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Background: Methicillin-resistant Staphylococcus aureus (MRSA)-USA300 is notorious for its ability to cause community- and healthcare-acquired infections, which are even more difficult to treat when associated with a biofilm phenotype. We aimed to characterize the genetic determinants of biofilm formation in a USA300 skin abscess isolate (UAS391) that formed prolific biofilms. Methods: USA300 S. aureus strains, TCH1516 and FPR3757, were found to be closely related based on whole genome mapping (Argus? Optical Mapping System, Opgen Inc, Gaithersburg, USA) to UAS391 (96.3-99.1% similarity, P=0.0151), however differed markedly in biofilm formation (P=0.0001) on a dynamic assay (BioFlux 200, Fluxion Biosciences, USA). Comparison of whole genome sequences of these strains identified differences in a total of 18 genes. Corresponding Tn (bursa aurealis-bearing) knockout mutants in these target genes were obtained from a publicly available mutant library of the same clonal lineage (USA300-JE2) and were characterized phenotypically for biofilm formation. Tn mutants showing significant differences in biofilm formation were utilized for transduction into a plasmid-cured erythromycin-sensitive derivative of UAS391 and for complementation experiments. All strains were tested on the dynamic assay, and 17h-biofilms were stained (SYTO9, Life Technologies) and fluorescence intensity quantified by microscopy (Zeiss, ImageJ). Gene expression levels in Tn and transduced mutants were studied by quantitative reverse transcriptase PCR (StepOnePlusTM, Applied Biosystems®). Results: Comparison of the sequenced genomes of TCH1516, FPR3757 and UAS391 yielded a limited number of variant genes (n=18) that were hypothesized to account for the observed difference in biofilm-forming capacity. Screening of Tn mutants disrupted in these target genes identified one mutant (NE229) bearing a transposon insertion in SAUSA300_1119 (fakA), which exhibited increased biofilm formation similar to UAS391 (P=0.9320). Transduction experiments confirmed that fakA::Tn corresponded to 1.9- to 4.6-fold increase in biofilm formation depending on the USA300 strain background (P≤0.0007), while complementation of the TCH1516 wild-type fakA allele in UAS391 resulted in a 4.3-fold reduction in biofilm formation (P<0.0001). Conclusions: This sequential approach, consisting of strain typing, genome comparison and functional genomics, identified fakA, a recently described fatty acid kinase in S. aureus that is essential for phospholipid synthesis and also impacts the transcription of numerous virulence factors, as a negative regulator of biofilm formation in S. aureus USA300. [ABSTRACT FROM AUTHOR]

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in livestock has refocused attention on S. aureus colonization and transmission in pigs. This study investigated the effect of the S. aureus colonization status of a sow on the colonization status of her piglets, and whether pigs carry the same strain of S. aureus throughout production. Nasal swabs were collected from the piglets of six healthy sows two days after birth and two days before and two days after they were moved into each production stage. The average prevalence of S. aureus colonization varied between 26% and 73%. The odds of being S. aureus positive were almost 12 times higher for piglets born to nasal-positive sows than for those born to nasal-negative sows, and three times higher again for piglets born to sows that were both nasal- and vaginal-positive. Isolates recovered from piglets immediately after birth were indistinguishable from those of the dam as determined by phenotypic and molecular typing, including microarray analysis and optical mapping. All isolates belonged to clonal complex 9 and the majority exhibited a novel spa type, t10449. The findings show that the S. aureus colonization status of the sow influences the colonization status of her piglets in the early production stages but strains carried by pigs change over time. Multiresistant S. aureus was detected, in particular post-weaning. Results suggest that sow status and management practices, including mixing of pigs and antimicrobial usage at weaning, should be considered when implementing control measures for S. aureus on a farm., (Copyright © 2014 Elsevier B.V. All rights reserved.)