Phenotype detection of biofilm from Serratia and Staphylococcus aureus isolated from clinical and subclinical mastitis of bovine in Diyala governorate

Abstract

Persistent intramammary infections caused by biofilm-producing bacterial pathogens represent a critical challenge for dairy cattle health and veterinary management. This research aimed to isolate Staphylococcus aureus and Serratia marcescens from clinical and subclinical bovine mastitis cases in Baqubah city within the Diyala governorate, evaluate their phenotypic and genotypic biofilm-forming profiles, and determine their susceptibility to Capsicum frutescens crude ethanolic extract. Out of 65 milk samples collected from lactating dairy cows exhibiting evident symptoms of mastitis, bacterial pathogens were successfully recovered from 58 samples, representing a 89.23 % isolation success rate, whereas 7 samples exhibited no pathogen development. Based on standard morphological characterization, conventional biochemical profiling, and definitive verification via the automated VITEK 2 identification system, 10 isolates were classified as Staphylococcus aureus, constituting 15.38 % of the total specimens, and 4 isolates were identified as Serratia marcescens, accounting for 6.15 % of the evaluated sample pool. Phenotypic quantification of biofilm production using the microtiter plate assay method revealed that among the 10 staphylococcal strains, 3 isolates (30 %) developed weak biofilms, 3 isolates (30 %) generated moderate biofilms, and 4 isolates (40 %) established strong, highly adherent biofilm architectures. For the 4 Serratia marcescens environmental-opportunistic strains, 1 isolate (25 %) exhibited moderate biofilm layers, while the remaining 3 isolates (75 %) demonstrated robust, strong biofilm matrix development. Molecular characterization via the Polymerase Chain Reaction verified the presence of the multidrug efflux pump gene norA at 391 bp in ciprofloxacin-resistant staphylococcal strains and the biofilm-associated genetic marker bsmA at 298 bp in Serratia marcescens strains. Evaluation of the alternative antibacterial efficacy of the crude Capsicum frutescens extract using the agar well diffusion assay at targeted concentrations of 500 and 1000 μg/mL demonstrated profound, dose-dependent inhibitory activity against both persistent bacterial pathogens, with the measured diameters of the inhibition zones expanding proportionally in direct correlation to the rising concentration of the agent. These findings confirm that biofilm matrix production directly accelerates chronic disease development and long-term bovine mastitis persistence within the region, establishing the crucial practical utility of implementing alternative biofilm-targeted control programs and natural phytotherapeutic alternatives in dairy herds.