Variation in staphyloxanthin production and biofilm formation by Staphylococcus aureus from chronic infections

RIVIERE AN; DOTTO C; SULIGOY LOZANO CM; LOMBARTE SERRAT A; SORDELLI DO; GIACOMODONATO MN; BUZZOLA FR

CORDOBA

Conferencia; Conferencia bianual de la Society for General Microbiology (SAMiGe-Argentina).; 2015

Society for General Microbiology (SAMiGe-Argentina).

Staphylococcus aureus is one of the most important humanpathogens both in the hospital and the community. This bacterium has theability to cause chronic infections due to the formation of surface-associatedaggregates or biofilms. The SigB alternative sigma factor is activated duringbiofilm formation and triggers the expression of genes associated withpigmentation and biofilm formation. The orange and yellow carotenoid pigmentsthat produce S. aureus are the products of the staphyloxanthin(STX) biosynthetic pathway. The carotenoid pigment biosynthesis genes areorganized in the operon crtOPQMN. STX is a virulence factor withantioxidant action and its production is positively regulated by the SigBfactor. To further study the contribution of STX in the fitness of S. aureus in chronic infections, we compared thelevel of pigments produced with the biofilm biomass formed by S. aureus isolated from patients with osteomyelitis.We selected 18 S. aureus isolates from our laboratorycollection of strains whose full genome was previously sequenced. The genotypiccharacterization (clonal complex, sequence typing, agr typing and spa typing) of each strain wasperformed by specific PCRs and sequence analysis. The pigment extracted wasquantified by evaluation of the optical density (OD) at 450 nm and bycorrelation pigment production with the culture OD. Biofilms formed by S. aureus were measured by static microtiter plateassays. The distribution of the average OD values from the 18 isolates intoquartiles permitted to classify the strains as high producers (HP: OD ³ 0.62), producers (P: OD ³ 0.4 and < 0.62), or non-producers (NP:OD < 0.4). The analysis of the levels of pigment extracted revealed thatfour strains were STX negative, other four isolates produced orange (A450 :0.44 -1.03) pigment and the rest of the strains showed yellow (A450: 0.28 -0.43) pigment. The NP biofilm isolates expressed only yellow levels of STX.However, almost 30% of the P and HP biofilm strains were STX negative. A coupleof genotypically identical (CC5, ST5, t002, agr type II) isolates that showed differences in pigment and biofilmproduction were selected for bioinformatical analysis. The SigB factorsequences were 100% identical among this pair of isolates whereas a singleaminoacidic change at the 108 position of the crtM gene was observed in the STX negative producer strainunder comparison. Taken together, these results indicate that STX production (orangepigment) correlates with increased biofilm formation in S. aureus isolates from patients with chronicosteomyelitis.  However, a low percentageof the P and HP biofilm isolates were unable to produce orange or yellowpigments suggesting the presence of a genetic defect in the crt operon.