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

A positive interaction between inhibitors of protein synthesis and cefepime in the fight against methicillin-resistant Staphylococcus aureus

Quinupristin-dalfopristin (Q-D) synergizes with cefepime for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Here, we studied whether the synergism was restricted to MRSA and if it extended to non-beta-lactam cell wall inhibitors or to other inhibitors of protein synthesis. Three MRSA and two methicillin-susceptible S. aureus (MSSA) strains were tested, including an isogenic pair of mecA −/mecA + S. aureus Newman. The drug interactions were determined by fractional inhibitory concentration (FIC) indices and population analysis profiles. The antibacterial drugs that we used included beta-lactam (cefepime) and non-beta-lactam cell wall inhibitors (D-cycloserine, fosfomycin, vancomycin, teicoplanin), inhibitors of protein synthesis (Q-D, erythromycin, chloramphenicol, tetracycline, linezolid, fusidic acid), and polynucleotide inhibitors (cotrimoxazole, ciprofloxacin). The addition of each protein inhibitor to cefepime was synergistic (FIC ≤ 0.5) or additive (FIC > 0.5 but < 1) against MRSA, but mostly indifferent against MSSA (FIC ≥ 1 but ≤ 4). This segregation was not observed after adding cotrimoxazole or ciprofloxacin to cefepime. Population analysis profiles were performed on plates in the presence of increasing concentrations of the cell wall inhibitors plus 0.25 × minimum inhibitory concentration (MIC) of Q-D. Cefepime combined with Q-D was synergistic against MRSA, but D-cycloserine and glycopeptides were not. Thus, the synergism was specific to beta-lactam antibiotics. Moreover, the synergism was not lost against fem mutants, indicating that it acted at another level. The restriction of the beneficial effect to MRSA suggests that the functionality of penicillin-binding protein 2A (PBP2A) was affected, either directly or indirectly. Further studies are necessary in order to provide a mechanism for this positive interactio

Efficacy of daptomycin in the treatment of experimental endocarditis due to susceptible and multidrug-resistant enterococci

Objectives: Daptomycin was tested in vitro and in rats with experimental endocarditis against the ampicillin-susceptible and vancomycin-susceptible Enterococcus faecalis JH2-2, the vancomycin-resistant (VanA type) mutant of strain JH2-2 (strain JH2-2/pIP819), and the ampicillin-resistant and vancomycin-resistant (VanB type) Enterococcus faecium D366. Methods: Rats with catheter-induced aortic vegetations were treated with doses simulating intravenously kinetics in humans of daptomycin (6 mg/kg every 24 h), amoxicillin (2 g every 6 h), vancomycin (1 g every 12 h) or teicoplanin (12 mg/kg every 12 h). Treatment was started 16 h post-inoculation and continued for 2 days. Results: MICs of daptomycin were 1, 1 and 2 mg/L, respectively, for strains JH2-2, JH2-2/pIP819 and D366. In time-kill studies, daptomycin showed rapid (within 2 h) bactericidal activity against all strains. Daptomycin was highly bound to rat serum proteins (89%). In the presence of 50% rat serum, simulating free concentrations, daptomycin killing was maintained but delayed (6-24 h). In vivo, daptomycin treatment resulted in 10 of 12 (83%), 9 of 11 (82%) and 11 of 12 (91%) culture-negative vegetations in rats infected with strains JH2-2, JH2-2/pIP819 and D366, respectively (P < 0.001 compared to controls). Daptomycin efficacy was comparable to that of amoxicillin and vancomycin for susceptible isolates. Daptomycin, however, was significantly (P < 0.05) more effective than teicoplanin against the glycopeptide-susceptible strain JH2-2 and superior to all comparators against resistant isolates. Conclusions: These results support the use of the newly proposed daptomycin dose of 6 mg/kg every 24 h for treatment of enterococcal infections in human

In vitro activities of tigecycline combined with other antimicrobials against multiresistant Gram-positive and Gram-negative pathogens

Objectives To test the activity of tigecycline combined with 16 antimicrobials in vitro against 22 Gram-positive and 55 Gram-negative clinical isolates. Methods Antibiotic interactions were determined by chequerboard and time-kill methods. Results By chequerboard, of 891 organism-drug interactions tested, 97 (11%) were synergistic, 793 (89%) were indifferent and 1 (0.1%) was antagonistic. Among Gram-positive pathogens, most synergisms occurred against Enterococcus spp. (7/11 isolates) with the tigecycline/rifampicin combination. No antagonism was detected. Among Gram-negative organisms, synergism was observed mainly with trimethoprim/sulfamethoxazole against Serratia marcescens (5/5 isolates), Proteus spp. (2/5) and Stenotrophomonas maltophilia (2/5), with aztreonam against S. maltophilia (3/5), with cefepime and imipenem against Enterobacter cloacae (3/5), with ceftazidime against Morganella morganii (3/5), and with ceftriaxone against Klebsiella pneumoniae (3/5). The only case of antagonism occurred against one S. marcescens with the tigecycline/imipenem combination. Selected time-kill assays confirmed the bacteriostatic interactions observed by the chequerboard method. Moreover, they revealed a bactericidal synergism of tigecycline with piperacillin/tazobactam against one penicillin-resistant Streptococcus pneumoniae and with amikacin against Proteus vulgaris. Conclusions Combinations of tigecycline with other antimicrobials produce primarily an indifferent response. Specific synergisms, especially against enterococci and problematic Gram-negative isolates, might be worth investigating in in vitro models and/or in animal models simulating the human environmen

Antibiotic Treatment of Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus epidermidis

The natural history and treatment of experimental endocarditis due to heterogeneous and homogeneous methicillin-resistant Staphylococcus epidermidis was investigated. Amoxicillin/clavulanate or vancomycin were administered for 3 days via a computerized pump to mimic human drug kinetics in animals. After challenge with the minimum inoculum producing 90% of infections (ID90) , bacteria in the vegetations grew logarithmically for 16 h. Then, bacterial densities stabilized (at ∼108 cfu/g) and growth rates sharply declined. Both regimens cured ⩾60% of endocarditis (due to heterogeneous or homogeneous bacteria) when started 12-16 h after infection, although the bacterial densities in the vegetations had increased by 20 times in between. In contrast, treatment started after 24 h failed in most animals, while bacterial densities had not increased any more. Thus, while both regimens were equivalent, the therapeutic outcome was best predicted by growth rates in the vegetations, not by bacterial densities. These observations highlight the importance of phenotypic tolerance developing in viv

Successful Single-Dose Amoxicillin Prophylaxis Against Experimental Streptococcal Endocarditis: Evidence for Two Mechanisms of Protection

Amoxicillin prophylaxis against experimental endocarditis due to one nontolerant and two tolerant strains of streptococci was studied in rats. Single-dose amoxicillin protected against the two tolerant strains in animals challenged with the 90% infective dose (ID90), but protection diminished with increasing inoculum sizes. Protection against the nontolerant strain was successful with inocula that were 100-and I,OOO-fold larger than the ID90. Close correlation existed between the speed of bacterial killing in vitro, the time of exposure to bactericidal levels in vivo, and the range of inocula against which prophylaxis was effective. Amoxicillin seemed to protect by at least two mechanisms. (1) When in vitro tests indicated adequate bacterial killing, protection was independent of the inoculum size and was probably conferred by bacterial killing. (2) When in vitro tests indicated bacterial inhibition but not. killing, protection was inoculum-dependent and was probably mediated by inhibition of bacterial adherenc