New kid on the block: First detection of Pseudomonas asiatica as an emerging mastitis pathogen in dairy cows
Original scientific paper: Sultana T, Rahman MM, Rahaman M, Arafat KY, Haider MG, Rahman ANMA, Talukder AK, Hoque MN, and Das ZC. (2025). Genomic characterisation of Pseudomonas asiatica as an emerging mastitis pathogen in dairy cows with resistance and virulence implications. J. Glob. Antimicrob. Resist. 44:21-29. doi: 10.1016/j.jgar.2025.05.022.
Background
Mastitis remains one of the most important diseases in dairy herds worldwide, with major economic and animal welfare impacts. While major mastitis pathogens include Staphylococcus aureus, Escherichia coli, and Streptococci, non-traditional Gram-negative opportunists are increasingly reported. Pseudomonas spp., particularly P. aeruginosa and P. putida, are known opportunistic mastitis pathogens. P. asiatica, a recently described member of the P. putida group, has been detected in human clinical samples and environmental sources but has not yet been described as a cause of bovine mastitis. Its potential for antimicrobial resistance (AMR) acquisition and environmental persistence makes it a concern for both animal and public health. This study was conducted to investigate the prevalence of P. asiatica in milk, feces, and soil from mastitis-affected dairy farms in Bangladesh, and to characterize its AMR profiles, explore phylogenetic relationships, and identify antibiotic resistance genes (ARGs) and virulence factor genes (VFGs) using whole-genome sequencing.
Materials and methods
A total of 110 samples - including 45 milk, 35 faeces, and 30 soil - were collected from 30 smallholder farms in the Gazipur district of Bangladesh. Bacterial identification was carried out using culture-based methods, VITEK-2 systems, and 16S rRNA gene sequencing. Antimicrobial susceptibility was assessed through the Kirby–Bauer disc diffusion method against 15 antibiotics commonly employed in both livestock and human medicine. Whole-genome sequencing was performed on four multidrug-resistant (MDR) isolates originating from milk (2M1), faeces (2F1 and 2F2), and soil (2S1), using the Illumina MiSeq platform. Bioinformatic analyses included genome assembly with SPAdes, annotation via PGAP and RAST, detection of ARGs using CARD, ResFinder, and AMRFinderPlus, VFGs prediction through VFDB, phylogenetic analysis with TYGS, and calculation of Average Nucleotide Identity (ANI).
Results
Out of 110 samples, 41 was positive to P. asiatica, with an overall prevalence of 37.3% across all samples, including 29.3% in milk, 34.2% in feces, and 36.6% in farm soil. Identification was confirmed through phenotypic characteristics - Gram-negative rods forming pale yellow to colourless mucoid colonies (Figure 1) and molecular methods. Antimicrobial susceptibility testing revealed high resistance to imipenem (95.1%), ampicillin (82.9%), sulphonamides (82.9%), oxacillin (80.49%), nitrofurantoin (78.1%), azithromycin (61.0%), tetracycline (51.2%), and cefoxitin (51.2%), while greater susceptibility was observed to streptomycin (78.1%) and gentamicin (73.2%), with moderate susceptibility to ciprofloxacin, nalidixic acid, and chloramphenicol. All of the 41 P. asiatica isolates were classified as MDR, with some resistant to up to 12 antibiotics. The assembled genome size of four MDR P. asiatica strains (2M1, 2F1, 2F2, and 2S1) is approximately 5.6 Mbp, with a GC content of 62.8% and a genome coverage of 50x. Phylogenetic analysis of P. asiatica strains revealed a close evolutionary connection with strains of Pseudomonas spp. isolated from diverse samples including bovine mastitis milk, with ANI value of >97%, confirming species identity. Each of the four P. asiatica strains harbored 11 ARGs, imparting resistance to multiple antibiotics, drugs, compounds and biocides; and 30 VFGs. The identified ARGs included mexB, mexE, mexF, mexK, mexI, mexW, ttgA, ttgB, ttgC, ttgR, and CpxR, conferring resistance to multiple antibiotic classes viz. β-lactams, aminoglycosides, fluoroquinolones, macrolides, tetracyclines, sulphonamides, and disinfectants through efflux pumps. VFGs were encoded by key functional groups related to motility (~40%), alginate biosynthesis and regulation (~21%), two-component regulatory systems (~9%), pyoverdine biosynthesis (~3.5%), and the Type VI Secretion System (T6SS), along with quorum sensing genes, supporting mechanisms for host interaction, biofilm formation, interbacterial competition, and coordinated expression of virulence traits.
Interpretation and implications
This study provides the first genomic evidence linking P. asiatica to bovine mastitis, identifying it as an emerging pathogen and expanding its known host range. The high levels of MDR and its ability to persist in farm environments raise significant public health concerns, highlighting the potential for zoonotic transmission and ARG flow across the animals, humans, and the environment. Therapeutic options are limited due to resistance to multiple frontline antibiotics, making current reliance on gentamicin and streptomycin a cautious necessity to avoid further resistance development. The detection of P. asiatica in milk, feces, and soil also underscores the role of farm environments as reservoirs for reinfection. These findings emphasize the urgent need for species-specific diagnostics, targeted antimicrobial stewardship, and robust biosecurity measures to monitor and control the spread of this pathogen in the dairy environment.
Conclusions
P. asiatica has emerged as a MDR mastitis pathogen in Bangladeshi dairy farms, possessing efflux pump-mediated resistance and a wide range of VFGs that facilitate environmental survival, biofilm formation, and host colonization. Phylogenetic analysis reveals its close genetic relatedness to Pseudomonas strains from various global sources, suggesting potential dissemination routes across regions. These findings highlight the urgent need to include P. asiatica in mastitis surveillance programs and to develop targeted interventions aimed at reducing AMR risks within livestock production systems.
Ziban Chandra Das and M. Nazmul Hoque - Molecular Biology and Bioinformatics Laboratory, Department of Gynecology, Obstetrics and Reproductive Health, Gazipur Agricultural University, Gazipur, Bangladesh
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