This study analyzed ten strains of coagulase-negative staphylococci (CNS) involved in nosocomial infections in three Brazilian hospitals. Their antibiotic susceptibility profile showed that most strains exhibited multiple antibiotic resistance and possessed the mecA gene. The ability of these strains to adhere to polystyrene microtiter plates was also tested and nine of them proved to be biofilm producers at least in one of the three conditions tested: growth in TSB, in TSB supplemented with NaCl, or in TSB supplemented with glucose. The presence of the bap gene, which codes for the biofilm-associated protein (Bap), was investigated in all ten strains by PCR. AU strains were bop-positive and DNA sequencing experiments confirmed that the fragments amplified were indeed part of a bap gene. The presence of the icaA gene, one of the genes involved in polysaccharide intercellular adhesin (PIA) formation, was also detected by PCR in eight of the ten strains tested. The two icaA-negative strains were either weak biofilm producer or no biofilm producer, although they were bop-positive. To our knowledge, this is the first report demonstrating the presence of the bap gene in nosocomial isolates of CNS, being also the first report on the presence of this gene in Staphylococcus haemolyticus and S. cohnii.

Aureocin A53 is an antimicrobial peptide produced by Staphylococcus aureus A53. The genetic determinants involved in aureocin A53 production and immunity to its action are organized in at least four transcriptional units encoded by the 10.4-kb plasmid pRJ9. One transcriptional unit carries only the bacteriocin structural gene, aucA. No immunity gene is found downstream of aucA, as part of the same transcriptional unit. Further downstream of aucA is found an operon which contains the three genes aucEFG, whose products seem to associate to form a dedicated ABC transporter. When aucEFG were expressed in RN4220, an aureocin A53-sensitive S. aureus strain, this strain became partially resistant to the bacteriocin. A gene disruption mutant in aucE was defective in aureocin A53 externalization and more sensitive to aureocin A53 than the wild-type strain, showing that aucEFG are involved in immunity to aureocin A53 by active extrusion of the bacteriocin. Full resistance to aureocin A53 was exhibited by transformants carrying, besides aucEFG, the operon formed by two genes, aucIB and aucIA, located between aucA and aucEFG and carried in the opposite strand. AucIA and AucIB share similarities with hypothetical proteins not found in the gene clusters of other bacteriocins. A gene disruption mutant in orf8, located upstream of aucA and whose product exhibits about 50% similarity to a number of hypothetical membrane proteins found in many Gram-positive bacteria, was strongly affected in aureocin A53 externalization but resistant to aureocin A53, suggesting that Orf8 is also involved in aureocin A53 secretion.

The action of lysostaphin, either alone or in combination with nisin, against established staphylococcal biofilm may represent an alternative to bovine mastitis control. However, the duration of the treatment should be considered for its application so that the best effectiveness can be achieved.

Staphylococcus aureus 4185 was previously shown to produce at least two bacteriocins. One of them is encoded by pRJ101. To detect the bacteriocin-encoding gene cluster, an~9160 kb region of pRJ101 was sequenced. In silico analyses identified 10 genes (aclX, aclB, aclI, aclT, aclC, aclD, aclA, aclF, aclG and aclH) that might be involved in the production of a novel cyclic bacteriocin named aureocyclicin 4185. The organization of these genes was quite similar to that of the gene cluster responsible for carnocyclin A production and immunity. Four putative proteins encoded by these genes (AclT, AclC, AclD and AclA) also exhibited similarity to proteins encoded by cyclic bacteriocin gene clusters. Mutants derived from insertion of Tn917-lac into aclC, aclF, aclH and aclX were affected in bacteriocin production and growth. AclX is a 205 aa putative protein not encoded by the gene clusters of other cyclic bacteriocins. AclX exhibits 50 % similarity to a permease and has five putative membrane-spanning domains. Transcription analyses suggested that aclX is part of the aureocyclicin 4185 gene cluster, encoding a protein required for bacteriocin production. The aclA gene is the structural gene of aureocyclicin 4185, which shows 65 % similarity to garvicin ML. AclA is proposed to be cleaved off, generating a mature peptide with a predicted M r of 5607 Da (60 aa). By homology modelling, AclA presents four a-helices, like carnocyclin A. AclA could not be found at detectable levels in the culture supernatant of a strain carrying only pRJ101. To our knowledge, this is the first report of a cyclic bacteriocin gene cluster in the genus Staphylococcus.

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