Human Pleural Fluid and Human Serum Albumin Modulate the Behavior of a Hypervirulent and Multidrug-Resistant (MDR) Acinetobacter baumannii Representative Strain

Abstract

Acinetobacter baumannii is a nosocomial pathogen capable of causing serious infections associated with high rates of morbidity and mortality. Due to its antimicrobial drug resistance profile, A. baumannii is categorized as an urgent priority pathogen by the Centers for Disease Control and Prevention in the United States and a priority group 1 critical microorganism by the World Health Organization. Understanding how A. baumannii adapts to different host environments may provide critical insights into strategically targeting this pathogen with novel antimicrobial and biological therapeutics. Exposure to human fluids was previously shown to alter the gene expression profile of a highly drug-susceptible A. baumannii strain A118 leading to persistence and survival of this pathogen. Herein, we explore the impact of human pleural fluid (HPF) and human serum albumin (HSA) on the gene expression profile of a highly multi-drug-resistant strain of A. baumannii AB5075. Differential expression was observed for ~30 genes, whose products are involved in quorum sensing, quorum quenching, iron acquisition, fatty acid metabolism, biofilm formation, secretion systems, and type IV pilus formation. Phenotypic and further transcriptomic analysis using quantitative RT-PCR confirmed RNA-seq data and demonstrated a distinctive role of HSA as the molecule involved in A. baumannii's response.

Keywords: Acinetobacter baumannii; huma serum albumin; human fluids; iron; quorum sensing.

Figure 1

Phenotypic and genetic analysis of quorum sensing coding genes. (A) Heatmap outlining the differential expression of genes associated with quorum sensing in presence of HPF or HSA. The majority of quorum sensing associated genes are up-regulated (green) in the presence of HPF and HSA. The asterisks represent the differentially expressed genes (DEGs) (adjusted p < 0.05 with log2fold change >1), one asterisks: p < 0.05; two asterisks: p < 0.01 and three asterisks: p < 0.001. (B) qRT-PCR of AB5075 strain genes associated with quorum sensing, aidA, kar, acdA, fadD, abaR and abaI expressed in LB or LB supplemented with HPF, dHPF, or dHPF + HSA. Fold changes were calculated using double ΔCt analysis. At least three independent samples were used. LB was used as the reference condition. (C) Agar plate assay for the detection of AHL using A. tumefaciens. The presence of AHL were determined by the development of the blue color. Quantification of 5,5’-dibromo-4,4’-dichloro-indigo were estimated as the percentage relative to C10-AHL standard, measured with ImageJ (NIH). The mean ± SD is informed. Statistical significance (p < 0.05) was determined by ANOVA followed by Tukey’s multiple-comparison test.

Figure 2

Phenotypic and genetic analysis of iron uptake genes. (A) Heatmap outlining the differential expression of genes (DEGs) associated with iron uptake in presence of HPF or HSA. The majority of iron uptake associated genes are down-regulated (red) in the presence of HPF and HSA. The asterisks represent the DEGs (adjusted p-value < 0.05 with log2fold change >1). (B) qRT-PCR of AB5075 strain genes associated with iron uptake, feoA, exbD, bauA, tonB, pfeA and bfd expressed in LB or LB supplemented with HPF, dHPF, or dHPF + HSA. Fold changes were calculated using double ΔCt analysis. At least three independent samples were used, and four technical replicates were performed from each sample. The LB condition was used as reference. Statistical significance (p < 0.05) was determined by ANOVA followed by Tukey’s multiple-comparison test, one asterisks: p < 0.05; two asterisks: p < 0.01 and three asterisks: p < 0.001.