| Objectives:1. In order to understand the drug-resistance of P. aeruginosa which were selected from hospital infections in Guangdong Province, graped differences among different regions and different time, then provide a better clinical selection of appropriate antibiotics.2. To explore and optimize pulsed-field gel electrophoresis method for P. aeruginosa operating, understand their genetic polymorphism and clarity genetic relationship between strains.3. Analysised the cluster outcomes of P. aeruginosa gene map together with epidemiological data. Distinguished dominant clonal strains as well as their distribution in different time, different regions and different human, then diagnosised whether they were outbreak strains.4. To understand the situations on P. aeruginosa resistance genes, the relationships among resistance spectrum, gene mapping and resistance genes carrying. Methods:1. Strains collection:during January 2008 to August 2010, selected samples including sputum, blood, Urine, faeces and other ones from nosocomial infection patients in four tertiary hospitals (Shantou Central Hospital, Shenzhen Children's Hospital, Guangzhou Railway Hospital and Guangzhou Chest Hospital), isolation and identification were carrying out by using VITEK232 automatic identification system (France Bio2Merieux), removed those repeated samples from the same patient.2. Susceptibility test:K-B disc diffusion method was used to detect the minimal inhibitory concentration (MIC) of 15 antimicrobial agents against isolates through referring antimicrobial agents susceptibility standards of CLSI 2007.3. Genotypic characterization:Built up a more appropriate operation for P. aeruginosa PFGE typing, with which we carried out operating on isolates; The un-weighted pair-group mean arithmetic (UPGMA) of Bionumerics V4.0software was used to cluster analysis.4. Resistant-genes detection:Used PCR amplification assay to detect the resistant genes of OprD2 as well as Metallo-β-lactamase.Results:1. Susceptibility testing results of P. aeruginosa from 2008-2010 in Guangdong area1.1 Susceptibility testing on P. aeruginosa islatesTotally we collected 376 P. aeruginosa from hospital infections, each strain showed varying degrees of drug-resistance among 15 common used antibiotics (Penicillins, cephalosporins, otherβ-lactams, aminoglycosides, quinolones, sulfonamides). No strain of pan-resistance or whole drug-sensitive were found in this experiment. The drug resistance situations were as follows:CZO (99.7%),SAM (99.5%),AMP (98.9%),SXT (98.7%),CTX (80.6%),GEN (50.8%),ATM (46.5%),TZP (45.1%),FEP (44.7%),LVX (39.9%),MEM (39.1%), CAZ (38.0%),AMK (37.5%),CIP (37.0%),IPM (34.6%).Almost all isolates were resistant to Penicillins,sulfonamides and the first generation cephalosporins. It showed lower resistant rate on aminoglycosides, quinolones, especially on carbapenem and the third generation cephalosporins.1.2 Region difference of drug resistance on P. aeruginosaSusceptibility testing showed there was a little difference among regions, almost all isolates got a higher drug resistance with time going on. In Shantou, the resistant rate of imipenem increased from 27.3% to 59.6% between 2008 and 2010;In Shenzhen, the rate of imipenem and ceftazidime were 4.2% and 4.2% in 2008 while they were 20% and 23.3% in 2010; As to Guangzhou, relatively, results appeared that it got a higher resistant rate between imipenem and ceftazidime in 2008, then decreased in 2009, but 2010 the number rose again. Shenzhen got a large growth rate of drug resistance between 2009 and 2010, most antibiotics were dubble times growing rate, especially AMK,GEN,IPM,MEM,CAZ,FEP,CIP as well as LVX.Statistics on drug-resistance showed that there were statistical difference among AMK,GEN,IPM and TZP in Shantou while AMK,GEN,IPN,CTX,FEP,CIP,LVX in Shenzhen and AMK,GEN,CAZ,CTX,TZP,SXT in Guangzhou.1.3 Time trends of antibiotics -resistance on P. aeruginosaShantou and Guangzhou got a common drug resistance, relatively, a higher drug sensitivity in Shenzhen.In Shenzhen there was no AMK,FEP,CIP,LVX resistant strain, but the number in Shantou was 33.3%,48.5%,45.5%,33.3% while 44.1%,73.5%,58.8%,64.7% in Guangzhou.Shantou and Guangzhou showed a slower or slightly declined on resistance change while Shenzhen increased rapidly with the years.In 2008, the resistant rate of imipenem were 27.3%,4.2% and 50.0%, ceftazidime were 57.6%,4.2% and 64.7% among Shantou,Shenzhen and Guangzhou, as time to 2010, the rate of imipenem increased to 59.6%,20.0% and 41.7% while 57.6%,4.2% and 64.7% among these three areas. Statistics on drug-resistance showed that there were statistical difference on antibiotics of AMK,GEN,IPM,MEM,CAZ,CTX,FEP,ATM,TZP,CIP,LVX in 2008 while AMK,GEN,IPM,MEM,CAZ,CTX,FEP,ATM,TZP,SXT,CIP,LVX in 2009 and AMK,GEN,IPM,MEM,FEP,ATM,AMP,CIP,LVX in 2010.1.4 The detection of multi-drug resistance P. aeruginosaThere were 84 strains of multi-drug resistance P. aeruginosa among total 376 ones, rate was 22.3% which was still increasing with the years. Statistics showed it got statistical difference among three regions on multi-drug resistance rate(χ2=39.29, P=0.000). Still in Shantou and Shenzhen, the rate were increasing, particularly in Shantou area, numbers revealed that in these three years, the multi-drug resistance rate were 18.2%,40.7% and 53.8%.In one area, statistics on multi-drug resistance rate showed Shantou (χ2=10.69, P=0.005) and Shenzhen (χ2=9.78, P=0.008) got a statistically significant change, as to make a statistical test in the same year, totally 2008 (χ2=6.30, P=0.043),2009 (χ2=24.39, P=0.000),2010 (χ2=21.15, P=0.000) had statistical difference.2. Genotyping of P. aeruginosa with Pulsed-fleld gel electrophoresis2.1 Appropriate operation for P. aeruginosa PFGE typing exploringWhen OD 5.2, cell disruption for 20h, Spel 20U digest 20h, initial switch time value of 2.16 sec, final switch time of 63.8sec at a gradient of 6 V/cm and an included angle of 120°, electrophoresed for19:30h in 0.5×TBE (Sigma) at 14℃. Results came out to be perfect(including band number,size,brightness and length).2.2 Genotyping results of P. aeruginosa and epidemiological investigationWith the same reagents,equipments and parameters, each strain showed 13-20 bands ranging from 30kb to 700kb after 20U Spel digestion.Clustered by Bionumerics V4.0, Similar values were among 34.2% and 100% with 357 types(consider to be a same type when similar value was 100%).Totally there were 16 pairs(35 strains) with a 100% similar value, among which Shantou got 4 pairs (8 strains), all occurred in wards of ICU and neurosurgery, specimen were sputum and blood from heart and brain injury patients, detection time span was no more than 2 months.Shenzhen got 7 pairs(17 strains), all detected from sputum, most were from ICU and internal medical department, detection time span was among 1 to 4 months, the longest was 8 months.Guangzhou got 3 pairs(6 strains), most strains were from respiratory patients of internal medical department, detection time span was between 1 and 2 months.Moreover, one was pair from Guangzhou Chest Hospital and Shenzhen Children's Hospital, detection time were 10.2010 and 6.2009, specimen were sputum and stool. Another was from Guangzhou Chest Hospital and Shantou Central Hospital, epidemiological investigation showed it was from the same patient but in different time and different places.2.3 Genotyping of 84 multiple antibiotic-resistant P. aeruginosaClustered by Bionumerics V4.0, similar values were among 62.5% and 100%, with 75 types and 6 pairs completely homologous. Cluster peaks appeared in different areas, especially in Shantou, with 7 strains owning 90.7% homologous or more. Another point,4 pairs with completely homologous came from Shantou, one from Shenzhen while the other was from two different places (Guangzhou and Shenzhen). Statistical test revealed that when similar value came to 90%,85% and 80%, there were statistical difference on PFGE patterns between groups of multiple antibiotic-resistant and total isolates.2.4 Genotyping of 34 resistance gene-positive P. aeruginosaSimilar values by Bionumerics V4.0 were from 64.6% to 100% and a totally 31 patterns among these 34 strains. Cluster peaks appeared in same areas, when similar values was 90%, PFGE patterns can be cutting up into 24 genotypes while to 80%, only 13 genotypes be clustered. Totally there were 2 pairs completely homologous from Shantou (2 strains) and Shenzhen (2 strains)3. Resistance mechanisms of multiple antibiotic-resistant P. aeruginosa3.1 Detection outcomes of OprD2Totally there were 30 strains with OprD2 negative, with a detection rate of 35.71% (30/84), among which Shantou was 13.35%(6/45), Shenzhen was 75% (3/4) and Guangzhou was 60%(21/35)3.2 Detection outcomes of metallo-β-lactamase.MBL was negative among Shantou strains; Guangzhou detected out 4 IMP and 2 VIM positive ones, with detection rates 11.4% and 5.7%; while Shenzhen only 2 VIM positive strains with a rate of 50%. No SPM positive strain was detected out in these three regions.Conclusions:1. Antibiotic-resistance experienced among P. aeruginosa strains was so common in Guangdong, almost all strains were resistant to CZO,SAM,AMP and SXT. As to aminoglycosides, quinolones, especially on carbapenem and ceftazidime, strains got a lower resistant rate. With time going on, almost all isolates got a higher drug resistance. Drug resistant appearance was very from different time and different places. From a regional viewpoint, Shantou and Shenzhen were more common, while with time going on, resistant growth seemed Shenzhen> Shantou> Guangzhou. Another point, multiple antibiotic-resistant rate was 22.3% and still increased, growth rate seemed Shantou> Shenzhen> Guangzhou.2. PFGE experimental technique has a strong awareness of the differences in molecular level. It is really a stable and accurate genotyping method which is suitable for homology analysis of nosocomial infections.3. In different regions of Guangdong province, dominant clone strains appeared among detected hospitals from 2008-2010. Small-scale spread of nosocomial infection took place in these three regions, especially in wards of ICU, still cross-infection can suffer different wards. In the other hand, completely homologous strains were found in different place which reminded us the importance of epidemiological investigation. PFGE patterns showed that multiple antibiotic-resistant P. aeruginosa, especially those carrying resistance gene strains, had a higher degree genetic homology, which reveal that it is more likely to carry out cross-infection.4. The most important mechanism of imipenem resistance P. aeruginosa is membrane porin OprD2 missing, follow up by IMP-1 and VIM-2 carrying, no other kinds of metallo-β-lactamase be detected out. Those two mechanisms can play a collaborative or independent role on imipenem resistant but still there exist other mechanisms.5. There are no inevitable associations among resistance patterns,PFGE patterns and resistance gene mapping. |
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