Investigation of Antibiotic Resistance and Biofilm Formation in Acinetobacter baumannii

Abstract

Acinetobacter baumannii is a multi-drug resistant Gram-negative bacterium of global concern. It commonly causes nosocomial infections such as ventilator-assisted pneumonia, bacteraemia, urinary tract infections, etc. The genome of AB5075 consists of three plasmids (83 kbp, 8.7 kbp and 2 kbp) and a ~4 Mbp chromosome. It was recognized as the number one priority pathogen by the World Health Organization (WHO) in 2017 due to its multi-drug resistant status. Efflux pumps play an important role in antibiotic resistance and can convey drug resistance to multiple antibiotics or selective resistance to a single antibiotic. Two efflux pump genes craA and cxpE were investigated in A. baumannii and were shown to play a significant role in chloramphenicol resistance. However, the function, importance and regulation of these efflux pumps are poorly understood and there is conflicting evidence on which genes in A. baumannii provide the largest contribution to chloramphenicol resistance in A. baumannii. In this study, we have determined that craA plays a role in the rapid adaptation to chloramphenicol exposure and that cxpE is required for the maintenance of growth in stationary phase in the presence of chloramphenicol. Transient chloramphenicol resistance was first identified in strain ATCC17978. In this study, the transient resistance phenotype was shown to be conserved in strain AB5075 with craA playing a key role in this transient resistance phenomenon. While CraA is proven to be present in the membrane throughout growth in minimal media, suggesting that CraA lacks the energy to rapidly and efficiently pump CHL out of the cell rendering a portion of the population sensitive to sub-inhibitory levels of chloramphenicol. The cxpE gene also appears to be involved in motility suggesting that cxpE has a broader role for A. baumannii physiology than just drug efflux. This study provides further insight into which genes are involved in chloramphenicol resistance in A. baumannii as well as a potential role for cxpE in the regulation of motility in A. baumannii.

The largest of the three plasmids "p1AB5075" harbors several antibiotic resistance genes (ARGs) e.g., aadB and strA and numerous unidentified genes. Unfortunately, this plasmid has only been studied in terms of (AR) antimicrobial resistance specifically, investigating (RI-2) resistance island 2 to date. In this research we have examined 8 p1AB5075 individually; aph(3')-VI, aaC(6')-Ib3, aadB, aadA2, strA, strB, and blaGES-11 for their contribution to overall A. baumannii AB5075 resistance. Also, we demonstrate that p1AB5075 contributes to overall virulence by significantly influencing biofilm formation, adhesion and motility. Upon the loss of p1AB5075 there is a reduction of motility, adhesion capability to polystyrene plastic and an increase in biofilm formation. We hypothesized that the second copy of H-NS (hns) located on p1AB5075 is responsible for the observed change in biofilm formation. Upon reintroduction of hns into the Δp1AB5075 background, biofilm formation levels are restored to wildtype levels. The addition of hns to Δp1AB5075 and wildtype strains reveals a significant growth defect in L-broth and minimal media. RNA-seq also revealed evidence of severe dysregulation of cellular processes that may explain this reduction in bacterial fitness. Overall, the p1AB5075-borne copy of H-NS influences biofilm production in A. baumannii. This is important as dissemination of this plasmid into the environment may not alter just the AR profile but also, virulence factors already present in the recipient strain due to the presence of the global transcriptional regulator HNS.