The Effectiong And Mechanism Of Environmental PH On The Drug-resistant Acinetobacter Baumannii Susceptibility

Acinetobacter baumannii is an important pathogen which causes nosocomialinfections, mainly about hospital-acquired pneumonia. It has the highest morbidity in ICU.In recent years, with constant generalization of clinical application and prolongation oftime to market of antibacterial agents, the drug resistance of Acinetobacter baumannii tocommonly used antimicrobial is increasing. The emergence of carbapenem-resistantAcinetobacter baumannii (CRAB) is considered to be resistant to the field of "sentinelevents” in the twenty-first century, and the clinical detection rate increased year by year.However, the speed of bacterial resistance is growing much faster than development ofnew antibacterial drugs. CRAB often shows clinical features of pan-resistant, andanti-infective options available to antimicrobial drugs are very limited to clinical efficacyand satisfactory. Thus this greatly extends acromegalic length of stay, increasesacromegalic mortality. Then, under the circumstance of effective antimicrobial drugschosen is very limited, could non-antibiotic therapy as adjuvant therapy improve the efficacy? Studies have shown that the bronchoalveolar lavage fluid of healthy human isalkalescence, it turns acidic when lungs got infected. Secondly, the pH of the environmentchanges has an impact on the biological activity of some bacteria. So, what changes willbe led to CRAB biological activity when environmental pH changes? No study has beenreported that. This study completed the following tasks:1. Analysis of distribution and drug resistance of acinetobacter baumanniiin ICU in some comprehensive hospitalObjective:The drug distribution and drug resistance to antimicrobial samples of various types ofinfections of Acinetobacter baumannii in all epidemiology specialists in ICU in xijinghospital for the last three years were analyzed. This as a guide is to provide theoreticalbasis for increased efficacy for rational development of anti-infective hospital clinicalprogram.Methods:Retrospectively analyze pathogen data in various infections samples of ICU from Jan2010to Dec2012．Results:There were1054Acinetobacter baumannii in4633strains. The proportion ofacinetobacter baumannii is20.57%,21.26%, and25.94%during2010to2012. The maindistribution of acinetobacter baumannii is in sputum specimens, which accounts for morethan80%in all infectious specimens. The results of drug sensitivity test showedPolymyxin B had the lowest drug resistance of acinetobacter baumannii. Minocycline andCefoperazone/sulbactam had the lower drug resistance of Acinetobacter baumannii. Theresistance of Acinetobacter baumannii to other antimicrobial drugs was higher and theresistance rates were all more than60%. The resistance rate of acinetobacter baumannii toimipenem and meropenem showed an increasing trend.Conclusion:Acinetobacter baumannii mainly causes respiratory infections. To most antimicrobial, the drug resistance of acinetobacter baumannii is very serious. The priority should begiven to those antibacterials of which the pathogens are susceptible．Attention should bepaid to use antibacterials rationally．2. The impact of carbon vinyl cyanide mildew resistant of Acinetobacterbaumannii in vitro proliferation under changes of pH in theenvironment.Objective:To understand the environmental pH proliferation in vitro activity of CRAB, and toprovide effective treatment for clinical reference.Methods42CRAB, which comes from clinical sputum specimens, are respectively formulatedinto5×105cfu/mL acterial suspension with pH6.9,7.4and7.9MH broth. Those threebacteria were added to sterilized96-well plates, each holes plus200μl. Then placed in anincubator at35℃overnight and cultured20h. OD of each well was measured using aplate reader at630nm.Results:Three different pH values bacterial suspension cultured for20h, OD mean valuesmeasured with a microplate reader were0.555±0.200at pH6.9group,0.523±0.187atpH7.4group,0.512±0.174at pH7.9group. As a control group to pH6.9, pH7.4and7.9,OD mean values group was statistically significant decreasing, P <0.05.Conclusion:Environmental pH has effect on CRAB in vitro proliferation. Compared with a pH of6.9acidic environment, pH=7.4,7.9neutral and alkaline environment significantlyinhibited the CRAB in vitro proliferation.3. Detection of resistance genes of carbapenem-resistant Acinetobacterbaumanni and the influence of pH value to the external drain pumpand outer membrane protein gene. Objective:To study the effects of pH on cefoperazone/sulbactam (2:1, CSF) combinedminocycline (MNO) in vitro chemosensitivity CRAB.Methods42CRAB and the pH value of6.9,7.4and7.9of MH broth were like experiment2.By using of the checkerboard method design, the minimum inhibitory concentration (MIC)of MH broth with different concentrations of cefoperazone/sulbactam, minocycline, andtheir combination were detected by broth dilution method in three different pH values.And the fractional inhibitory concentration (FIC) index was calculated.Results:At pH increased from6.9to7.4and7.9, the two drugs alone, MICG value of CSFdecreased gradually (each group P <0.05), while the MICG MNO’s value is graduallyincreased (each group P <0.05). Combination of two drugs, FIC index≤1percentage were23.81%,11.90%,28.59%,1<FIC index≤2percentage is73.81%, respectively,88.10%,71.43%.Conclusion:In the alkaline environment of pH7.9, CSF alone or in combination with MNO, theactivity of antimicrobial was enhanced. Two drugs together on CRAB mainly hasnon-related effects.4. Discussion of molecular mechanisms of carbapenem-resistantAcinetobacter baumannii and effects of environmental pH on geneexpression levels of resistanceObjective:Understands carrying cases of resistance genes in CRAB carbapenemase OXA-23,OXA-51and efflux pump system adeB, outer membrane protein carO. Explore the effectof pH value on gene expression quantity of external drain pump adeB and outer membraneprotein carO. Methods:By ordinary PCR,42CRAB resistance gene carbapenemase OXA-23, OXA-51andthe efflux pump system adeB, outer membrane protein carO were detected, then analyzedand sequenced. Selected10CRAB with adeB and carO,4Ab sensitive clinical isolates, astandard strain (ATCC BAA-747). Cultured with6.9,7.4,7.9pH values in MH brothovernight for20-24h. Using real-time quantitative PCR measure adeB, gene Ct value carO,calculate the relative expression levels of the gene using2-CT method.Results:42CRAB carO genes detected40positive, rate was95.2%; adeB, OXA-23, OXA-51gene testing positive rate was100%. Standard strain BAA-747as a control,10CRABrelative expression with four clinical isolates adeB sensitive genes were respectively:37.34±41.89,1.55±1.77; relative gene expression carO volume were:0.38±0.34,4.80±3.87. Compared with the sensitive strains, CRAB adeB expression increased, carOdecreased expression levels, and were statistically significant, P <0.05. PH values betweendifferent groups, with a pH7.4as a control group, pH values of6.9and pH7.9group adeBgroup relative gene expression levels were:1.20±3.23,1.26±0.97; carO relative geneexpression was:1.07±1.18,1.26±1.02. No statistically significant between the twogroups (P>0.05).Conclusion:Producing OXA-23, OXA-51-type carbapenemase and over-expression of effluxpump adeB, the decreasing of outer membrane protein carO both exist in most CRAB inthe hospital. This coexistence of multiple resistance mechanisms is the important reason ofdrug resistance of CRAB in lower respiratory tract infection. When the environmental pHis at6.9,7.4and7.9, there is no significant effect on gene expression levels of CRABadeB and carO.These results indicate that the environmental pH has effects on both proliferationactivity and drug sensitivity of CRAB. Compared with pH6.9acidic environment, pH7.4and7.9neutral and alkaline environment has significantly inhibited CRAB proliferativeactivity. At pH7.9in an alkaline environment, antimicrobial activity are enhanced by CSF alone or in combination with MNO. At pH6.9,7.4and7.9, there was no obvious effect ongene level expression of CRAB adeB and carO.