Carbapenem resistance genes and biofilm-virulence uncoupling in Acinetobacter baumannii

Acinetobacter baumannii has earned its reputation as one of the most formidable hospital pathogens on Earth. Listed by the World Health Organization as a critical-priority bacterium in both its 2017 and 2024 bacterial priority pathogen lists, this Gram-negative coccobacillus causes ventilator-associated pneumonia, bloodstream infections, urinary tract infections, and wound infections that are notoriously difficult to treat. Now, a comprehensive molecular epidemiology study from Haryana, India, has delivered a finding that could reshape how clinicians and diagnostic developers think about this pathogen: carbapenem resistance and biofilm formation, long assumed to travel together, largely behave as functionally independent adaptive traits.

The research, published in Frontiers in Antibiotics, analyzed 200 non-duplicate clinical and environmental isolates of A. baumannii collected between 2022 and 2024 from a hospital network in the Delhi-NCR region. The team, led by Shyam Tripathi and V. Samuel Raj of SRM University, subjected every isolate to antimicrobial susceptibility testing by broth microdilution against 17 antibiotics, interpreted according to the Clinical and Laboratory Standards Institute M100 guidelines. Biofilm-forming capacity was quantified using the standardized microtiter plate crystal violet assay, while polymerase chain reaction detected six carbapenemase genes, three beta-lactamase genes, and three virulence genes. Sample size adequacy was verified using Buderer’s formula, ensuring the collection was statistically powered for the genotype-phenotype comparisons at the heart of the study.

The resistance landscape the researchers uncovered was stark. Among the 200 isolates, 54 percent were multidrug-resistant, 34 percent were extensively drug-resistant, and 61 percent were carbapenem-resistant. Fluoroquinolone resistance was near-universal, with 84 percent of isolates resistant to both ciprofloxacin and levofloxacin. Cephalosporin resistance ranged from 74.5 to 75.5 percent, and aminoglycoside resistance reached 67.5 percent for amikacin. Colistin, one of the last-resort polymyxin antibiotics, retained activity against 90 percent of isolates according to EUCAST criteria, while tigecycline remained active against 62 percent. These figures align with regional surveillance data from the ATLAS network, which identified nearly 87 percent carbapenem-resistant A. baumannii across South and Southeast Asia, far exceeding the roughly 40 percent rates observed in Europe.

At the genetic level, one carbapenemase gene towered above all others. The blaOXA-23 gene, an Ambler class D oxacillinase frequently mobilized by the insertion sequence ISAba1 and disseminated globally within the international clonal complex 2 lineage, was present in 61 percent of isolates. Other acquired carbapenemase genes, including blaOXA-24, blaOXA-58, blaNDM-1, blaVIM, and blaIMP, were detected only sporadically at frequencies of 3 percent or less. The intrinsic blaOXA-51 gene, used for species confirmation, was carried by all 200 isolates, while AmpC was widespread at 79 percent and blaPER-1 appeared in 60.5 percent of the collection.

The statistical concordance between blaOXA-23 carriage and phenotypic carbapenem resistance was remarkable. Cohen’s kappa reached 0.81, classified as almost-perfect agreement, with 92.6 percent of gene-positive isolates resistant to carbapenems compared with just 11.5 percent of gene-negative isolates. The odds ratio was 96.26 with a 95 percent confidence interval of 36.45 to 254.24. When the researchers considered composite carbapenemase carriage, defined as the presence of at least one carbapenemase gene, concordance improved further to kappa 0.88, with sensitivity of 98.4 percent and specificity of 88.5 percent. Carbapenemase gene burden also correlated strongly with meropenem MIC escalation, with a Spearman rank correlation coefficient of 0.816. From a diagnostic standpoint, these findings powerfully support integrating blaOXA-23 PCR into frontline resistance screening, ideally through rapid molecular platforms that can deliver results within one to two hours of isolate recovery.

Yet the study’s most provocative finding concerns what did not correlate. Biofilm-forming capacity, measured by crystal violet staining of attached biomass in 96-well polystyrene plates, was completely unrelated to cumulative antimicrobial resistance burden. Resistance burden, defined as the number of antibiotic classes showing resistance, did not differ significantly across weak, moderate, and strong biofilm producers, with median values of 12, 12, and 11 respectively. Kruskal-Wallis analysis confirmed no significant group difference, and Spearman correlation showed no monotonic association. XDR and PDR status likewise showed no significant relationship with biofilm phenotype. This null result challenges the widely assumed coupling between multidrug resistance and enhanced biofilm production in A. baumannii, and it converges with independent evidence from Chennai, India, where 247 isolates showed biofilm formation in 99 percent of strains without direct correlation to resistance phenotype.

There was one notable exception to this decoupling. Strong biofilm producers showed significantly higher colistin MIC values compared with weak producers, with a p-value of 0.001. The researchers offer a mechanistically coherent explanation rooted in the chemistry of polymyxin action. Colistin exerts its bactericidal effect by electrostatic exchange of divalent cations such as calcium and magnesium from the lipid A portion of lipopolysaccharide, destabilizing the bacterial outer membrane. The anionic extracellular polymeric substance matrix of A. baumannii biofilms, which contains poly-beta-(1,6)-N-acetylglucosamine, alginate-like polysaccharides, and extracellular DNA, sequesters these cations and physically prevents colistin from reaching its site of action at the bacterial surface. This represents a biofilm-associated phenotypic tolerance mechanism rather than confirmed genetic colistin resistance, as pmrAB mutations and lipid A modifications were not investigated. The practical implication is direct: routine colistin MIC testing performed on planktonic bacteria will systematically underestimate the effective concentration required to eradicate biofilm-associated infections, particularly in device-associated settings or ventilator-associated pneumonia.

The virulence genes told an equally compelling story of functional separation. All three virulence determinants analyzed, the biofilm-associated protein gene bap, the outer membrane protein A gene ompA, and the chaperone-usher pili gene csuE, were strongly and independently associated with strong biofilm formation. Among bap-positive isolates, 78.8 percent were strong biofilm producers compared with just 4.1 percent of bap-negative isolates. Similar patterns held for ompA and csuE, and blaPER-1 carriage was also linked to enhanced biofilm capacity. Critically, however, none of these virulence genes were associated with increased antimicrobial resistance burden. Mann-Whitney U analysis showed no significant difference in resistance burden according to bap, ompA, csuE, or blaPER-1 carriage, all with p-values above 0.05. These findings support the concept that virulence and antimicrobial resistance may evolve independently under different hospital selection pressures, with the biofilm-virulence axis establishing a distinct persistence mechanism that does not expand the spectrum of conventional resistance.

Three clinically actionable recommendations emerge from this work. First, blaOXA-23 PCR may serve as a useful rapid screening tool in high-carbapenem-resistance settings, though it should complement rather than replace phenotypic susceptibility testing because a small subset of blaOXA-23-positive isolates remained carbapenem-susceptible, possibly reflecting absence of upstream ISAba1 promoter activation or low-level gene expression. Second, phenotypic biofilm quantification may be considered as a supplementary risk-stratification tool in device-associated A. baumannii infections, particularly where colistin therapy is contemplated, given the elevated colistin MICs among strong biofilm formers. Third, the high prevalence of virulence-gene-driven biofilm capacity underscores the need for anti-biofilm adjunct strategies, including biofilm-disrupting agents, improved device coatings, and combination regimens pairing newer agents such as cefiderocol or sulbactam-durlobactam with biofilm-disrupting compounds.

The researchers acknowledge important limitations that temper the strength of their conclusions. The retrospective design using archived isolates did not allow longitudinal monitoring of resistance trends or correlation with patient outcomes. Advanced molecular typing approaches such as multilocus sequence typing and whole-genome sequencing were not undertaken, meaning clonal relationships, international lineage assignments, and plasmid-mediated transmission could not be fully delineated. ISAba1 mapping upstream of blaOXA genes and gene-expression analysis were also not performed, leaving the mechanisms underlying blaOXA-23-positive but carbapenem-susceptible discordant isolates unresolved. Growth kinetics were not assessed in parallel with the biofilm assay, so growth-rate effects on optical-density-based biofilm biomass cannot be fully excluded. Despite these constraints, the study demonstrates with statistical precision that biofilm formation and carbapenem resistance diverge as functionally independent adaptive strategies in A. baumannii. Resistance genotyping and biofilm phenotyping provide complementary rather than redundant information, and understanding this decoupling may prove essential for optimizing infection severity stratification and therapeutic decision-making in the ongoing battle against this critical-priority pathogen.

Subject of Research: Microbiology

Article Title: Carbapenem resistance genes and biofilm−virulence uncoupling in Acinetobacter baumannii

Article References: Tripathi, S., Rani, K., Berlia, R., Sharma, A., Hari, A., Gaur, P., Raj, P. S. S., & Raj, V. S. (2026). Carbapenem resistance genes and biofilm−virulence uncoupling in Acinetobacter baumannii. Frontiers in Antibiotics, 5, Article 1888133. https://doi.org/10.3389/frabi.2026.1888133

Image Credits: AI Generated

DOI: 10.3389/frabi.2026.1888133

Keywords: Acinetobacter baumannii, carbapenem resistance, blaOXA-23, biofilm, virulence genes, antimicrobial resistance, colistin, genotype-phenotype concordance, Frontiers in Antibiotics, India, Carbapenem, resistance