Molecular Epidemiological Characterization Of Acinetobacter Baumannii Strains Isolated From CSF And Global Gene Expression Profile Of Acinetobacter Baumannii In CSF

Acinetobacter baumannii meningitis is a serious threat to neurosurgical patients with high mortality rate.Outbreaks of nosocomial meningitis caused by A.baumannii have been reported world-widely,mostly with multidrug-resistant(MDR)strains.But there is a dearth of literature for the molecular epidemiology of A.baumannii causing meningitis/ventriculitis.In the first part,a total of 54 unique clinical A.baumannii strains isolated from cerebral-spinal fluid together with 21 bloodstream isolates collected from 5 tertiary hospitals of East China between April 2013 and November 2016 were studied for susceptible profiles,the prevalence of resistant genes.Molecular epidemiology of all the isolates and comparison between isolates of different source were assessed using multi-locus sequence(MLST),pulsed-field gel electrophoresis(PFGE),and core genome MLST(cgMLST).94.4%(50/54)CSF isolates were blaOXA-23-carrying CRAB.Their average rate of resistance to tested antibiotics was extremely high(>90%),except for Tigcycline and Colistin.According to Oxford MLST schemes all of CSF isolates fell into 10 defined STs and 4 novel STs.ST 195 and ST208 were the leading 2 STs in either source of isolates.50 isolates were assigned to clonal complex 92(CC92),revealing a wild distribution of CC92 in the hospitals of China.All of the CC92 isolates shared high similarity in PFGE patterns.Subsequently,we applied cgMLST genotyping approach using the publicly available core genome MLST scheme for A.baumannii(2390 targets)to explore the potential nosocomia outbreaks.Based on epidemiological data linked in time and space,we demonstrated that using A.baumannii cgMLST scheme we were able to distinguish different outbreaks in the same hospitals from the 4 clusters of the minimum spanning tree.We also observed isolates from the different source could be involved in the same nosocomial outbreaks in some cases,illustrating that epidemic clones have the potential to cause a range of human disease with no tissue tropsim as previously reported.According to cgMLST,we still found no correlation between phylogeny and body sites of isolation.Using Scoary,no significant genes were identified that were different between the CSF versus bloodstream groups of isolates.These results emphasize that the genetic potential of this pathogen is vast enough to infect multiple human body sites.In the second part we performed an ex vivo transcriptomic analysis of A.baumannii in pooled human CSF.We observed that A.baumannii alters the expression of about 40%of ORFs of the genome.And analysis of the differentially expressed genes by COG category identified a high proportion with decreased expression that encoded proteins involved in translation and ribosomal function(J),intracellular trafficking,secretion,and vesicular transport(U).Together these data suggest a decreased growth rate for the bacteria in ex vivo compared with that in LB broth culture.In contrast,genes function unknown(S)were over represented in the up-regulated set,suggesting that they are more important for adaptation to the challenges of growing in ex vivo.In particular,we found that 2 recognized siderophore iron uptake clusters moderately up-regulated,reflecting the iron-restrictive environment in ex vivo.Genes encoding AdeFGH efflux pumps and quorum sensing components also showed increased expression.Many genes that have been reported as essential for virulence showed reduced or unchanged expression in human pooled CSF,especially genes involved in outer membrane proteins,lipopolysaccharide and capsule biosynthesis showed reduced expression.This first gene expression analysis of A.baumannii in CSF significantly increases our knowledge of how this bacterium responds to human CSF and causes meningitis.Our results also provide new information on gene function and may ultimately help in the development of diagnostic and therapeutic strategies to control this devastating disease.In conclusion,this study analyzed the characteristics of antimicrobial resistance patterns,distribution of drug resistance genes and molecular typing of the strains isolated from CSF.We also explored the transmission mechanism of the meningitis nosocomial outbreaks using cgMLST scheme and comparisons with bloodstream isolatates were made simutaniously.This is the first gene expression analysis of A.baumannii in pooled human CSF,which significantly increases our knowledge of how this bacterium responds to human CSF and causes meningitis.