specific PCR.Among156clinical isolated strains,21H. parasuis isolates showed higher MIC values of nalidixic acid, enrofloxacin and ciprofloxacin. No strains possessed qnrA, qnrB, qrnC, qnrS, aac(6')-Ib-cr and qepA plasmid and efflux pump mediated quinolone resistance. Mutation analysis of QRDRs showed that the lower susceptibility strains carried at least one mutation in GyrA (at codon83or87), mutation occurred in ParC(at codon73and77) followed by mutation in gyrA.Key findings were that the initial QRDR changes always occurred in GyrA and were the predominant influence on phenotypic expression of quinolone resistant. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in H. parasuis. GyrA alterations at Ser-87in H. parasuis were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on quinolone.This thesis provides a comprehensive analysis of the relationship between QRDR alterations and quinolone resistance in H. parasuis and offers insights into the potential for quinolone development in H. parasuis.These data provide important insights into the mechanism of fluoroquinolone resistance in H. parasuis, thereby highlighting the usefulness of quinolones for the treatment and control of this infection.2. Plasmid-borne antibacterial resistance in H. parasuisH. parasuis isolate from pigs were investigated for the genetic basis of antibacterial resistance.156H. parasuis isolate strains were screened for plasmid content. A4.2kb plasmid, designated pHPS-A67, was extracted from strain A67.R-plasmid isolated as described above was digested with Sau3AI according to the manufacturer's recommendations. Restriction fragments were purified using QIAquick Spin columns (Qiagen) and ligated into vector pUC19which previously digested with BamHI. Ligation reactions were used to transform E.coli BL21chemically competent cells. Recombinant plasmids were purified with QIAprep Spin columns (Qiagen) and inserts were sequenced using the vector's internal sequencing primers(M13forward and reverse primer). According to the sequence of inserts design primers used for further sequence of the whole sequence of the plasmid.The plasmid is a4.2kb double DNA. It is comprised with mobA, mobB, mobC, sul2and strA genes. mobA, mobB and mobC coded the mobilization protein for plasmid transport, while sul2and strA coded protein mediated resistance to streptomycin and sulfonamide. Strain A67, which contained the isolated plasmid, has streptomycin and sulfonamide MIC128and512μg/mL, respectively.The results of this study showed that a plasmid-borne sul2and strA gene was responsible for streptomycin and sulfonamide resistance in the swine respiratory tract pathogen. This is, to the best of our knowledge, the first report of an antibiotic resistance plasmid in H.parasuis from China. 3. Transcriptional responses of H. parasuis to subinhibitory concentration of florfenicolThe response of H. parasuis to subinhibitory concentration antibiotics is similar to mimic in vivo conditions encountered during infectious disease treatment. The transcriptional profile of H. parasuis to subinhibitory concentrations of florfenicol was investigated using the H. parasuis microarray developed by Agilent Corporation.All experiments were carried out in biological triplicate. An overnight liquid culture was inoculated into TSB with and without0.25μg/mL florfenicol. The cells were harvested16h latter. RNA was extracted using a TRIzol Max bacterial RNA isolation kit (Qiagen) according to the manufacturer's instructions, and then DNase treated. A16S rRNA PCR amplification was carried out using0.5mL RNA as template to ensure there was no residual DNA.Significantly regulated genes and operons were identified that are unique to or common between antibiotic stresses. The microarray chip demonstrate that a total of163genes were identified to be differentially regulated(FC>1.5, p<0.05),96of which were upregulated, and67down-regulated. Florfenicol-induced transcriptional changes mainly occurred in genes responsible for carbon utilization, ion uptake and virulence factor. The results reveal general changes in gene expression that are inconsistent with known mechanisms of action of florfenicol.This study provides a starting point for detailed analyses of numerous genes whose expression is affected by florfenicol and may therefore be involved in adaptation to the host environment.