Study On Antimicrobial Resistance And Homology Of Haemophilus Influenzae

BackgroundHaemophilus influenzaee generally live in respiratory tract without causingdisease. Haemophilus influenzae, which is an opportunistic pathogen, infection ratescontinue to rising as result of the extensive use of antimicrobial agents for the pastfew years, especially for child and elderly patients with viral infection, invasiveoperation, using immunity inhibitor and so on. Under this background Haemophilusinfluenza easily creates serious diseases such as meningitis, bacteria,community-acquired pneumonia, acute sinusitis, and acute otitis media and so on.Haemophilus influemzae is secondarily common pathogen after S. pneumonia forcommunity-acquired pneumonia. Ampicillin and ciprofloxacin are the commonantibiotics in respiratory infection. The long-term surveillances of Haemophilusinfluenzae around the world show that the resistance rate of Ampicillin andSulfamethoxazole–Trimethoprim increases year by year and differs from area to area.β-lactamase-negative and ampicillin-resistant strain (BLNAR) also increases.Fluoroquinolone-resistant influenzae has been reported in our country．In Haemophilus influenzae, the resistance to ampicillin and other β-lactams is theproduction of β-lactamase which has two genotypes of TEM-1and ROB-1, TEM-1type accounting for great majority. In the case of β-lactamase-negativeampicillin-resistant isolates, the presence of altered PBPs low affinity with β-lactams.The mechanism of Haemophilus influenzae resistance to fluoroquinolones antibioticsis the amino acids mutation in quinolone resistance determining region (QRDR). Thetarget site of fluoroquinolones is topisomerases Ⅱ coded by gyr gene andtopisomerases Ⅳcoded by par gene, which are related to DNA replication andtranscription. The mutations of the QRDR region inhibit bacteria DNA replication,finally leading to bacteria death. There are different kinds of methods of molecular biology typing such asenterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), pulse field gelelectrophoresis (PFGE), which identify the homology between strains throughstudying genome DNA. PFGE is commonly considered a gold standard inepidemiological studies of pathogenic organisms for strong resolution, and highrepeatability. PFGE assay is a ideal tool in monitoring bacterial transmission，trackingsource of infection，controlling prevalence.ObjectiveTo investigate the distribution and antimicrobial resistance of Haemophilusinfluenzae strains, to examine genotypes and molecular epidemiology of Haemophilusinfluenzae in this area so as to effectively guide clinical rational drug use and provideevidences for the clinical therapy.MethodKirby-Bauer disk diffusion method was applied for the drug susceptibility tests,and antibiotics test results were judged by CLSI20. Nitrocefin slide test was used todetect β-lactamase. genes TEM, ROB, gyrA, gyrB, parC, parE, qnrA, qnrB, qnrS andaac（6’）-Ib-cr were amplified by polymerase chain reaction(PCR), and PCR productswere sequenced. DNA homology was analyzed by pulsed-field gel electrophoresismethod (PFGE). A total of52clinical isolates were analyzed based on WHONET5.4software.ResultsA total of52strains of Haemophilus influenzae were collected from January2010to July2011,42strains from clinical sputum samples and10strains fromoutpatient. These strains were isolated from patients whose ages were39～85（66±4.9）. The percentage of isolated bacteria in respiratory department, outpatientservice and other department was53.85%,19.23%and26.92%respectively. The resistance rate of Haemophilus influenzae isolates to ampicillin was19.23%,Sulfamethoxazole–Trimethoprim65.38%, Aztreonam and Amoxicillin/ClavulanatePotassium11.54%. The susceptible rates of Haemophilus influenzae isolates to thirdand fourth generation cephalosporins, ampicilin-sulbactam azithromycin andchloromycetin were above90%. Haemophilus influenzae strains were not resistant topiperacillin-tazobatam and imipenem. The10resistant strains to ampicillin allproduced β-lactamases. The prevalence of β-lactamase was19.23%.Polymerase chain reaction (PCR) indicated:10strains of Haemophilusinfluenzae β-lactamases–producing had PCR products of TEM gene, TEM-1genotypecompared to GenBank, but no products of ROB.2strains of Haemophilus influenzaeresistant to Ciprofloxacin agents had PCR products of gyrA, gyrB, parC and parEgenes, but no products of qnrA, qnrB, qnrS and aac（6’）-Ib-cr genes on plasmid. Bothstrains of resistant to ciprofloxacin produced No.84and88amino acid mutation ingyrA gene, namely Ser84Phe and Asp88Tyr. One ciprofloxacin resistant isolateproduced Ser84Ile mutation, another not.51different PFGE bandage patterns were presented in the total52isolates.2Haemophilus influenzae strains had same types and had only one distinct bandage.ConclusionCurrently, the susceptible rates of Haemophilus influenzae to commomantibiotics remain relatively high except ampicillin and Sulfamethoxazole–Trimethoprim in HeFei area of China. TEM-1β-lactamase leads to emergence ofHaemophilus influenzae resistance to ampicillin. The resistance to ciprofloxacin isrelated to the amino acid mutation in gyrA and parC genes. PFGE results show thatHaemophilus influenzae strains are mostly different clones which are distributeddiffusedly in the region. The cloning spread is not prevalent yet.