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Antimicrobial resistance in bacteria is an ancient phenomenon that emerged as a serious threat to humans and animals within only the last few decades. Nowadays, multi-drug resistant bacteria cause severe diseases in humans as well as animals worldwide, leaving few therapeutic options. Among these, Acinetobacter (A.) baumannii is of increasing importance, especially with regard to the epidemic clonal lineages IC I-III, which are particularly associated with carbapenem and multi-drug resistance. Moreover, these clonal lineages could already be detected among A. baumannii of animal origin, indicating a zoonotic potential of this pathogen. The current work contributes to two aspects of current A. baumannii research: i) the occurrence of A. baumannii in animal clinical specimens, especially concerning the occurrence of antimicrobial resistance and ii) the identification of factors, e.g. specific antimicrobial compounds, which contribute to the enrichment of the resistome and clinical success of A. baumannii.
A total of 642 clinical human and animal Acb-complex isolates were collected for a one-year time-period starting in February 2013. Identification to species level was performed using restriction fragment length polymorphism (RFLP) of the 16S-23S IGS, as introduced in the present study and was verified by means of partial rpoB and 16S-23S IGS sequencing. A. baumannii was the predominant species among animal Acb-complex isolates, accounting for a proportion of 44.41% and originating from various host species, with a considerably high proportion of 50.92% of isolates being multi-drug resistant (compared to 15.52% of human A. baumannii isolates). This clearly points towards a role of animals in the reinforcement and dissemination of antimicrobial resistances in A. baumannii. Subsequently, 27 clinical human and animal A. baumannii isolates were chosen for whole genome sequencing in order to gain insight in their genomic diversity and relatedness. Additionally, ten published complete genome sequences of human A. baumannii isolates were included in the analysis. Based on SNP analysis of the maximum common genome, a maximum likelihood tree and a distance matrix were generated, revealing a clear separation into a closely related cluster of human multi-drug resistant ST2 isolates and a heterogeneous group of human and animal antimicrobial susceptible non-ST2 isolates. In accordance with previous studies we therefore hypothesize that an ancestral ST2 isolate split from the heterogeneous group in the recent past followed by subsequent adaption to the hospital environment.
Moreover, we hypothesized that DNA damaging antimicrobials like fluoroquinolones may play a crucial role in adaption of A. baumannii to antimicrobial selective pressure and new ecological niches. Thus, spontaneous enrofloxacin (ENR) resistant mutant isolates were generated using sublethal ENR concentrations. Comparative genomic analysis of the whole genome sequences of the mutant and their respective wild-type isolates revealed novel mutations in the DNA gyrase encoding genes causing ENR resistance. Furthermore, an association of ENR selective pressure and mutations in the AdeFGH and AdeIJK efflux pump regulator genes adeL and adeN could be demonstrated.
Although the present work provides evidence that fluoroquinolone selective pressure can cause a multi-drug resistant phenotype in A. baumannii, no direct association of the resistance phenotype and genomic mutations could be proven. Future research should investigate the role of altered regulatory processes under antimicrobial stress conditions, e.g. due to ROS and sRNAs, in order to understand the mechanisms underlying the rapid evolution and clinical success of specific antimicrobial resistant A. baumannii lineages. Moreover, comprehensive epidemiological studies are urgently needed to assess the potential role of animals as reservoir for antimicrobial resistant A. baumannii and infection source for humans.