Targeted isolation of bacteria with potential to competitively exclude Staphylococcus aureus in the upper respiratory tract of pigs

Considering global antimicrobial resistance (AMR) prevalence, alternative or complementary strategies to antimicrobial use, are of interest. Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is of particular interest as despite significant AMU reduction, LA-MRSA prevalence in pig husbandry has not decreased. We performed targeted isolation of bacterial species with potential antagonism against LA-MRSA in pig farms. Duplicate piglet nasal swabs from three European countries (Germany, Ireland and The Netherlands) were taken longitudinally from birth up to 10 weeks, one for amplicon sequencing and qPCR, and the other was cryopreserved for culturing. We identified potential probiotic species by anticorrelation analysis of bacterial abundance from amplicon sequencing data with quantitative S. aureus estimates from qPCR data from the samples. A literature-screen was performed on the species identified, to determine their probiotic potential. Following this, 1302 isolates were grown from selected cryopreserved swabs and identified using MALDI-TOF and additional 16S rRNA gene sequencing to isolate the anticorrelating species. Ninety-five isolates of interest were screened for absence of tetracycline resistance and hemolytic activity and whole genome sequencing was conducted to verify their taxonomy and to assess their AMR and virulence gene profile. Additional phenotypic antimicrobial resistance testing selected three different Lactococcus lactis strains. During an in vitro challenge using spent medium, all three strains demonstrated inhibition against two S. aureus strains. These L. lactis strains may have the ability to be used safely to reduce LA-MRSA carriage in the nasal passages of pigs but further in vivo testing is necessary to confirm this potential. ImportanceAn approach to tackle antimicrobial resistance in livestock is to use competitive exclusion agents. We employed a sequencing guided isolation approach to identify bacterial species with in silico antagonism to livestock-associated Staphylococcus aureus (LA-MRSA) in the nasal microbiome of piglets. Based on this, three Lactococcus lactis isolates were found to be suitable for further probiotic testing. This strategy can be used to effectively identify, isolate, and screen bacteria that live in antagonism with opportunistic pathogens such as LA-MRSA in a complex microbiome.