Genomic and phenotypic characterization of six multidrug-resistant Acinetobacter pittii isolates

Characterization and relatedness of the isolates

The susceptibility profiles of six clinical A. pittii isolates are presented in Table 1. All isolates were susceptible to aminoglycosides, tetracyclines and fluoroquinolones. All isolates were susceptible to ceftolazone/tazobactam, colistin, ampicillin/sulbactam, trimethoprim/sulfamethoxazole and nitrofurantoin; and resistant to ampicillin, first generation cephalosporin cefazolin, 3rd generation cephalosporins ceftriaxone and cefotaxime and aztreonam. Two isolates (AP290R and AP5092) were intermediate to piperacillin/tazobactam and resistant amoxicillin/clavulanic acid; only one isolate (AP290R) was resistant to all carbapenems tested and had elevated levels of ceftazidime (8 ug/ml).

The six A. pittii isolates and reference strain ATCC19606 were analyzed by FT-IR to determine phenotypic similarity. No clusters (indicating similar isolates) were evident (Fig. 1A). To determine whether FT-IR could distinguish between A. pittii and A. baumannii, we added 11 A. baumannii strains, belonging to different sequence types (STs), to the FT-IR analysis. Still no clusters were identified, and A. pittii isolates were not always more similar to each other than to A. baumannii isolates (Fig. 1B).

All 6 A. pittii strains were sequenced and their genomes were analyzed. The isolates belonged to different STs (Table 2) with different KL types. We constructed a phylogenetic tree based on pangenome analysis (Supplementary Fig. 1). The tree revealed distinct separation, with consistent and even distances observed between each isolate. Next, we conducted core genome alignment of the six A. pittii isolates compared to 64 publicly available complete genomes of A. pittii (Supplementary Table 1) from different countries and different years. The genomes represent a temporal range spanning from 2011 to 2024, allowing for examination of potential genomic changes over nearly three decades. This analysis placed AP5092 and MML4 (isolated in 2023, Hong Kong) on one branch close to each other. Other four isolates were located on a second branch, with AP5047 and AP290R most closely related to each other (Fig. 2). Isolate AP290R most close to isolate HCG18 isolated in Mexico in 2023.

Phylogenetic tree of six A. pittii isolates together with 64 publicly available genomes of A. pittii.

Antibiotic resistance genes

Genomic analysis revealed the presence of several antimicrobial resistance determinants among the isolates (Table 3).

All isolates carried at least two genes conferring β-lactam resistance: a variant of ampC cephalosporinase (ADC) and a variant of OXA-type β-lactamase. Notably, isolate AP290R carried three bla_OXA genes – bla_OXA−272, bla_OXA−255 and bla_OXA−72 carbapenemases. Correspondingly, this was the only isolate to display carbapenem resistance.

In addition, all six study isolates harbored genes conferring quinolone resistance (adeF and abaQ), efflux pump components (abeS, adeF and adaQ), and a gene involved in colistin resistance (lpsB). Three isolates (AP4773, AP4968, and AP5092) contained a single putative aminoglycoside resistance gene, ant(3’’)-IId, with relatively low sequence homology (69%) to the closest match in the Comprehensive Antibiotic Resistance Database (CARD).

Virulence studies

In vitro phenotype

We next evaluated characteristics of the A. pittii isolates which are classically associated with bacterial virulence – growth, motility and biofilm formation. A. baumannii reference strain ATCC19606 served as a reference strain. The growth of all isolates was similar to that of the control strain (p > 0.05; Supplementary Fig. 2). Three A. pittii isolates demonstrated significantly higher motility than the control stain (p < 0.0001) (Fig. 3A), and four isolates produced significantly less biofilm (p < 0.001) (Fig. 3B).

In vitro phenotype of six A. pittii studied isolates. (A) Motility. Columns show average length of tentacle formation for each isolate. (B) Biofilm formation. Quantification of biofilm mass by crystal violet. (*) p-value < 0.001 compared to ATCC19606 values, bar represents standard error mean (SEM). (C) Survival of A. pittii isolates in 80% normal human serum (NHS) and heat inactivated NHS (as a control, strains were grown without serum in BHI medium and their growth was measured). Bar represents standard error mean (SEM). Each isolate is represented in a different color. ATCC19606 used as reference strain.

All A. pittii isolates were serum sensitive (Fig. 3C), displaying either death (4/6 isolates) or reduced growth (2/6 isolates) after 4 h exposure to NHS. Heat inactivation of the complement system reduced the serum sensitivity of 5/6 isolates (except for AP5091), with one isolate (AP5092) displaying full resistance to the inactivated serum.

In vivo virulence

We next evaluated the virulence of the A. pittii isolates in vivo, using killing assay in Z. morio larvae. In this model, AP290R exhibited high virulence potential (Fig. 4), killing 70% of infected Z. morio larvae within 24 h. The other A. pittii strains were less lethal, with a 10%−20% lethality rate 24 h post infection and a 25%−35% lethality rate 7 days post infection.

In vivo virulence of six A. pittii studied isolates. Kaplan- Meier survival curves for Z. morio larvae (30 per group) infected with 1 × 10⁷ CFU of each isolate of A. pittii. G257 – Acinetobacter baumannii clinical strain used as reference.

Virulence factors

AP290R possessed the largest number of virulence genes, and AP4773 and AP5092 – the smallest. Nine virulence genes (cpaA, entE, gspDE1E2FINO, bauCDF, pilBCFGHMTU2D, pbpG, lpxABCL, barB, lbsB) were present in all 6 isolates (Fig. 5). These genes are related to various virulence functions, including biofilm formation, adherence, coagulation (cpaA), siderophore biosynthesis (entE), motility, and others. Several differences in genomic content were evident. The cluster of three genes – pilA, pilQ (surface motility) and basI (siderophore biosynthesis) – was missing from AP290R, AP4773, AP4968 and AP5091. However, only pilA was missing in isolate AP5092, and only basI was missing in isolate AP5047. The iron acquisition gene barA was missing in AP5047.

Heatmap of virulome analysis of six A. pittii studied isolates. Comparison of genomes to the virulence factor database (VFDB) was complete, the existence of genes represented by blue color, the absence in white. Hierarchical clustering of the isolates based on the presence or absence of the virulence factors shown at the top. .

Plasmid content

Whole genome analysis revealed that two A. pittii isolates carried plasmids. Properties of these plasmids are described in Table 4 and the specific ORFs – in Supplementary Table 3. Plasmid p290R (Fig. 6A) carried ten hypothetical proteins and 4 genes of known function: ydhP (associated with glycosidase and hydrolase activity), azoR1 (azoreductaze), gcvA (regulates the glycine cleavage system, transcriptional activator), and most significantly – the carbapenemase bla_OXA−72 gene. The much larger plasmid p5092 (Fig. 6C) carried 119 genes, among them genes involved in different metabolic pathways, transporters and transcriptional regulators, and several insertion sequences (ISAba46, ISAba22, ISAba23, ISAha3, ISAcsp3, ISAba26 and IS1301), and 79 hypothetical proteins.

Plasmid analysis. Circular maps of plasmid pAP290R from isolate AP290R (A) and pAP5092 from isolate AP5092 (C). The open reading frames are marked along the map in blue. tRNA genes found only in pAP5092 are indicated in pink. The bla_OXA-72 gene found in pAP290R is indicated in red. Phylogenetic analysis of A. pittii plasmids pAP290R (B) and pAP5092 (D). Scale indicates branch length (nucleotide substitution per site). Plasmid maps were generated via Proksee.

BLASTn analysis revealed a significant number of comparable plasmids within the public database, all reported in Acinetobacter spp. From this pool, we selected the ten most closely related plasmids for each of the plasmids (pAP5092 and pAP290R) and constructed a phylogenetic tree (Fig. 6B, D). The plasmid closest to pAP290R was pSU8507_OXA-2 (accession number LC777725.1) isolated from A. pittii in Japan in 2023, followed by 5 closely related plasmids from the US, China and Spain isolated in 2017–2021 (Fig. 6B). The plasmid closest to pAP5092 found in the database was pML4_1 (accession number CP118934.1) isolated from A. pittii in Hong Kong in 2023 (Fig. 6D). Additional information regarding accession numbers, year and country of isolation can be found in Supplementary Table 2.

Plasmid pAP290R contained the bla_OXA−72 gene (an OXA-24 family carbapenemase). To test the inter-species transferability of this plasmid, it was extracted from A. pittii AP290R and electroporated it into AB2142, a carbapenem-susceptible A. baumannii strain. Plasmid pAP290R conferred resistance to beta-lactams and carbapenems (Supplementary Tables 4 and Supplementary Fig. 3). However, resistance to ceftazidime in AP290R seems to be intrinsic; it was not transferred to AB2142 by the pAP290R plasmid.

Crucially, the plasmid also significantly increased the virulence of AB2142 (Fig. 7), suggesting that it contributes to the virulence of AP290R, raising the possibility that some of the hypothetical genes it carries are in fact virulence factors.

Kaplan- Meier survival curves for Z. morio larvae (30 per group) infected with 1 × 10⁷ CFU of AB2142 – not virulent A. baumannii strain, AP290R -study strain and AB2142 containing plasmid from AP290R.