Detection Of Carbapenem Gene And Multilocus Sequence Typing Of Multi-drug Resistance Acinetobacter Baumannii

Objective: Acinetobacter baumannii is a group of gram-negative non-fermenting bacteria widely distributed in hospital environments,waters,soil and the surface of human skin,which makes an important category of opportunistic pathogens for nosocomial infections.Individuals infected by Acinetobacter baumannii are prone to a variety of nosocomial infections including septicemia,endocarditis,secondary meningitis,aquired pneumonia,infection of wounds,and infection of the urinary tract.With extensive application of immunosuppressants and broad spectrum antibiotics such as carbapenems,cephalosporins,β-lactams,aminoglycosides and quinolonos,numerous strains resistant to three or more categories of the aforementioned antibiotics have sprung up in recent years.An increasingly higher detection rate of multidrug-resistant Acinetobacter baumannii marks its significant pandemicity in nosocomial environments,posing immense challenges for practitioners in prevention and treatment of infections.This study aims for a test on susceptibility of Acinetobacter baumannii to common antibiotics,measurement of drug-resistance genes,and an analysis on phylogenetic relationships between different strains,which show peculiar materiality in researches on strain homology.The ensuing outcomes are expected to provide a theoretical basis for sensible choices of antibiotics as well as prevention and control of nosocomial outbreaks and infections caused by Acinetobacter baumannii;and lay the experimental foundation for molecular epidemiology research on multidrug-resistant Acinetobacter baumannii.Methods: Eighty-seven laboratory strains of Acinetobacter baumannii derived from 34 hospitals in Anhui Province during clinical work in 2015 were put into experiment.With employment of the two-fold agar dilution method,the strains were first put into a series of tests to assess their respective susceptibility to 11 common antibiotics.The PCR method was then employed to detect the primary drug-resistance genes(such as OXA-23,OXA-51,OXA-24,OXA-58,etc)that render Acinetobacter baumannii resistant to carbapenems.An analysis on the homology between the 87 strains of clinically derived Acinetobacter baumannii was achieved with the usage of multilocus sequence typing,and thus the allelic profiles and their corresponding sequence types of each strain were confirmed.On the basis of the aquired allelic profiles and sequence types,the study was successful in the construction of minimum spanning tree graphs and cluster dendrograms using Bionumerics software,and pertainance of the respective strains to different clone complexes was analysed and determined with the aid of e BURST.Results: The drug resistance rates of the 87 strains of multidrug-resistant Acinetobacter baumannii to meropenem and imipenem were 66.7% and 74.7%,respectively.Sixty-eight strains were resistant to gentamycin,62 strains resistant to piperacillin/tazobactam,47 strains resistant to levofloxacin,60 strains resistant to ceftriaxone;and all 87 strains showed substantial drug resistance to other common antibiotics.All of the 87 Acinetobacter baumannii strains were found to carry the oxa-51 gene,82 strains(which take up 94.3% of the 87 strains)carried oxa-23,and no multidrug-resistant Acinetobacter baumannii carrying oxa-58 and/or oxa-24 genes were found.The 87 strains researched in the MLST(multilocus sequence typing)experiment were subdivided into 42 ST types,6 of which were already involved in the database,and 36(temporarily dubbed STnew01-STnew36)of which were newly discovered subtypes.Thirty-seven strains of the tested Acinetobacter baumannii are preponderant subtypes and share a high-degree homology in sequence typing with epidemic Acinetobacter baumannii clones observed in a number of nations around the world,accounting for 42.5% in all epidemic clones of the 87 strains derived from the specimen locality in 2015,whose allelic profile was ST2(2-2-2-2-2-2-2).The other 41 ST types each involve 1-4 strains,and 35 of the 41 ST types involve a single strain,namely the35 strains each correspond with a unique ST type.After all the allelic profiles on the website of Pasteur Institute(http://www.pasteur.fr/mlst/)were retrieved and analysed with e BURST,the experiment succeeded in confirming that the ST2 Acinetobacter baumannii undergoing the experiment belong in the clone complex CC1.The ST types classified as STnew05,STnew01,STnew29,STnew06,STnew04,STnew28,STnew21,ST724 and ST570 showed only minute differences from ST2 Acinetobacter baumannii in a single allelic gene,and thus they collectively belong in clone complex CC1.An ST105 strain observed in this study belongs in clone complex CC4,ST63 belongs in CC6,and ST235 is an independent ST type.The allelic profile of 36 new found ST type o f A c i n e t o b a c t e r b a u m a n n i i w a s S T n e w 0 1(2-2-2-2-2-2-4 9)、STnew02(13-4-40-1-42-1-1)、STnew03(1-3-2-1-3-1-46)、STnew04(2-2-2-2-2-2-44)、STnew05(2-2-2-2-2-2-20)、STnew06(2-2-2-2-2-2-2-97)STnew07(13-4-40-1-42-1-98)、STnew08(13-4-40-1-42-1-2)、STnew09(3-2-5-1-3-1-2)、STnew10(2-2-2-2-3-1-2)、STnew11(13-4-2-3-6-1-2)、STnew12(8-5-5-1-7-1-2)、STnew13(3-4-4-2-9-1-2)、STnew14(3-1-2-1-18-1-2)、STnew15(3-1-7-1-7-2-2)、STnew16(17-20-23-10-20-13-2)、STnew17(20-26-26-14-26-16-2)、STnew18(23-20-28-10-25-18-2)、STnew19(45-20-44-67-25-29-2)、STnew20(27-2-110-57-121-56-2)、STnew21(2-2-2-2-2-2-3)、STnew22(1-3-40-2-7-1-4)、STnew23(3-1-2-2-3-2-4)、STnew24(3-5-5-1-7-1-2 6)、 STnew25(1-4-2-2-7-1-29)、STnew26(22-2 6-90-14-23-16-48)、 STnew27(3-3-3-4-7-4-4 9)、STnew28(2-2-2-2-2-2-51)、STnew29(2-2-2-2-2-2-63)、STnew30(97-2-13-2-3-2-2)、STnew31(97-2-13-2-3-2-2)、STnew32(3-2-2-2-2-2-5)、STnew33(1-4-2-2-7-1-2)、STnew34(22-26-90-14-23-16-2)、STnew35(3-2-2-2-3-4-2)、STnew36(3-3-2-5-7-2-44).Conclusion: All multidrug-resistant Acinetobacter baumannii showed substantially high drug resistance to 11 common antibiotics in clinical antibiotic application.It is affirmed that 100% of the 87 strains of Acinetobacter baumannii carry the OXA-51 gene,94.3% carry OXA-23,while no multidrug-resistant Acinetobacter baumannii carrying OXA-51 and/or OXA-24 were detected.The ST2 strains are the primary epidemic multidrug-resistant Acinetobacter baumannii clones in Anhui Province,and finally,a high degree homology was observed between epidemic ST2 strains in Anhui Province and the primary epidemic clones around the world,who collectively belong in clone complex CC1.