Daptomycin Resistance and Tolerance Due to Loss of Function in Staphylococcus aureus dsp1 and asp23.

Lipopeptide daptomycin is a last-line cell-membrane-targeting antibiotic to treat multidrug-resistant Staphylococcus aureus Alarmingly, daptomycin-resistant S. aureus isolates have emerged. The mechanisms underlying daptomycin resistance are diverse and share similarities with resistances to cationic antimicrobial peptides and other lipopeptides, but they remain to be fully elucidated. We selected mutants with increased resistance to daptomycin from a library of transposon insertions in sequent type 8 (ST8) S. aureus HG003. Insertions conferring increased daptomycin resistance were localized to two genes, one coding for a hypothetical lipoprotein (SAOUHSC_00362, Dsp1), and the other for an alkaline shock protein (SAOUHSC_02441, Asp23). Markerless loss-of-function mutants were then generated for comparison. All transposon mutants and knockout strains exhibited increased daptomycin resistance compared to those of wild-type and complemented strains. Null and transposon insertion mutants also exhibited increased resistance to cationic antimicrobial peptides. Interestingly, the Δdsp1 mutant also showed increased resistance to vancomycin, a cell-wall-targeting drug with a different mode of action. Null mutations in both dsp1 and asp23 resulted in increased tolerance as reflected by reduced killing to both daptomycin and vancomycin, as well as an increased tolerance to surfactant (Triton X-100). Neither mutant exhibited increased resistance to lysostaphin, a cell-wall-targeting endopeptidase. These findings identified two genes core to the S. aureus species that make previously uncharacterized contributions to antimicrobial resistance and tolerance in S. aureus.

Phenotypic and genotypic characterization of biofilm formation in Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most frequently isolated coagulase-negative staphylococci. The ability to produce biofilm has contributed to its emergence as a nosocomial pathogen. In this study, some growth conditions were tested to determine their influence on biofilm formation. Brain-heart infusion (BHI) broth containing glucose was used to screen 64 clinical strains. A strong biofilm producer strain showed cells surrounded by a thick layer of extracellular matrix. The presence of atlE, fbp, bap, and icaA genes was analyzed. We concluded that S. haemolyticus biofilm production can be increased with cells grown in BHI, and highlighted that it could be an ica-independent process.

shsA: A novel orthologous of sasX/sesI virulence genes is detected in Staphylococcus haemolyticus Brazilian strains.

The surface protein SasX, has a key role in methicillin-resistant Staphylococcus aureus (MRSA) colonization and pathogenesis, and has been associated with the epidemic success of some MRSA clones. To date, only one SasX homologous protein, named SesI, has been described in Staphylococcus epidermidis. In this work, we analyze the occurrence of the sasX gene and its genetic environment in Staphylococcus haemolyticus S. haemolyticus clinical strains (n = 62) were screened for the presence of the sasX gene and its carrier, the prophage Φ SPß-like. A deep characterization was done in one strain (MD43), through which we determined the complete nucleotide sequence for the S. haemolitycus sasX-like gene. Whole genome sequencing of strain MD43 was performed, and the gene, termed here because of its unique attributes, shsA, was mapped to the Φ SPß-like prophage sequence. The shsA gene was detected in 33 out of 62 strains showing an average identity of 92 and 96% with the sasX and sesI genes and at the amino acid level, 88% identity with SasX and 92% identity with SesI. The ~124Kb Φ SPß-like prophage sequence showed a largely intact prophage compared to its counterpart in S. epidermidis strain RP62A, including the sesI insertion site. In conclusion, we identified a new sasX ortholog in S. haemolyticus (shsA). Its horizontal spread from this reservoir could represent an emergent threat in healthcare facilities since so far, no S. aureus sasX+ strains have been reported in Brazil.

Methicillin-resistant Staphylococcus epidermidis carrying biofilm formation genes: detection of clinical isolates by multiplex PCR.

Staphylococcus epidermidis is the most prevalent coagulase-negative Staphylococcus (CNS) and is a major cause of hospital bacteremia. Based on 18 reference strains and 149 Staphylococcus clinical strains, used in a novel multiplex PCR method, the aim of this study was to identify S. epidermidis with respect to the sequence of three genes: recN, which encodes a recombination/repair protein, mecA (methicillin resistance), and icaAB, which is involved in biofilm formation. Amplicons of 219 bp (S. epidermidis-recN gene), 154 bp (mecA gene), and 546 bp (icaAB genes) were obtained. Reliable results were achieved for 100% of the evaluated strains, suggesting that this new multiplex-PCR approach could be useful for the accurate identification of methicillin-resistant S. epidermidis with the potential to produce biofilm.

Identification of coagulase-negative Staphylococcus saprophyticus by polymerase chain reaction based on the heat-shock repressor encoding hrcA gene.

Staphylococcus saprophyticus is an uropathogen belonging to the human microbiota and is responsible for community-acquired infections of the urinary tract. Identification of Staphylococcus species by biochemical tests is laborious and costly when compared to routine laboratory tests. Because of their high sensitivity and specificity, molecular methods are better suited for accurate identification of Staphylococcusspp. Therefore, the goal of this work was to standardize a polymerase chain reaction (PCR) protocol using species-specific primers, based on the heat-shock repressor coding hrcA gene, for the identification of S.saprophyticus. A total of 142 S. saprophyticus strains were obtained from different sources, including clinical, environmental, and foodborne strains. We also included 98 strains of Staphylococcus spp. to further validate the proposed method. Reliable results for the detection of S. saprophyticus isolates were obtained for 100% of the strains evaluated. The results were in accordance with matrix-assisted laser desorption ionization-time of flight mass spectrometry identification, thus highlighting the applicability of species-specific PCR for the molecular identification of S. saprophyticus.

Daptomycin and methicillin-resistant Staphylococcus aureus isolated from a catheter-related bloodstream infection: a case report.

BACKGROUND: Daptomycin is an alternative option for the treatment of catheter-related bloodstream-infections caused by methicillin-resistant Staphylococcus aureus. This study reports a case of a daptomycin and methicillin-resistant Staphylococcus aureus isolate recovered from the blood of a Brazilian patient undergoing hemodialysis. CASE PRESENTATION: A 64-year-old white male patient suffering from diabetes mellitus, systolic hypertension, heart disease with a coronary stent, obesity and chronic renal failure and on use of permcath catheter developed a catheter-related bloodstream-infection by a daptomycin-methicillin-resistant Staphylococcus aureus isolate after one month of daptomycin therapy. The isolate was identified as the SCCmec II/USA100/sequence type 5 lineage by molecular techniques. CONCLUSIONS: In this work we described a Brazilian patient with bloodstream infection caused by a daptomycin and methicillin-resistant Staphylococcus aureus belonging to the lineage USA100/sequence type 5. Our case highlights the careful management of bloodstream infections and the importance of the judicious use of antimicrobials due the possibility of daptomycin-resistance developing among S. aureus isolates, especially in patients under hemodialysis, which are frequently exposed to vancomycin and daptomycin therapy.