In Vitro Activity of Iclaprim against Methicillin-Resistant Staphylococcus aureus Nonsusceptible to Daptomycin, Linezolid, or Vancomycin: A Pilot Study.

Iclaprim is a bacterial dihydrofolate reductase inhibitor in Phase 3 clinical development for the treatment of acute bacterial skin and skin structure infections and hospital-acquired bacterial pneumonia caused by Gram-positive bacteria. Daptomycin, linezolid, and vancomycin are commonly used antibiotics for these indications. With increased selective pressure to these antibiotics, outbreaks of bacterial resistance to these antibiotics have been reported. This in vitro pilot study evaluated the activity of iclaprim against methicillin-resistant Staphylococcus aureus (MRSA) isolates, which were also not susceptible to daptomycin, linezolid, or vancomycin. Iclaprim had an MIC ≤ 1 µg/ml to the majority of MRSA isolates that were nonsusceptible to daptomycin (5 of 7 (71.4%)), linezolid (26 of 26 (100%)), or vancomycin (19 of 28 (66.7%)). In the analysis of time-kill curves, iclaprim demonstrated ≥ 3 log10 reduction in CFU/mL at 4-8 hours for tested strains and isolates nonsusceptible to daptomycin, linezolid, or vancomycin. Together, these data support the use of iclaprim in serious infections caused by MRSA nonsusceptible to daptomycin, linezolid, or vancomycin.

Minor groove binders as anti-infective agents.

Minor groove binders are small molecules that form strong complexes with the minor groove of DNA. There are several structural types of which distamycin and netropsin analogues, oligoamides built from heterocyclic and aromatic amino acids, and bis-amidines separated by aromatic and heterocyclic rings are of particular pharmaceutical interest. These molecules have helical topology that approximately matches the curvature of DNA in the minor groove. Depending upon the precise structure of the minor groove binder, selectivity can be obtained with respect to the DNA base sequence to which the compound binds. Minor groove binders have found substantial applications in anti-cancer therapy but their significance in anti-infective therapy has also been significant and is growing. For example, compounds of the bis-amidine class have been notable contributors to antiparasitic therapy for many years with examples such as berenil and pentamidine being well-known. A recent growth area has been inreased sophistication in the oligoamide class. High sequence selectivity is now possible and compounds with distinct antibacterial, antifungal, antiviral, and antiparasitic activity have all been identified. Importantly, the structures of the most active compounds attacking the various infective organisms differ significantly but not necessarily predictively. This poses interesting questions of mechanism of action with many different targets involved in DNA processing being candidates. Access of compounds to specific cell types also plays a role and in some cases, can be decisive. Prospects for a range of selective therapeutic agents from this class of compounds are higher now than for some considerable time.

In vivo efficacy of the antimicrobial peptide ranalexin in combination with the endopeptidase lysostaphin against wound and systemic meticillin-resistant Staphylococcus aureus (MRSA) infections.

New treatments are urgently required for infections caused by meticillin-resistant Staphylococcus aureus (MRSA) as these strains are often resistant to multiple conventional antibiotics. Earlier studies showed that ranalexin, an antimicrobial peptide (AMP), in combination with lysostaphin, an antistaphylococcal endopeptidase, synergistically inhibits the growth of MRSA, meaning that it deserved consideration as a new anti-S. aureus therapy. Using haemolysis and Vero cell viability assays, ranalexin with lysostaphin is proven to be non-toxic at antibacterial concentrations. In human serum, ranalexin with lysostaphin is significantly more effective against MRSA than treatment with either component alone. In a rabbit model of wound infection, ranalexin with lysostaphin reduced MRSA in the wound by ca. 3.5log(10) colony-forming units (CFU) compared with the untreated control. The combination is significantly more effective than treatment with ranalexin or lysostaphin alone. In a mouse model of systemic infection, ranalexin with lysostaphin reduced MRSA kidney burden by ca. 1log(10)CFU/g compared with untreated controls or treatment with ranalexin or lysostaphin alone. Importantly, the combination is synergistically bactericidal against various S. aureus isolates in vitro, including those with reduced susceptibility to lysostaphin or vancomycin. Ranalexin and lysostaphin could be incorporated in wound dressings for the prevention and treatment of topical S. aureus infections. That AMPs can enhance the antibacterial effectiveness of lysostaphin in vivo highlights a new avenue of research in the fight against drug-resistant staphylococci.

Influence of tigecycline on expression of virulence factors in biofilm-associated cells of methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are complicated by the ability of the organism to grow in surface-adhered biofilms on a multitude of abiotic and biological surfaces. These multicellular communities are notoriously difficult to eradicate with antimicrobial therapy. Cells within the biofilm may be exposed to a sublethal concentration of the antimicrobial due to the metabolic and phenotypic diversity of the biofilm-associated cells or the protection offered by the biofilm structure. In the present study, the influence of a sublethal concentration of tigecycline on biofilms formed by an epidemic MRSA-16 isolate was investigated by transcriptome analysis. In the presence of the drug, 309 genes were upregulated and 213 genes were downregulated by more than twofold in comparison to the levels of gene regulation detected for the controls not grown in the presence of the drug. Microarray data were validated by real-time reverse transcription-PCR and phenotypic assays. Tigecycline altered the expression of a number of genes encoding proteins considered to be crucial for the virulence of S. aureus. These included the reduced expression of icaC, which is involved in polysaccharide intercellular adhesin production and biofilm development; the upregulation of fnbA, clfB, and cna, which encode adhesins which attach to human proteins; and the downregulation of the cap genes, which mediate the synthesis of the capsule polysaccharide. The expression of tst, which encodes toxic shock syndrome toxin 1 (TSST-1), was also significantly reduced; and an assay performed to quantify TSST-1 showed that the level of toxin production by cells treated with tigecycline decreased by 10-fold (P < 0.001) compared to the level of production by untreated control cells. This study suggests that tigecycline may reduce the expression of important virulence factors in S. aureus and supports further investigation to determine whether it could be a useful adjunct to therapy for the treatment of biofilm-mediated infections.

Comparison of biofilm-associated cell survival following in vitro exposure of meticillin-resistant Staphylococcus aureus biofilms to the antibiotics clindamycin, daptomycin, linezolid, tigecycline and vancomycin.

The efficacy of commonly used antistaphylococcal antimicrobials (clindamycin, linezolid and vancomycin) and recently developed antibiotics (daptomycin and tigecycline) was compared against clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA). Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations, time-kill kinetics and biofilm-associated cell survival were examined for 12 clinical isolates of MRSA treated with each antibiotic. The MIC ranges for daptomycin, linezolid, tigecycline, clindamycin and vancomycin were 0.06-0.25, 1-2, 0.06, 0.125-1024 and 0.5-1 microg/mL, respectively. Daptomycin and vancomycin were bactericidal following 6h of incubation with planktonic cells, whilst clindamycin, linezolid and tigecycline were bacteriostatic. None of the antibiotics killed 100% of biofilm-associated cells. Mean cell survival in biofilms treated with clindamycin, daptomycin, linezolid, tigecycline and vancomycin was 62%, 4%, 45%, 43% and 19%, respectively. Although all antibiotics were effective against planktonic staphylococcal populations, vancomycin and daptomycin possessed superior activity against biofilm-associated cells.

Biofilm formation by Scottish clinical isolates of Staphylococcus aureus.

The biofilm-forming capacity of 972 clinical isolates of Staphylococcus aureus was tested using a high-throughput polystyrene 96-peg plate format. Isolates of S. aureus were collected from patients in hospitals throughout Scotland from 2004 to 2006; 763 of these were meticillin-resistant S. aureus (MRSA) and 209 were meticillin-sensitive S. aureus (MSSA). The biomass of each biofilm was quantified using a crystal violet staining technique. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The majority of MRSA (53.8 %) and MSSA (43.5 %) isolates formed moderately attached biofilms. Fully established biofilms were formed by 20.5 % of MRSA isolates and 28.0 % of MSSA isolates, whilst 25.7 % of MRSA isolates and 28.5 % of MSSA isolates formed negligible biofilms. There was no significant correlation between susceptibility to meticillin and biofilm formation (P=0.77). MRSA isolates were divided into clonal types (EMRSA-15, EMRSA-16 and sporadic isolates) based on PFGE genotyping results. EMRSA-15 isolates formed significantly more moderately and fully established biofilms than EMRSA-16 isolates (P<0.001). S. aureus strains isolated from the skin of patients had a significantly greater capacity to form biofilms than isolates from other body sites, including the blood. Microscopic examination of biofilms by scanning electron microscopy (SEM) revealed that poorly adherent biofilm formers failed to colonize the entire surface of the peg, whilst moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group that formed fully established biofilms confirmed that this isolate developed a dense biofilm with a textured, multi-layered, three-dimensional structure.

The association between biocide tolerance and the presence or absence of qac genes among hospital-acquired and community-acquired MRSA isolates.

OBJECTIVES: The MBCs of three commonly used hospital biocides [containing quaternary ammonium compounds (QACs), chlorhexidine gluconate and triclosan] were determined for clinical isolates of Staphylococcus aureus, which were also screened for genes encoding Qac efflux pumps. METHODS: MBCs were determined by broth microdilution for 94 clinical isolates of S. aureus, including 38 hospital-acquired methicillin-resistant S. aureus (HA-MRSA), 25 community-associated methicillin-resistant S. aureus (CA-MRSA), 25 methicillin-susceptible S. aureus (MSSA) and 6 with intermediate resistance to vancomycin (VISA). All isolates were screened by PCR for the presence of qacA, B, C, G, H and J. RESULTS: Biocides had MBCs 10-1000-fold lower than the concentration recommended for use by the manufacturer. HA-MRSA isolates developed significantly enhanced tolerance to QACs following repeat exposure to subinhibitory concentrations. Ten HA-MRSA and four VISA isolates carried qacA. Two HA-MRSA isolates, one MSSA isolate and one VISA isolate carried qacC. One VISA isolate carried qacA and qacC. The CA-MRSA isolates did not carry qac genes. qacG, H and J were not detected in any HA-MRSA. Isolates with qac genes had significantly (P < 0.0001) higher MBCs for biocides containing QACs and chlorhexidine gluconate. These biocides induced expression of qac genes when assayed with a luciferase reporter. CONCLUSIONS: Biocides commonly used in the hospital environment should be effective against clinical isolates of S. aureus if used at concentrations recommended by the manufacturer. However, isolates have the potential to develop increased tolerance to these agents and the expression of Qac efflux pumps results in isolates with a selective advantage when challenged with biocides containing QACs and chlorhexidine gluconate.

Antimicrobial lexitropsins containing amide, amidine, and alkene linking groups.

The synthesis and properties of 80 short minor groove binders related to distamycin and the thiazotropsins are described. The design of the compounds was principally predicated upon increased affinity arising from hydrophobic interactions between minor groove binders and DNA. The introduction of hydrophobic aromatic head groups, including quinolyl and benzoyl derivatives, and of alkenes as linkers led to several strongly active antibacterial compounds with MIC for Staphylococcus aureus, both methicillin-sensitive and -resistant strains, in the range of 0.1-5 microg mL-1, which is comparable to many established antibacterial agents. Antifungal activity was also found in the range of 20-50 microg mL-1 MIC against Aspergillus niger and Candida albicans, again comparable with established antifungal drugs. A quinoline derivative was found to protect mice against S. aureus infection for a period of up to six days after a single intraperitoneal dose of 40 mg kg-1.

Guidelines for the prophylaxis and treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections in the UK.

These evidence-based guidelines have been produced after a literature review of the treatment and prophylaxis of methicillin-resistant Staphylococcus aureus (MRSA) infection. The guidelines were further informed by antibiotic susceptibility data on MRSA from the UK. Recommendations are given for the treatment of common infections caused by MRSA, elimination of MRSA from carriage sites and prophylaxis of surgical site infection. There are several antibiotics currently available that are suitable for use in the management of this problem and potentially useful new agents are continuing to emerge.

Glycopeptide resistance in Staphylococcus aureus: is it a real threat?

Following the discovery in 1992 that resistance to vancomycin could be transferred in vitro from a strain of Staphylococcus haemolyticus to Staphylococcus aureus, the threat that a similar event could happen in vivo has existed. In 1996, the threat became a reality in Japan followed by reports of low-level heteroresistant S. aureus from elsewhere in the world. However, the threat has not gone away; indeed it has become more intimidating within the past 2 years with the isolation of higher-level resistance to vancomycin transferred on a plasmid from Enterococcus faecalis. The mechanisms of resistance are different in each of the types of S. aureus with reduced susceptibility to the glycopeptides: in one hetero-vancomycin intermediate-susceptible S. aureus (hVISA) and vancomycin intermediate-susceptible S. aureus (VISA), cell wall biosynthesis is altered, hindering glycopeptide reaching its target, and in the other vancomycin-resistant S. aureus (VRSA) the target is altered. This review attempts to place into context the threat posed to patients by the appearance of strains of S. aureus more resistant to one of the key therapeutic agents used in our hospitals. It will do so by raising a number of important questions followed by the presentation of experimental evidence that may or may not provide answers that heighten or lower the threat.

Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of bacillus species associated with nongastrointestinal infections.

With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential. However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized. Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors. PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T (BceT) in five B. cereus strains and in Bacillus coagulans NB11. Enterotoxin HBL was also harbored by Bacillus polymyxa NB6. After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin. Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacillus licheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive. This study constitutes the first demonstration that Bacillus spp. associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B. cereus.

Cell wall thickening is a common feature of vancomycin resistance in Staphylococcus aureus.

We have previously shown that a thickened cell wall is responsible for the vancomycin resistance of vancomycin-resistant Staphylococcus aureus (VRSA) (equivalent to vancomycin-intermediate S. aureus and glycopeptide-intermediate S. aureus) strain Mu50 (L. Cui, H. Murakami, K. Kuwahara-Arai, H. Hanaki, and K. Hiramatsu, Antimicrob. Agents Chemother. 44:2276-2285, 2000). However, the mechanism of vancomycin resistance in other VRSA strains remained unclear. In this study, 16 clinical VRSA strains from seven countries were subjected to serial daily passage in drug-free medium. After 10 to 84 days of passage in the nonselective medium, passage-derived strains with decreased MICs of vancomycin (MIC, <4 mg/liter) were obtained. However, all of the passage-derived strains except one (15 of 16) still possessed subpopulations that were resistant to vancomycin as judged by population analysis, and vancomycin-resistant mutant strains were selected from the passage-derived strains by one-step vancomycin selection with a frequency of 4.25 x 10(-6) to 1.64 x 10(-3). The data indicated that vancomycin-resistant cells are frequently generated from the passage-derived strains even after vancomycin selective pressure is lifted. Cell wall thicknesses and MICs of glycopeptides (vancomycin and teicoplanin) and beta-lactams (imipenem and oxacillin) were determined for a total of 48 strains, including 15 sets of three strains: the clinical VRSA strain, the passage-derived strain, and the vancomycin-resistant mutant strain obtained from the passage-derived strain. No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin (r = 0.679; P < 0.001) and between MICs of imipenem and oxacillin (r = 0.787; P < 0.001) were recognized. Moreover, all of the VRSA strains had significantly thickened cell walls, which became thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains. The data showed that cell wall thickness had high correlation with the MICs of the two glycopeptides (correlation coefficients, 0.908 for vancomycin and 0.655 for teicoplanin) but not with those of the beta-lactam antibiotics tested. These results together with coupled changes of cell wall thickness and vancomycin MICs in 16 isogenic sets of strains indicate that thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for vancomycin resistance in S. aureus.

Virulence factor expression by Gram-positive cocci exposed to subinhibitory concentrations of linezolid.

Linezolid is a new oxazolidinone with potent antibacterial activity against Gram-positive cocci; it uniquely inhibits bacterial translation through inhibition of 70S initiation complex formation. The effects of sub-growth-inhibitory concentrations of linezolid on the expression of various structural and soluble virulence factors of Staphylococcus aureus and Streptococcus pyogenes were examined. For S. aureus, strains Wood 46 and Cowan 1 (NCTC 8532) were used to measure protein A, coagulase, alpha-haemolysin (hla) and delta-haemolysin (hld). For S. pyogenes, strain NCTC 9994 was used to measure M protein, streptolysin O (SLO) and DNase. Coagulase was assayed by clotting of citrated rabbit plasma, and hla, hld and SLO by lysis of rabbit, human and horse erythrocytes, respectively. Protein A and M protein were measured indirectly using bacterial susceptibility to phagocytic ingestion of radiolabelled bacteria by human neutrophils. When S. aureus was grown in 1/2, 1/4 and 1/8 MIC, linezolid, coagulase, hla and hld production were impaired. Susceptibility to phagocytosis was changed by growth in the presence of 1/2 MIC linezolid compared with that in its absence (50.8 +/- 4.1% versus 38.9 +/- 2.9%; P