Joint Transcriptional Control of Virulence and Resistance to Antibiotic and Environmental Stress in Acinetobacter baumannii

Abstract

The increasing emergence of antibiotic-resistant bacterial pathogens represents a serious risk to human health and the entire health care system. Many currently circulating strains of Acinetobacter baumannii exhibit resistance to multiple antibiotics. A key limitation in combating A. baumannii is that our understanding of the molecular mechanisms underlying the pathogenesis of A. baumannii is lacking. To identify potential virulence determinants of a contemporary multidrug-resistant isolate of A. baumannii, we used transposon insertion sequencing (TnSeq) of strain AB5075. A collection of 250,000 A. baumannii transposon mutants was analyzed for growth within Galleria mellonella larvae, an insect-based infection model. The screen identified 300 genes that were specifically required for survival and/or growth of A. baumannii inside G. mellonella larvae. These genes encompass both known, established virulence factors and several novel genes. Among these were more than 30 transcription factors required for growth in G. mellonella. A subset of the transcription factors was also found to be required for resistance to antibiotics and environmental stress. This work thus establishes a novel connection between virulence and resistance to both antibiotics and environmental stress in A. baumannii.

Importance: Acinetobacter baumannii is rapidly emerging as a significant human pathogen, largely because of disinfectant and antibiotic resistance, causing lethal infection in fragile hosts. Despite the increasing prevalence of infections with multidrug-resistant A. baumannii strains, little is known regarding not only the molecular mechanisms that allow A. baumannii to resist environmental stresses (i.e., antibiotics and disinfectants) but also how these pathogens survive within an infected host to cause disease. We employed a large-scale genetic screen to identify genes required for A. baumannii to survive and grow in an insect disease model. While we identified many known virulence factors harbored by A. baumannii, we also discovered many novel genes that likely play key roles in A. baumannii survival of exposure to antibiotics and other stress-inducing chemicals. These results suggest that selection for increased resistance to antibiotics and environmental stress may inadvertently select for increased virulence in A. baumannii.

FIG 1

G. mellonella differentiates pathogenic and nonpathogenic Acinetobacter strains. (A) G. mellonella larvae were inoculated with 10⁶ CFU of the strains indicated. Larvae were homogenized and bacteria were quantified immediately following infection (t = 0) and after 4 h at 37°C (t = 4). Data from a representative experiment are presented as the ratio of the number of CFU recovered at t = 4 to the number of CFU recovered at t = 0 (error bars, 1 standard deviation). (B) G. mellonella larvae were inoculated with 10⁶ CFU of the strains indicated. Survival was monitored daily for 6 days. ****, P < 0.0001; ***, P < 0.001. ADP1, A. baylyi ADP1; 17978, A. baumannii ATCC 17978; AB5075, A. baumannii AB5075.

FIG 2

TnSeq experiment. (A) TnSeq experiment data. The outermost ring depicts the AB5075 chromosome and plasmids. The three middle rings depict the numbers of hits per gene in the pregrowth, LB growth, and Galleria growth samples (green, blue, and red, respectively). The innermost ring is a heat map of the RR of the Gm to the LB samples (Gm-essential genes are red). Essential genes are white, and genes with a general growth defect are black. The image was created with Circos (61). (B) Pie chart depicting Gm-essential hits grouped by COG categories. AA, amino acid; T/M, transport and metabolism; Coenz, coenzyme; metab, metabolism; Carb, carbohydrate; div, division; Chrm Part., chromosome partitioning; env., envelope; mot & sec, motility and secretion; Intracell. trfck & sec, intracellular trafficking and secretion.

FIG 3

Growth of selected mutants in G. mellonella larvae. The mutant strains indicated were inoculated into G. mellonella larvae. Data are presented as described in the legend to Fig. 1. Panels: A, stress response genes; B, transcriptional regulators.

FIG 4

Osmotic stress genes are required for virulence in G. mellonella. (A) Growth of osmotic stress mutants in freshly collected hemolymph monitored over time. (B) G. mellonella larvae (n = 16) were infected with 10⁵ CFU of wild-type AB5075 or isogenic mutants. Larval survival was monitored daily for 6 days. *, P < 0.05.

FIG 5

Stress response genes are required for G. mellonella killing and growth in Galleria. (A) G. mellonella larvae were infected with the strains indicated as described in the legend to Fig. 1. (B) Growth of wild-type AB5075 (gray bar) or the strains indicated harboring a hygromycin resistance gene (white bars) or a wild-type copy of the deleted gene at the Tn7 locus (gray bars) following inoculation of G. mellonella larvae as described in the legend to Fig. 1. ****, P < 0.0001.

FIG 6

Gig genes are required for virulence in G. mellonella. (A) G. mellonella larvae were infected with the strains indicated and monitored for survival as described in Fig. 1. (B) Growth of AB5075 with the empty vector (left gray bar) or the gigB mutant harboring the empty vector (white bar) or a complementing clone of gigB (right gray bar). (C) Growth of wild-type AB5075 (left gray bar) or the strains indicated harboring a hygromycin resistance gene (white bars) or a wild-type copy of the deleted gene at the Tn7 locus (center and right gray bars). In panels B and C, growth is depicted as described in the legend to Fig. 1. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01.

FIG 7

Genes required for growth in Galleria are also required for growth in subinhibitory concentrations of antibacterials. The strains indicated were grown for 24 h at 37°C in LB with or without 625 µg/ml kanamycin (Kan). Growth was measured by determining the OD₆₀₀ every 10 min in a Tecan 96-well plate reader.