The study, led by Johanna Rühl-Teichner and colleagues at Justus Liebig University Giessen together with the Robert Koch Institute in Berlin, drew on isolates submitted to a veterinary diagnostic laboratory between 2008 and 2024. Most samples came from Germany, with smaller numbers from France, Italy, the Netherlands, and Belgium. The team identified each bacterium using MALDI-TOF mass spectrometry and confirmed species identity through ribosomal multilocus sequence typing. What they found was sobering: nearly 80 percent of the isolates were multidrug-resistant, meaning they withstood agents from three or more antibiotic classes, and resistance rates in cats and dogs were dramatically higher than in horses.

Resistance patterns varied sharply by host species. Every single isolate was either intrinsically or acquired resistant to ampicillin, amoxicillin-clavulanate, cephalexin, cefovecin, ceftiofur, nitrofurantoin, and chloramphenicol. Resistance to cefpodoxime reached 98.6 percent across the board. Yet amikacin remained remarkably effective, with 97.8 percent of isolates still susceptible. The most striking host difference emerged with imipenem, a last-line carbapenem antibiotic: 49.3 percent of feline isolates and 31.1 percent of canine isolates were resistant, compared with just 1.2 percent of equine isolates. Fluoroquinolone resistance told a similar story, with nearly 90 percent of cat and dog isolates resistant to enrofloxacin and marbofloxacin, while horse isolates lagged far behind at around 40 percent.

Biofilm formation proved equally widespread but far more nuanced. The researchers used a crystal violet staining assay to quantify how much biofilm each isolate produced after 24 and 48 hours of incubation, normalizing the results to bacterial growth to calculate a specific biofilm formation value. Canine isolates showed progressive maturation over time, with the number of strong biofilm producers nearly doubling from 46 to 85 between the two time points. Feline isolates remained largely stable, while equine isolates displayed a delayed but dramatic shift, jumping from just one strong producer at 24 hours to twenty by 48 hours. This temporal dimension suggests that biofilm development follows distinct trajectories depending on the host environment the bacteria have adapted to.

Clinical origin mattered just as much as host species. Isolates recovered from wounds and abscesses, urinary tract infections, and medical devices such as catheters and implants consistently showed the strongest biofilm production. In contrast, isolates from the genital tract were predominantly weak or non-biofilm producers, and respiratory tract isolates fell somewhere in between. The researchers observed a significant shift toward stronger biofilm formation between 24 and 48 hours, particularly among wound, urinary, and device-associated isolates, suggesting that these infection sites may select for bacteria with enhanced persistence capabilities.

Genomic analysis revealed that clonal lineage was perhaps the most powerful predictor of biofilm behavior. The team assigned each isolate to one of twelve known international clones using multilocus sequence typing and whole-genome comparison with reference genomes. IC1 and IC2 dominated the collection, accounting for 181 and 118 isolates respectively, and both lineages exhibited the strongest biofilm-forming capacity. IC1 isolates produced strong biofilms in 42.5 percent of cases at 24 hours, rising to nearly 60 percent by 48 hours. IC2 isolates showed no strong biofilm production at 24 hours but developed substantial strong production by 48 hours. Other clones, including IC3, IC7, and IC8, showed weaker or delayed biofilm formation, and 138 isolates could not be assigned to any known international clone.

The relationship between biofilm-associated genes and actual biofilm production proved surprisingly complex. Most of the eighteen genes the team screened were nearly universal, including the regulatory pair bfmR and bfmS, the quorum-sensing system abaI and abaR, the pgaABCD cluster encoding a biofilm matrix polysaccharide, and the csu pilus genes. Yet the presence of these conserved genes alone could not explain the wide variation in biofilm phenotypes. Two genes stood out for their lineage-specific distributions: bap, which encodes a large surface protein, was found almost exclusively in IC2 isolates and showed an inverse association with biofilm strength, while blp1, a Bap-like protein gene, was present in all IC1, IC2, IC3, and IC5 isolates but absent from IC7 and IC8.

When the researchers applied a multivariable logistic regression model to disentangle these relationships, host species and clonal lineage emerged as the dominant independent predictors of strong biofilm formation. Equine isolates showed nearly eight times higher odds of strong biofilm production compared with feline isolates, while IC1 membership remained strongly associated with the phenotype. Notably, the apparent association between blp1 and biofilm formation disappeared after adjustment, suggesting that the gene’s distribution simply mirrors the clonal background rather than acting as an independent driver. The inverse association between bap and biofilm production persisted, contradicting its commonly described role as a biofilm promoter and underscoring how context-dependent these genotype-phenotype relationships can be.

The convergence of multidrug resistance and strong biofilm formation is particularly concerning from a clinical perspective. Highly resistant isolates, those withstanding eight to ten antimicrobial agents, were strongly enriched among moderate and strong biofilm producers, and MDR isolates showed a pronounced shift toward strong biofilm production over time. This combination likely promotes chronic and recurrent infections, reduces antimicrobial penetration, and favors the emergence of persister cells that survive antibiotic exposure. In veterinary medicine, where treatment options are already limited, the findings support the consideration of anti-biofilm strategies as adjuncts to conventional therapy.

The study also carries broader implications for public health. Previous comparative genomic work has demonstrated close relationships between A. baumannii isolates from humans and animals, and the predominance of IC1 and IC2 in companion animals mirrors the global epidemiology of hospital-acquired infections. While the observational nature of the study precludes conclusions about causality, and the authors caution that sampling bias and confounding between host species, clinical origin, and clonal lineage may influence the observed patterns, the message is clear: biofilm formation is a central feature of A. baumannii epidemiology in companion animals, closely linked to the same clonal lineages and resistance phenotypes that make this pathogen so formidable in human medicine. Understanding these connections may prove essential for developing targeted therapeutic strategies that work across both veterinary and human clinical settings.

Subject of Research: Biofilm formation and antimicrobial resistance in Acinetobacter baumannii from companion animals and horses

Article Title: Biofilm formation in Acinetobacter baumannii isolates from companion animals and horses: associations with host, clinical origin, antimicrobial resistance, and clonal lineage

Article References: Biofilm formation in Acinetobacter baumannii isolates from companion animals and horses: associations with host, clinical origin, antimicrobial resistance, and clonal lineage. (n.d.). https://doi.org/10.3389/fbrio.2026.1874938

Image Credits: AI Generated

DOI: 10.3389/fbrio.2026.1874938

Keywords: Acinetobacter baumannii, biofilm formation, antimicrobial resistance, multidrug resistance, companion animals, international clones, veterinary medicine, genomics, IC1, IC2, One Health, biofilm-associated genes