Acinetobacter baumannii Virulence Traits: A Comparative Study of a Novel Sequence Type with Other Italian Endemic International Clones

Abstract

Carbapenem-resistant Acinetobacter baumannii (CRAb) have emerged in recent decades as major causes of nosocomial infections. Resistance is mainly due to overexpression of intrinsic and/or acquired carbapenemases, especially oxacillinases (OXA). In Italy, although the sequence type (ST) 2 and the ST78 are the most frequently detected, we recently reported ST632, a single locus variant of ST2. Therefore, this study was aimed at unraveling common bacterial surface virulence factors involved in pathogenesis and antibiotic resistance in representative CRAb of these ST genotypes. Outer membrane protein (OMP) composition together with motility, biofilm formation, in vitro adherence to, invasion of, and survival within pneumocytes were analyzed. Differently from the carbapenem-susceptible reference strain ATCC 17978, either overexpressed OXA-51 or both OXA-23 and OXA-51 co-purified with OMPs in CRAb. This tight association ensures their maximal concentration on the inner surface of the outer membrane to provide the best protection against carbapenems. These findings led us to propose for the first time a common behavior of OXA enzymes in CRAb. Despite the presence of both OmpA and phosphorylcholine-porinD and the ability of all the strains to adhere to cells, invasion, and survival within pneumocytes was shown only by ST2 and ST78 isolates, sharing the highest number of identified OMPs. Conversely, notwithstanding genetic and OMPs similarities with ST2, ST632 was unable to invade and survive within epithelial cells. Overall, our study shows that different STs share a specific OMP composition, also shaped by overexpressed OXA, that is needed for invasiveness and survival of CRAb.

Keywords: Acinetobacter baumannii; biofilm; host-pathogen interactions; motility; oxacillinases.

FIGURE 1

Protein profiles and analysis of the outer membrane (OM) fractions from carbapenem-resistant Acinetobacter baumannii (CRAb). Proteins present in WCEs and in the outer membrane protein (OMP) fractions were stained with Coomassie brilliant blue (A) or transferred to a PVDF membrane and probed using anti-OmpA and anti-ChoP TEPC 15 antibodies (B). Size markers (kDa), the four gel slices excised and subjected to in-gel digestion (complete data are presented in Supplementary Table S2) and the positions and names of OmpA and ChoP are indicated.

FIGURE 2

Venn diagram showing the number of proteins shared among A. baumannii strains. Each A. baumannii isolate is depicted by a different oval color, as indicated. Proteins included were those identified by MS (cut-off: protein confidence = 95%) in the 20–36 kDa range from the Coomassie-stained gel shown in Figure 1, and having at least 95% = 4 minimum peptides. Numbers in the non-overlapping regions show the number of proteins unique to that strain. Supplementary Table S2 contains the information shown in this diagram at the protein level.

FIGURE 3

Motility of CRAb on agar plates. A representative picture of the motility pattern of each isolate is reported (A). The distance migrated (diameter) for each strain was measured (B); data are means ± standard deviation from at least three independent experiments, in duplicate (P-value < 0.01).

FIGURE 4

Biofilm formation of CRAb. Quantification of biofilm formation in A. baumannii clinical isolates. Data are means ± standard deviation from at least three independent experiments, eight wells per strain (P-value < 0.0001).

FIGURE 5

Ability of CRAb to adhere to, be internalized, and survive within cultured epithelial cells. Semi-confluent A549 cell monolayers were infected with each isolate or ATCC 17978 reference strain using a multiplicity of infection (MOI) of 100. Cell-surface-adherent bacteria were enumerated after 1-h incubation, whereas intracellular bacteria were counted after additional 12, 24, and 48-h of incubation in the presence of colistin (5 μg/ml). Data (CFU/ml) are means ± standard deviation from at least three independent experiments, in duplicate. Asterisks (^∗) above the bars indicates a statistically significant difference among isolates (P-values < 0.01). Uninfected cells were used as control.

FIGURE 6

Intracellular localization of A. baumannii in epithelial cells. Semi-confluent A549 cell monolayers were infected as reported before. At 12-h post-infection, cells were fixed and actin was stained with rhodamine-conjugated phalloidin (red), while nuclei and bacterial DNA were stained with DAPI (blue). Images, representatives of at least three independent experiments, in duplicate, were captured using a Leica camera and processed using Qwin software (Leica). Arrows point representative intracellular bacteria. Uninfected cells were used as control (CC). Scale bar, 20 μm.