Abstract
BackgroundMethicillin-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.MethodsUSA300 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®).ResultsComparison 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).ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1956-8) contains supplementary material, which is available to authorized users.
Highlights
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
Typing and phenotypic analysis of USA300 isolates Whole genome mapping (WGM) allowed typing and grouping of strain UAS391 with sepsis strain USA300_TCH1516 (TCH1516) isolated at Texas Children’s Hospital in Houston [21] along with two other S. aureus USA300 strains, USA300-FPR3757 (FPR3757) and its plasmid-cured laboratory derivative USA300-JE2 (JE2), both belonging to the USA300 clonal lineage
According to the WGM similarity cut-off recently established for USA300 isolates [22], a WGM-based clonal cluster is defined as a set of isolates having a whole genome map similarity of >95 %, which assigned all four isolates discussed here to the same WGM clonal cluster (Fig. 1)
Summary
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. It is generally accepted that biofilms, comprising conglomerations of cells attached to a solid support and embedded in a matrix of extracellular polymers, represent a major problem in clinical practice, due to their formation on implanted medical devices [3] and their intrinsic enhanced resistance to antibiotics that are otherwise efficacious against the bacterium’s planktonic life forms [4]. These biofilm-associated complications have triggered the search for potential genes and/or metabolic pathways, interruption of which could represent new therapeutic or preventive interventions targeting this bacterial life-style. Subsequent proteomic studies performed by Resch et al [6] showed still more stringently that biofilm-induced proteins are involved in cell attachment and peptidoglycan synthesis, in pyruvate and formate metabolism, as well as in regulatory processes, in particular those exerted by the staphylococcal accessory regulator A protein
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