Antimicrobial Resistance Analysis Of Acinetobacter Baumannii And In Vitro And In Vivo Studies On Vancomycin-colistin Combination Therapy

Acinetobacter baumanii have been increasingly recognized as major pathogens implicated in hospital-acquired and healthcare-associated infections worldwide.This organism has been implicated in infections at a variety of sites,including the respiratory tract,bloodstream,skin and soft tissues,and prosthetic devices,and has become a particular problem in the intensive care unit setting.The combination of intrinsic and increasing acquired resistance to various antimicrobial agents often limits the therapeutic options.Successful multidrug-resistant clones have disseminated worldwide and often remain susceptible only to agents such as tigecycline and polymyxins,which are usually considered treatments of “last resort”.Evidence to date suggests that colistin constitutes the backbone of therapy,but the unique pharmacokinetic properties of colistin have led many to suggest the use of combination antimicrobial therapy.On the other hand,with few new agents in the pipeline,clinicians are increasingly turning to combinations of antimicrobials in the hope that they may act synergistically together.However,the combination of agents and dosing regimens that delivers the best clinical efficacy while minimizing toxicity is yet to be defined.The development of new strategies to effectively control drug-resistant bacterial infections has become imperative aim of research for us.The main contents are divided into two parts as follows: Part Ⅰ Distribution and antimicr obial resistance of Acinetobacter baumannii infection in Anhui area Objective:To analyze of clinical distribution and drug resistance of A.baumannii in Anhui area;To prevent and control of nosocomial infection and therefore provide theoretical basis to selection of clinical drugs.Materials and MethodsA total of non-duplicate 981 strains of A.baumannii were collected from various clinical specimens from 35 hospitals in Anhui,China from 2010 to 2013 were analysed.Escherichia coli ATCC 25922 was used as a quality control strain for antimicrobial susceptibility testing.The MICs of 17 antimicrobial agents including colistin were determined by agar plate dilution method.The results were judged according to the criteria recommended by Clinical Laboratory Standard Institute(2012).The MIC of tigecycline was determined by broth microdilution method,while the results were judged according to the suggestion of U.S.Food and Drug Administration.The ward distribution,the drug resistances rates and the changing trend were analyzed by using the WHONET 5.6 software.ResultsAbout 73.3%(719/981)of A.baumanii were isolated from sputum samples,16.2%(159/981)from secretion and pus samples,and 6.0%(59/981)from blood.The most common ward was Intensive Care Unit(ICU)(46.7%),followed by the department of respiratory(12.3%)and neurosurgery(10.6%).More than 70% were resistant to more than three antimicrobials.The drug resistances rates of A.baumanii to the most commonly used antibiotics including piperacillin-tazobactam,amikacin,imipenem and meropenem were more than 40%.The drug resistance rates of cefoperazone-sulbactam were more than 20%,however,there was obviously increasing overall trend in drug resistance rates from 2010 to 2013.The drug resistance rate of minocycline was ~30%.Only tigecycline and colistin showed high sensitivity rates.ConclusionA.baumannii mainly cause lower respiratory tract infection,and distributed in the ICU.The results of drug susceptibility testing show that the bacterial resistance situation is still quite serious.It is urgent to enhance the surveillance of bacterial resistance to guide the appropriate use of antibiotics in clinical.Part Ⅱ In vitro and in vivo eff cacy of vancomycin-colistin combination therapies for Acinetobacter baumannii infection Objective:We assessed the activities of vancomycin-colistin combination both in vitro and using a Galleria mellonella caterpillar model of A.baumannii infection in an attempt to gain further insights into whether these therapies should be explored further for the treatment of multidrug-resistant(MDR)A.baumannii.Materials and MethodsA.baumannii ATCC 19606 was used as an antibiotic-susceptible type strain,while a clinical isolate GN 2231 was used as an MDR isolate.The MICs of 9 antimicrobial agents including colistin and vancomycin were determined by agar dilution method.We utilized G.mellonella larvae to determine the utility of this infection model to study antibacterial efficacy.G.mellonella killing with heat-killed or live isolates was tested.We also investigated the effect of postinoculation incubation temperature on the survival of infected larvae.The protection of administration of antibiotics to infected larvae was investigated.Finally,we determined the G.mellonella hemolymph burden of A.baumannii after administration of different antibiotics.Synergy when colistin was combined with vancomycin was assessed in standard checkerboard assays in microtiter plates with wells containing doubling dilutions of each agent in MH broth.Time–kill assays were conducted for each strain using colistin alone and in combination with vancomycin.The in vivo activity of vancomycin/colistin combination was assessed using a G.mellonella larvae model.Caterpillars were infected with a lethal dose of A.baumannii ATCC 19606 or GN2231.Antibiotics simulating human doses were administered within 2h of inoculation.Treatment was given only once.Uninoculated and mock-inoculated(sterile PBS)caterpillars were used as controls.The caterpillars were observed for survival every 24 h for 4 days.ResultsWith live bacterial inocula,G.mellonella killing was significantly dependent on the number of A.baumannii cells injected in a dose-dependent manner.Further,we observed that survival was reduced with increasing the postinoculation temperature.Treatment of a lethal A.baumannii infection with antibiotics that had in vitro activity significantly prolonged the survival of larvae compared with treatment with antibiotics to which the bacteria were resistant.In chequerboard assays,vancomycin was highly active against A.baumannii when combined with colistin [fractional inhibitory concentration(FIC)of <0.5].In the time-kill assays,the vancomycin/colistin combination displayed both rapid and sustained bactericidal activity at clinically relevant serum concentrations over the time course of the assay for each of the strains.Treatment of G.mellonella larvae infected with lethal doses of A.baumannii resulted in significantly enhanced survival rates when vancomycin was given with colistin compared with colistin treatment alone(P < 0.05).ConclusionThe G.mellonella infection model has the potential to be used to facilitate the in vivo study of hostepathogen interactions in A.baumannii and the efficacy of antibacterial agents.In vitro and in vivo findings reported here suggest that combination therapy consisting of colistin and vancomycin may be an effective therapeutic option for the treatment of MDR-A.baumannii infections,although additional studies are required prior to clinical trials.Part Ⅲ In vitro study on mutant prevention concentrations of colistin alone and in combinati on with vancomycin against Acinetobacter baumannii infection Objective:To test the mutant selection window(MSW)hypothesis in vitro with Escherichia coli and Pseudomonas Aeruginosa exposed to fosfomycin.Materials and MethodsThe effects of colistin concentration on colony-forming ability of resistant mutant subpopulations of ATCC 19606 were measured.Cells were applied to drug-containing agar plates and,at the same time,the cell density of the culture was determined retrospectively by applying serial dilutions to drug-free agar.In vitro bactericidal activities of colistin against high-inoculum of A.baumannii were assessed by modified time-kill assays.The drugs were added to the bacterial cultures at concentrations corresponding to 0.5×,4×,16×,64×,and 128× MIC.Bacterial cultures and antibiotics were incubated at 35°C.Surviving bacteria were counted after 0,4,8,12,24 and 48 hours of incubation at 35 oC by subculturing 100 μl serial dilution of every flask on MH agar plates.Mutant prevention concentrations of colistin or combined with vancomycin for 30 strains of A.baumannii were detected with agar dilution method.ResultsIn the in vitro time-kill studies,colistin exhibit rapid bactericidal activity against high-inoculum planktonic A.baumannii.At antibiotic concentrations of 4 ×,16× and 64 × MIC,there was a temporary inhibitory effect until 12 h and then quickly re-grew;moreover,the mutants of A.baumannii with increased MICs were readily selected at the endpoint of the time-kill studies.At both the lowest(0.5 × MIC)and the highest(128 × MIC)concentrations,no development of resistance against fosfomycin was observed.The MPC of colistin alone for 30 strains of A.baumannii were 64~>512μg/mL,MPC50 and MPC90 were 256 and >512μg/mL,respectively.MPC of the combination application with vancomycin for A.baumannii were16~>512μg/mL;MPC50 and MPC90 were 128 and >512μg/mL,respectively.ConclusionAgar plate determinations are fit the MSW for colistin against A.baumannii.Drug concentration fell in the MSW when colistin was used alone.The MPC values were decreased when colistin combined with vancomycin,however,resistance development still can not be prevented.