Study On The Relationship Between Biofilm Formation Ability And Drug Resistance Of Clinical Acinetobacter Baumannii Isolates

Objective:Acinetobacter baumannii is a Gram-negative bacterium that is frequent isolatedfrom clinic.It is highly drug-resistant and often has clonal transmission in the hospital.The formation of bacterial biofilm is one of the important virulence factors and resistance mechanisms.However,the relationship between biofilm formation ability and bacterial resistance is still inconclusive.The purpose of this study was to investigate the relationship between the biofilm formation ability and drug resistance of clinical Acinetobacter baumannii isolates and the related regulatory genes of biofilm formation.Furthermore,the homology of bloodstream infection strains was analyzed.We aimed to provide a theoretical basis for the research of drug resistance mechanism of Acinetobacter baumannii,the regulation of biofilm formation and the control of nosocomial infection.Methods:1.The isolated strains identified as Acinetobacter baumannii by VITEK 2-Compact from July 2016 to June 2018 in Affiliated Hospital of Jiangsu University were randomly collected.The multiplex PCR method was used to confirm that the strains collected were Acinetobacter baumannii.The antimicrobial susceptibility of the strains to 15 commonly used antibiotics was tested by VITEK 2-Compact and K-B method.2.A biofilm model of Acinetobacter baumannii was constructed in vitro usinga 96-well cell culture plate.Then the biofilm formation ability of all strains was quantitatively determined by crystal violet staining.Afterwards the differences of biofilm formation ability of strains from different samples and different antimicrobial susceptibility results were analyzed.3.Biofilm-related genes,such as bap,abaI,bfs,bfmS,bfmR,csuA,csuE,csuC,csuD,csuAB and ompA were detected by PCR,then the PCR products were verified by Sanger sequencing.Furtherly,real-time fluorescence quantitative reverse transcription PCR（RT-qPCR）was used to detect the mRNA expression level of genes csuA,csuE,bfs,ompA and bfmS in different groups with different biofilm formation ability.We analyzed the differences of biofilm formation ability of strains from different groups of biofilm-related genes and different antimicrobial susceptibility results.Therefore,the mechanism of biofilm formation could preliminarily be discussed.4.The homology of Acinetobacter baumannii strains isolated from bloodstreaminfection was analyzed by Pulsed-field gel electrophoresis（PFGE）.Results:1.A total of 107 strains of Acinetobacter baumannii were identified bymultiplex PCR.These strains were isolated from blood,urine and sputum specimen.Sputum is the largest proportion of the specimens.All these strains were distributed in various departments of the hospital.The drug resistance rate of the strains to CIP and FEP was high,both 67.29%,and the resistance rate to IPM was 63.55%.The resistance rate to TGC（14.02%）,SCF（24.30%）and MNO（25.23%）was relatively low.MDRAB accounted for 64.49%,while non-MDRAB accounted for 35.51%.2.Crystal violet staining results showed that the biofilm formation abilities of107 strains of Acinetobacter baumannii were all positive,the test results were among0.24±0.02 to 3.17±0.14,the median and interquartile range M(P₂₅,P₇₅)was 0.98（0.74,1.40）.There were no significant differences in biofilm formation ability among different specimens（P>0.05）.For the overall drug resistance of bacteria,the biofilm formation ability of non-MDRAB group was stronger than that of MDRAB group（P<0.05）,at the same time,for the specific antibacterial drug susceptibility results,the bacterial biofilm formation abilities of the 15 antibiotic non-resistant groups were stronger than that of the resistant groups（all P<0.05）.3.The detection rates of ompA,csuAB,csuA,csuC,csuD,csuE,bfmR,bfmS,bap,abaI and bfs were 89.72%,89.72%,85.98%,85.95%,86.92%,86.92%,98.13%,95.33%,75.70%,85.98%,90.65%,respectively.The Sanger sequencing splicing results of the PCR products were confirmed to be Acinetobacter baumannii biofilm-related gene by GenBank-BLASTn online alignment analysis.The detection rates of biofilm-related genes were different between the MDRAB group and the non-MDRAB group.More specifically,the detection rates of the genes bap,abaI,bfs and bfmS in the MDRAB were higher than those in the non-MDRAB group（all P<0.05）.On the contrary,the detection rates of the genes csuC,csuD,bfmR,csuA,csuE,csuAB,and ompA were no significant differences between the two groups（all P>0.05）.On the one hand,for biofilm-related genes bfmR,csuA,csuE,csuAB,csuC andcsuD,the biofilm formation ability of the genes detection group was stronger than that of the genes undetected group（all P<0.05）;on the other hand,for the biofilm-related genes bap and abaI,the biofilm formation ability of the genes undetected group was stronger than that of the genes detection group（all P<0.05）.However,for the biofilm-related genes bfs,bfmS and ompA,there were no significant differences of biofilm formation ability between these two groups（all P>0.05）.Furtherly,the results of RT-qPCR analysis showed that the expression levels of mRNA in group with relatively strong biofilm formation ability（Group A）were significantly higher than those in group with relatively weak biofilm formation ability（Group B）for the genes ompA,bfmS and csuE（all P<0.05）.In the meanwhile,there were no significant differences between Group A and Group B for the genes csuA and bfs（both P>0.05）.4.Totally,30 strains of Acinetobacter baumannii isolated from bloodstreaminfection could be divided into 16 clonotypes（A^P clonotypes）by PFGE.To be more specific,the main clonotypes were A^F.Clonotype A accounted for 16.67%,clonotypes B and C accounted for 13.33%,respectively,clonotype D accounted for10.00%,clonotypes E and F accounted for 6.67%,respectively,while the remaining 10clonotypes were sporadic,each group contained only one strain of bacteria.Clonotype A distributed in multiple departments in the hospital,and the proportion of MDRAB is80%.For clonotypes B,C,D and E,they existed only in ICU,whereas clonotype F distributed in ICU and general surgery department,the proportion of MDRAB is 100%.Conclusions:1.Clinical isolates of Acinetobacter baumannii are highly drug-resistant,onlymaintain high sensitivity to TGC,SCF and MNO.Consequently,antibiotics should be rationally selected for clinical anti-infective therapy.2.All strains used for this study have the ability to form biofilm in vitro,butthere is no significant difference of the biofilm formation ability with the different specimens.The biofilm formation ability of non-MDRAB is stronger than that of MDRAB.3.The formation process of bacterial biofilm is not controlled by only one singlegene,but involves the participation of multiple genes,among which the bacterial pili system,ompA and two-component regulation system play a vital role in the formation of bacterial biofilm.Genes bap and abaI not only participate in the formation of biofilm,but also may affect bacterial resistance by other mechanisms besides controlling biofilm formation.However,the specific molecular mechanism of biofilm formation remains to be further studied.4.MDRAB has a phenomenon of clonal transmission in the hospital,thus strictinfection control measures must be taken to prevent further spread and dissemination.