Excision of staphylococcal cassette chromosome mec in methicillin-resistant Staphylococcus aureus assessed by quantitative PCR.

BACKGROUND: Methicillin-resistance in staphylococci is conferred by the mecA gene, located on the genomic island Staphylococcal Cassette Chromosome mec (SCCmec). SCCmec mobility relies on the Ccr recombinases, which catalyze insertion and excision form the host's chromosome. Although being a crucial step in its horizontal transfer, little is known about the dynamics of SCCmec excision. RESULTS: A quantitative PCR-based method was used to measure the rate of SCCmec excision by amplifying the chromosome-chromosome junction and the circularized SCCmec resulting from excision. SCCmec excision rate was measured in methicillin-resistant Staphylococcus aureus (MRSA) strain N315 at various growth times in broth cultures. In the present experimental settings, excision of SCCmec occurred at a rate of approximately 2 × 10(-6) in MRSA N315. CONCLUSION: This work brings new insights in the poorly understood SCCmec excision process. The results presented herein suggest a model in which excision occurs during a limited period of time at the early stages of growth

Expression of SCCmec cassette chromosome recombinases in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

Objectives Methicillin resistance in staphylococci is mediated by the mecA gene, which is carried on the staphylococcal cassette chromosome mec (SCCmec). SCCmec is responsible for vertical and horizontal transfer of methicillin resistance. Horizontal transfer implies first SCCmec excision from the chromosome. Site-specific excision is catalysed by the Ccr recombinases, which are encoded by ccrAB genes located on the cassette. The aim of this study is to determine the promoter activity of ccrAB genes in individual cells of methicillin-resistant Staphylococcus aureus (N315, COL and MW2) and Staphylococcus epidermidis (RP62A). One mutant cured of its SCCmec (N315EX) was also used. Exposure to various stresses was included in the study. Methods For each strain, translational promoter-green fluorescent protein (gfp) fusions were used to assess the levels of ccr promoter activity in individual cells. Analyses were performed using epifluorescence microscopy and flow cytometry. Results ccr promoter activity was observed in only a small percentage of cell populations. This ‘bistable' phenotype was strain dependent (GFP was expressed in N315 and RP62A, but not in COL and MW2) and growth dependent (GFP-expressing cells decreased from approximately 3% to 1% between logarithmic and stationary growth phases). The ccr promoter of strain N315 displayed normal promoter activity when expressed in SCCmec-negative N315EX. Likewise, the ccr promoter of strain COL (which was inactive in COL) showed normal N315-like activity when transformed into N315 and N315EX. Conclusions SCCmec excision operates through bistability, favouring a small fraction of cells to ‘sacrifice' their genomic islands for transfer, while the rest of the population remains intact. Determinants responsible for the activity of the ccr promoter were not located on SCCmec, but were elsewhere on the genome. Thus, the staphylococcal chromosome plays a key role in determining SCCmec stability and transferabilit

Comparison of Single Doses of Amoxicillin or of Amoxicillin-Gentamicin for the Prevention of Endocarditis Caused by Streptococcus faecalis and by Viridans Streptococci

Recent recommendations for the prophylaxis of endocarditis in humans have advocated single doses or short courses of antibiotic combinations (β-lactam plus aminoglycoside) for susceptible patients in whom enterococcal bacteremia might develop or for patients at especially high risk of developing endocarditis (e.g., patients with prosthetic cardiac valves). We tested the prophylactic efficacy (in rats with catheter-induced aortic vegetations) of single doses of amoxicillin plus gentamicin against challenge with various streptococcal strains (two strains of Streptococcus faecalis, one of Streptococcus bovis, and three of viridans streptococci); we then compared this efficacy with that of single doses of amoxicillin alone. Successful prophylaxis against all six strains was achieved with single doses of both amoxicillin alone and amoxicillin plus gentamicin. This protection, however, was limited, for both regimens, to the lowest bacterial-inoculum size producing endocarditis in 90% of control rats and was not extended to higher inocula by using the combination of antibiotics. We concluded that a single dose of amoxicillin alone was protective against enterococcal and nonenterococcal endocarditis in the rat, but that its efficacy was limited and could not be improved by the simultaneous administration of gentamici

Loss of penicillin tolerance by inactivating the carbon catabolite repression determinant CcpA in Streptococcus gordonii

Objectives Antibiotic tolerance is a phenomenon allowing bacteria to withstand drug-induced killing. Here, we studied a penicillin-tolerant mutant of Streptococcus gordonii (Tol1), which was shown to be deregulated in the expression of the arginine deiminase operon (arc). arc was not directly responsible for tolerance, but is controlled by the global regulator CcpA. Therefore, we sought whether CcpA might be implicated in tolerance. Methods The ccpA gene was characterized and subsequently inactivated by PCR ligation mutagenesis in both the susceptible wild-type (WT) and Tol1. The minimal inhibitory concentration and time-kill curves for the strains were determined and the outcome of penicillin treatment in experimental endocarditis assessed. Results ccpA sequence and expression were similar between the WT and Tol1 strains. In killing assays, the WT lost 3.5 ± 0.6 and 5.3 ± 0.6 log10 cfu/mL and Tol1 lost 0.4 ± 0.2 and 1.4 ± 0.9 log10 cfu/mL after 24 and 48 h of penicillin exposure, respectively. Deletion of ccpA almost totally restored Tol1 kill susceptibility (loss of 2.5 ± 0.7 and 4.9 ± 0.7 log10 cfu/mL at the same endpoints). In experimental endocarditis, penicillin treatment induced a significant reduction in vegetation bacterial densities between Tol1 (4.1 log10 cfu/g) and Tol1ΔccpA (2.4 log10 cfu/g). Restitution of ccpA re-established the tolerant phenotype both in vitro and in vivo. Conclusions CcpA, a global regulator of the carbon catabolite repression system, is implicated in penicillin tolerance both in vitro and in vivo. This links antibiotic survival to bacterial sugar metabolism. However, since ccpA sequence and expression were similar between the WT and Tol1 strains, other factors are probably involved in toleranc

Experimental Bacterial Endocarditis After Dental Extractions in Rats with Periodontitis

The development of bacterial endocarditis was analyzed after dental extractions in rats with or without periodontal disease. Periodontal disease was produced in rats by tying silk ligatures around the two maxillary first molars and placing the animals on a high sucrose diet for 14 weeks. Sterile aortic valve vegetations were produced by means of a transaortic catheter, and 24 hr later the maxillary first molars were extracted. The animals were killed 72 hr after the extractions. In rats with periodontal disease, extractions resulted in a 48% (14 of 29) incidence of bacterial endocarditis, most cases of which were due to Streptococcus spp. (one was caused by Staphylococcus aureus). In contrast, when the teeth with a healthy periodontium were extracted, only 6% (one of 15) of the rats developed endocarditis. When catheters were placed in anim, tis with periodontal disease but no extractions were performed, no endocarditis occurre

Les nouvelles lignes directrices du European Network of Forensic Sciences Institut en matière d’évaluation et de communication des résultats d’analyses et d’expertises scientifiques

Au printemps 2015, le European Network of Forensic Science Institutes a publié des lignes directrices visant à unifier les méthodes de travail des scientifiques agissant comme auxiliaires de la justice, plus précisément l’évaluation des résultats d’analyses scientifiques (logique du raisonnement) et la communication de ces résultats aux magistrats. Cette contribution a pour but d’expliciter ces lignes directrices et d’expliquer en quoi il est primordial que la justice suisse les adopte. — Im Frühjahr 2015 publizierte das ENFSI (European Network of Forensic Science Institutes) Leitlinien für die Vereinheitlichung wissenschaftlicher Arbeitsmethoden, insbesondere für Bewertung von wissenschaftlichen Untersuchungsergebnissen (Logik der Befundbewertung) und die Berichterstattung zuhanden der Justiz. Dieser Beitrag erläutert diese Richtlinien und erklärt, weshalb es wichtig ist, dass sie in die Schweizer Justizpraxis Eingang finden

New concepts in the pathophysiology of infective endocarditis

Endocarditis pathogens colonize valves with pre-existing sterile vegetations or valves with minimal endothelial lesions. Inflamed endothelia produce cytokines, integrins, and tissue factor, which in turn attract fibronectin, monocytes, and platelets. Bacteria attaching to such structures further activate the cascade, becoming embedded and protected from host defenses. Staphylococcus aureus also actively invade the endothelium, causing apoptosis and endothelial damage. Knowledge of this interplay identifies host factors as potential therapeutic targets. Blocking infection by modulating host factors might be opportune because host factors are conserved. In contrast, interfering with bacterial virulence factors might be more complicated because they vary among different bacteri

β-Lactam Resistance Mechanisms of Methicillin-Resistant Staphylococcus aureus

In vitro and in vivo activity of amoxicillin and penicillin G alone or combined with a penicillinase inhibitor (clavulanate) were tested against five isogenic pairs of methicillin-resistant Staphylococcus aureus (MRSA) producing or not producing penicillinase. Loss of the penicillinase plasmid caused an eight times or greater reduction in the MICs of amoxicillin and penicillin G (from ⩾64 to 8 µg/ml), but not of the penicillinase-resistant drugs methicillin and cloxacillin (⩾64 µg/ml). This difference in antibacterial effectiveness correlated with a more than 10 times greater penicillin-binding protein 2a affinity of amoxicillin and penicillin G than of methicillin and a ⩾90% successful amoxicillin treatment of experimental endocarditis due to penicillinasenegative MRSA compared with cloxacillin, which was totally ineffective (P < .001). Amoxicillin was also effective against penicillinase-producing parent MRSA, provided it was combined with clavulanate. Penicillinase-sensitive β-lactam antibiotics plus penicillinase inhibitors might offer a rational alternative treatment for MRSA infection