Maria do Carmo de Freire Bastos

Detection of ileS-2 gene encoding mupirocin resistance in methicillin-resistant Staphylococcus aureus by multiplex PCR

The presence of the ileS-2 gene, responsible for mupirocin resistance, in clinical isolates of methicillin-resistant Staphylococcus aureus was determined by multiplex polymerase chain reaction. Three pairs of primers were used, which yielded specific fragments of femA (encoding a unique feature of S. aureus), mecA (encoding resistance to methicillin) and ileS-2 genes. The multiplex polymerase chain reaction system is an easy and time-saving technique that, together with a rapid method for DNA extraction by boiling, may be incorporated as a routine analysis in clinical diagnostic laboratories.

Biochemical characterisation and genetic analysis of aureocin A53, a new, atypical bacteriocin from Staphylococcus aureus.

Aureocin A53 is produced by Staphylococcus aureus A53. It is encoded on a 10.4 kb plasmid, pRJ9, and is active against Listeria monocytogenes. Aureocin A53 is a highly cationic 51-residue peptide containing ten lysine and five tryptophan residues. Aureocin A53 was purified to homogeneity by hydrophobic-interaction, cation-exchange, and reverse-phase chromatography. MALDI-TOF mass spectrometry yielded a molecular mass of 6012.5 Da, which was 28 Da higher than predicted from the structural gene sequence of the bacteriocin. The mass increment resulted from an N-formylmethionine residue, indicating that the aureocin A53 is synthesised and secreted without a typical bacteriocin leader sequence or sec-dependent signal peptide. The structural identity of aureocin A53 was verified by Edman sequencing after de-blocking with cyanogen bromide and extensive mass spectrometry analysis of enzymatically and laser-generated fragments. The complete sequence of pRJ9 was determined and none of the open reading frames identified in the vicinity of the structural gene aucA showed similarity to genes that are typically found in bacteriocin gene clusters. Thus, neither a dedicated protease or transporter, nor modifying enzymes and regulatory elements seemed to be involved in the production of aureocin A53. Further unique features that distinguish aureocin A53 from other peptide bacteriocins include remarkable protease stability and a defined, rigid structure in aqueous solution.

Staphylococcus haemolyticus as an Important Hospital Pathogen and Carrier of Methicillin Resistance Genes

ABSTRACT Phenotypic and molecular methods were used to characterize the antibiotic resistance of 64 clinical isolates of Staphylococcus haemolyticus. By PCR of the mecA gene, 87% were found to be methicillin resistant. Approximately 55% harbored staphylococcal cassette chromosome mec element (SCCmec) type V, and only one SCCmec type IV. Many isolates (75%) displayed multiresistance, and pulsotype analysis showed a high diversity.

Lysostaphin: A Staphylococcal Bacteriolysin with Potential Clinical Applications.

Lysostaphin is an antimicrobial agent belonging to a major class of antimicrobial peptides and proteins known as the bacteriocins. Bacteriocins are bacterial antimicrobial peptides which generally exhibit bactericidal activity against other bacteria. Bacteriocin production is a self-protection mechanism that helps the microorganisms to survive in their natural habitats. Bacteriocins are currently distributed into three main classes. Staphylococcins are bacteriocins produced by staphylococci, which are Gram-positive bacteria of medical and veterinary importance. Lysostaphin is the only class III staphylococcin described so far. It exhibits a high degree of antistaphylococcal bacteriolytic activity, being inactive against bacteria of all other genera. Infections caused by staphylococci continue to be a problem worldwide not only in healthcare environments but also in the community, requiring effective measures for controlling their spread. Since lysostaphin kills human and animal staphylococcal pathogens, it has potential biotechnological applications in the treatment of staphylococcal infections. In vitro and in vivo studies performed with lysostaphin have shown that this staphylococcin has potential to be used, solely or in combination with other antibacterial agents, to prevent or treat bacterial staphylococcal infectious diseases.

Antimicrobial resistance and plasmid profiles of Campylobacter jejuni and Campylobacter coli from human and animal sources

Aims: The purpose of this study was to determine the susceptibility of Campylobacter jejuni and Campylobacter coli isolates to antimicrobial agents and to investigate the presence of plasmid DNA.

Molecular Characterization and Transfer Among Staphylococcus Strains of a Plasmid Conferring High-Level Resistance to Mupirocin

Abstract In this work, mupirocin resistance was correlated with the presence of plasmids in methicillin-resistant Staphylococcusaureus (MRSA) strains isolated in the Rio de Janeiro Federal University Hospital in Brazil, where topical mupirocin has been used extensively since 1990. Of 19 strains studied, those exhibiting high-level resistance carried a large and relaxable plasmid of about 35 kb. Mupirocin-sensitive derivatives, obtained by growth at 42  °C of a strain exhibiting high-level resistance, were devoid of the large plasmid, which was designated pMG1. Mupirocin resistance was transferred to strain RN8411 during overnight filter-matings at low frequencies (7.0×10–9/donor). The pMG1 plasmid was shown to be responsible for high-level mupirocin resistance in our isolates and to be incompatible with pGO1. Hybridization experiments suggested that mupirocin resistance in pMG1 is due to the presence of the ileS-2 gene. The pMG1 plasmid was successfully and bidirectionally transferred from Staphylococcus aureus to Staphylococcusepidermidis, suggesting that the latter may be a reservoir of this resistance plasmid. No transfer was detected to Staphylococcushaemolyticus. The development of self-transferable high-level mupirocin resistance should be considered when using mupirocin to control the spread of MRSA in hospitals.

The gene bap, involved in biofilm production, is present in Staphylococcus spp. strains from nosocomial infections

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.

Bacteriocins as alternative agents for control of multiresistant staphylococcal strains

Aims:  To investigate the activity of seven staphylococcins, bacteriocins produced by staphylococci, against multiresistant Staphylococcus aureus and coagulase‐negative staphylococci (CNS) involved in human infections.

Identification of coagulase-negative staphylococci from bovine mastitis using RFLP-PCR of the groEL gene

Coagulase-negative staphylococci (CNS) have become the predominant pathogens causing bovine mastitis in many countries. CNS infections are associated with damage to milk secretory tissue of the mammary gland by increased connective tissue stroma, moderate increases of somatic cells count in milk and significant production decreases. These consequences impose serious economic losses for the farmers and the dairy industry. Routine veterinary laboratories do not usually identify CNS at the species level. Thereby, the aims of this study were to identify the most common staphylococcal pathogens involved in bovine mastitis using PCR-restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and to compare our results with the identification carried out by the conventional method. A total of 54 isolates of Staphylococcus, involved in bovine mastitis, were analyzed by this method. The size and number of the fragments obtained by either AluI or HindIII/PvuII digestions made possible to form clear patterns differentiating, among the isolates, 11 of the most common species of animal staphylococcal pathogens. Most of the isolates clustered together with the reference strain of Staphylococcus chromogenes (28) and the type strain of Staphylococcus epidermidis (8). Besides, some isolates clustered together with the type strain of Staphylococcus aureus (5). All patterns were confirmed by the conventional biochemical method, showing concordant results. Thus, the PCR-RFLP of the groEL gene constitutes a reliable and reproducible molecular method for identification of CNS species responsible for bovine mastitis.

Mode of action of the antimicrobial peptide aureocin A53 from Staphylococcus aureus.

ABSTRACT We investigated the mode of action of aureocin A53 on living bacterial cells and model membranes. Aureocin A53 acted bactericidally against Staphylococcus simulans 22, with >90% of the cells killed within a few minutes. Cell death was followed by lysis, as indicated by a clearing of the cell suspension and Gram staining. Aureocin A53 rapidly dissipated the membrane potential and simultaneously stopped biosynthesis of DNA, polysaccharides, and protein. Aureocin A53 induced a rapid release of preaccumulated glutamate and Rb+. Experiments on model membranes demonstrated that aureocin A53 provoked significant leakage of carboxyfluorescein (CF) exclusively from acidic liposomes but only at relatively high concentrations (0.5 to 8 mol%). Thus, the bactericidal activity of aureocin A53 derives from membrane permeation via generalized membrane destruction rather than by formation of discrete pores within membranes. Tryptophan emission fluorescence spectroscopy demonstrated interaction of aureocin A53 with both acidic and neutral membranes, as indicated by similar blue shifts. Since there was no significant aureocin A53-induced CF leakage from neutral liposomes, its appears that the peptide does interact with neutral lipids without provoking membrane damage.