Study On The Prevalance And Determinants And Molecular Feature Of Pseudomonas Aeruginosa Isolates With Drug-resistant Status From Nosocomial Infection Patients In Five Hospital Of Guangzhou

Objective to analyze the prevalance and risk factors of1-2class drug-resistantstrains、multidrug resistant strains and pandrug resistant strains of Pseudomonasaeruginosa(PA). To explore the beta-lactamase enzyme、genes of distribution andgeotyping conditions.To analyze the possible resistance mechanisms of PA.Methods The Pseudomonas aeruginosa isolates were Collected from fivehospitals in Guangzhou from July2008to December2012. The strains have beenidentified. The clinical data were collected by the retrospective review of cases method.PA were sensitive assays for antibiotics.Classified them into sensitive strains,1-2class drug resistant strains, multi-drug resistant strains and pan-drug resistant strainswere selected. To Analyze the infection status of PA and single variable analysis wasused by Chi-square test and Multivariable analysis was used by multinomial logisticregression to analyze the infected risk factors of1-2class drug resistant strains,MDRPA and PDRPA.Screening the beta-lactamase (ESBLEs, MBL and AmpC) produced by PA withthe double disk compound test and double disk synergy test and implement PCRdetection to the encoding gene of beta-lactamase （NDM-1, TEM, PER, OXA-10,IMP and VIM）、encoding gene of aminoglycoside modifying enzyme（ant (3″)- Ⅰand aac (6′)-Ⅱ）、outer membrane porin Oprd2、Integron and house-keeping genes ofgyrA and parC. Then, PCR-RFLP method were applied to detect the Integron and themutation of QRDR segment of house-keeping genes of gyrA and parC. At last, thedifferences of resistance mechanism among isolates from five hospitals was made bydescriptive study method and Chi-square test was used to analyze the relationshipbetween drug-resistant strains and resistance mechanism.Molecular typing of PA strains was made by PFGE,Bionumerie6.0software wasused to make a cluster analysis of genetic relationship of PA strains.To identify thesporadic or outvreak trends of strains.Results During2008to2012，348cases of PA infection have been collected with149casesof MDRPA whose multiple resistant rate is42.8%(149/348) and39cases of PDRPA whose pandrug resistant rate is11.2%(39/348). Among the patients infected with MDRPA, male patientsoccupy63.3%with the median age of73. The male patients infected with PDRPA occupy59.0%(23/39) with the median age of74. PDRPA patients are mainly distributed in ICU (12/39),Respiratory Medicine Dept (11/39) and Neurology Dept (8/39) and so is MDRPA. The source ofspecimens of MDRPA and PDRPA mainly come from phlegm which have reached81.1%(146/180) and82.1%(32/39) respectively. The Drug resistant rate of13antibacterial drugs exceptLEV, ATM are showing an upward tendency with the change of time.The PA has been Divided into four groups according to the resistance spectrum:Sensitive Group,1-2class Resistant Group, Multidrug Resistant Group and Pan DrugResistant Group. Compared each of the factors in the four groups and the factors withstatistical differences are: surgical operation, being sent into ICU before the detection,using carbapenems antibiotics, using the second generation cephalosporins antibioticdrugs, the total number of days using antibacterial drugs, hospital days beforeinfection, using antibacterial drugs more than3kinds, mixed infection, aspiration ofsputum, mechanical ventilation, catheter intubation and nasal feeding stomach tubeintubation. From the factor analysis, we come to a conclusion that the risk factor of1-2class Resistant Group is the aspiration of sputum （AdjustedOR=2.79,95%CI=1.09-7.12） compared with Sensitive Group as the control group. The risk factors of Multidrug Resistant Group are using carbapenems antibiotics（Adjusted OR=2.29,95%CI=1.02-5.15） and being sent into ICU before thedetection（Adjusted OR=2.90，95%CI=1.22-6.91）. And the risk factors of Pan DrugResistant Group are using carbapenems antibiotics （Adjusted OR=5.94，95%CI=1.97-17.85） and mechanical ventilation （Adjusted OR=11.78，95%CI=3.14-44.20）.Detection results of Beta-Lactamase and gene:In the384cases beta-lactamaseproduced by PA, the generation of MBL is12.9%(45/384), AmpC is12.9%(45/384)and ESBLs is12.1%(42/384). The each condition of beta-lactamase generated by thefive hospitals is different from one other. The differences of each enzyme productionrate and the in four groups have some statistical significance. The encoding geneNDM-1of beta-lactamase has not been detected yet. The relevance ratio of encodinggene of ESBLs is43.4%(151/348), the ratio of PER is22.7%(79/348) and the ratioof OXA-10is23.2%(81/348). The encoding gene IMP of MBL is15.2%(53/348)and the encoding gene VIM is4.6%(16/348). The each condition of beta-lactamasegenerated by the five hospitals is different from one other. Analyzed by chi-squaretest, the differences among pandrug resistant groud and sensitive groupof TEM,OXA-10and VIM have some statistical significance.The Permeability of Outer Membrane Decreases: The missing rate of OprD2genein the348cases of PA reaches19.5%(68/348). The difference of missing rate ofOprD2gene in pandrug resistant Group and Sensitive Group has statisticalsignificance.The Encoding Gene of Aminoglycoside Modifying Enzyme: PCR amplifies348PA bacterial strains with aac(6＇)-Ⅱ reaching24.7%(86/348) and ant(3”)-Ⅰreaching32.2%(112/348). The relevance ratio of aac(6＇)-Ⅱ、ant(3”)-Ⅰ in pandrug resistanGroup、multidrug resistant Group and inSensitive Group,the differences havestatistical significance.Detection of Integron:there are172strains ofⅠclass Integron in the348strainsof PA, which occupy49.4%(172/348) but there are neither Ⅱ or Ⅲ class Integron detected. There are80strains of Ⅰclass Integron detected in the149strains ofMDRPA, which occupy53.7%(80/149). And33strains ofⅠ class Integron aredetected in39strains of pana-drug resistance with the occupation of84.6%(33/39).The highest relevance ratio of PA Integron in the five hospitals is68.7%(79/115) andthe lowest is28.6%(16/56). From the comparison between positive and negativeresults of I class Integron in PA drug sensitivity, we can observe that antibacterialdrugs like GEN, TOB, AK, PIP, TIC, IMP, MEN, LEV, CIP, ATM, CAZ and FEP havea higher drug resistance rate in the positive group of Integron than that in the negativegroup. The differences have statistical significance (P<0.05). The relevance ratio ofIntegron in MDRPA group、PDRPA Group is higher than that in Sensitive Group afterChi-square test and have statistical significance.The Mutation of QRDR: In the162strains of PA quinolone resistance, gyrA genemutation occupies40.7%(66/162) and parC mutation occupies17.3%(28/162). Theresult of sequencing and comparing PCR production shows that the QRDR segmentmutation of gyrA gene mainly happens on ACC�'ATC of the83rdamino codon. Theencoding amino acid is The�'Ⅱe. At the same time, there is a static mutation ofdrug-resistant strain in the132ndamino codon (CAC�'CAT). This mutation does notcause the change of amino acid. However, the parC sequencing result shows thatQRDR segment mutation is mainly located on the TCC�'GCC mutation happened onthe79thamino codon. The mutation on this site causes the chance of amino acidSer�'Ala. After Chi-square test analysis, QRDR segment mutation of gyrA and parCin PDRPA Group is obviously higher than that in Sensitive Group with statisticalsignificance.PFGE Gene Typing：To emerge eight couples of bacteria with the homology of100%After PFGE Gene Typing for the348strains. the patients with the same typebacterial strains with PFGE of type A, C, D, E, and F not only have the overlappinghospitalization time, but also have the same department of the same hospital. With thesimilar resistance spectrum, we consider that it is the mutual infection betweenpatients with the same department. However, patients with the bacterial strains ofsame type of B, G and H all come from the same hospital but different departments. There may be possibility of cross infection within the department but the route ofinfection remains to be investigated.Gene typing rate of PDRA is between44.3%and97.1%and there are five groups of homologous strains with more than80%typingrate. The bacterial strains of each group come from the same hospital involving11strains of PDRPA and the rest28strains are distributed.Conclusion The infection of PA drug resistance is common in hospitals ofGuangzhou. The resistant rate of13antibacterial drugs except LEV, ATM are showingan upward tendency with the change of time.the risk factor of1-2class Resistant Group is the aspiration of sputum.The riskfactors of Multidrug Resistant Group are using carbapenems antibiotics and beingsent into ICU before the detection. And the risk factors of Pan Drug Resistant Groupare using carbapenems antibioticsand mechanical ventilation.Beta-lactamase and its gene (TEM, OXA-10, VIM), the miss of aminoglycosidemodifying enzyme encoding gene aac(6＇)-Ⅱand OprD2gene, and Integron, QRDRmutation of gyrA and ParC gene may be the major resistance mechanism produced bypan drug resistance strains.There is no pandemic of monoclone of PA.Most of the bacterial strains are insporadic epidemic trend while there is slight cloning spread in the same hospital orsame infected patch. The frequency of occurrences is relatively high in ICU andRespiratory Medicine Dept. There is also cross infection among differentdepartments.