BIOCHEMICAL CHARACTERIZATION AND CLINICAL SIGNIFICANCE OF COAGULASE-NEGATIVE STAPHYLOCOCCI

Although coagulase-negative staphylococci (C-NS) have been implicated in certain human infections, they are generally regarded as contaminants and their clinical significance is questioned. To assess their role as pathogens, 205 isolates of C-NS from wounds, and body fluids (blood, urine, pleural and peritoneal fluids, etc.) were studied. Patient's charts were reviewed and using strict criteria a determination was made regarding the clinical significance of these isolates. The organisms were then identified using the scheme of Kloos and Schleifer to determine if certain species of C-NS were associated with specific infections. S. epidermidis sensu stricto accounted for 81% of the C-NS isolated; the frequency of other species was S. haemolyticus (6%), S. hominis (5%), S. capitis (4%), S. warneri (3%), and others (1%). Only two isolates were novobiocin resistant; neither was identified as S. saprophyticus. Using these criteria, 22% of C-NS were considered to be clinically significant and the majority of these (93%) were due to S. epidermidis. The most common source of the clinically relevant C-NS isolates was from wounds. These data suggest that identifying C-NS species other than S. epidermidis may be of limited value in predicting clinical significance.^ In addition, selected pathogenic and non-pathogenic strains of C-NS were compared for their ability to adhere to human cells in vitro. Although the results were not conclusive, it appeared that pathogenic C-NS adhered more avidly than non-pathogenic C-NS to buccal cells. Experiments with HeLa cells showed no difference between pathogenic and non-pathogenic C-NS in adherence abilities. ^

A family of fibrinogen-binding MSCRAMMs from Enterococcus faecalis.

We report that three (EF0089, EF2505 and EF1896, renamed here Fss1, Fss2 and Fss3, respectively, for Enterococcus faecalis surface protein) of the recently predicted MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) in E. faecalis strain V583 bind fibrinogen (Fg). Despite an absence of extensive primary sequence homology, the three proteins appear to be related structurally. Within the N-terminal regions of the three enterococcal proteins, we identified pairs of putative IgG-like modules with a high degree of predicted structural similarity to the Fg-binding N2 and N3 domains of the staphylococcal MSCRAMMs ClfA and SdrG. A second N2N3-like segment was predicted in Fss1. Far-UV circular dichroism spectroscopy revealed that all four predicted N2N3-like regions are composed mainly of beta-sheets with only a minor proportion of alpha-helices, which is characteristic of Ig-like folded domains. Three of the four identified enterococcal N2N3-like regions showed potent dose-dependent binding to Fg. However, the specificity of the Fg-binding MSCRAMMs differs, as indicated by far-Western blots, which showed that recombinant segments of the MSCRAMMs bound different Fg polypeptide chains. Enterococci grown in serum-supplemented broth adhere to Fg-coated surfaces, and inactivation in strain OG1RF of the gene encoding Fss2 resulted in reduced adherence, whilst complementation of the mutant restored full Fg adherence. Thus, E. faecalis contains a family of MSCRAMMs that structurally and functionally resemble the Fg-binding MSCRAMMs of staphylococci.

Clinical and microbiological aspects of linezolid resistance mediated by the cfr gene encoding a 23S rRNA methyltransferase.

The cfr (chloramphenicol-florfenicol resistance) gene encodes a 23S rRNA methyltransferase that confers resistance to linezolid. Detection of linezolid resistance was evaluated in the first cfr-carrying human hospital isolate of linezolid and methicillin-resistant Staphylococcus aureus (designated MRSA CM-05) by dilution and diffusion methods (including Etest). The presence of cfr was investigated in isolates of staphylococci colonizing the patient's household contacts and clinical isolates recovered from patients in the same unit where MRSA CM-05 was isolated. Additionally, 68 chloramphenicol-resistant Colombian MRSA isolates recovered from hospitals between 2001 and 2004 were screened for the presence of the cfr gene. In addition to erm(B), the erm(A) gene was also detected in CM-05. The isolate belonged to sequence type 5 and carried staphylococcal chromosomal cassette mec type I. We were unable to detect the cfr gene in any of the human staphylococci screened (either clinical or colonizing isolates). Agar and broth dilution methods detected linezolid resistance in CM-05. However, the Etest and disk diffusion methods failed to detect resistance after 24 h of incubation. Oxazolidinone resistance mediated by the cfr gene is rare, and acquisition by a human isolate appears to be a recent event in Colombia. The detection of cfr-mediated linezolid resistance might be compromised by the use of the disk diffusion or Etest method.

Eluding antibiotic resistance: capitalizing on antimicrobial peptides interaction with the lipid bilayer

It is widely accepted that the emergence of drug-resistant pathogens is the result of the overuse and misuse of antibiotics. Infectious Disease Society of America, Center for Disease Control and World Health Organization continue to view, with concern, the lack of antibiotics in development, especially those against Gram-negative bacteria. Antimicrobial peptides (AMPs) have been proposed as an alternative to antibiotics due to their selective activity against microbes and minor ability to induce resistance. For example, the Food and Drug Administration approved Daptomycin (DAP) in 2003 for treatment of severe skin infections caused by susceptible Gram-positive organisms. Currently, there are 12 to 15 examples of modified natural and synthetic AMPs in clinical development. But most of these agents are against Gram-positive bacteria. Therefore, there is unmet medical need for antimicrobials used to treat infections caused by Gram-negative bacteria. In this study, we show that a pro-apoptotic peptide predominantly used in cancer therapy, (KLAKLAK)2, is an effective antimicrobial against Gram-negative laboratory strains and clinical isolates. Despite the therapeutic promise, AMPs development is hindered by their susceptibility to proteolysis. Here, we demonstrate that an all-D enantiomer of (KLAKLAK)2, resistant to proteolysis, retains its activity against Gram-negative pathogens. In addition, we have elucidated the specific site and mechanism of action of D(KLAKLAK)2 through a repertoire of whole-cell and membrane-model assays. Although it is considered that development of resistance does not represent an obstacle for AMPs clinical development, strains with decreased susceptibility to these compounds have been reported. Staphylococci resistance to DAP was observed soon after its approval for use and has been linked to alterations of the cell wall (CW) and cellular membrane (CM) properties. Immediately following staphylococcal resistance, Enterococci resistance to DAP was seen, yet the mechanism of resistance in enterococci remains unknown. Our findings demonstrate that, similar to S. aureus, development of DAP-resistance in a vancomycin-resistant E. faecalis isolate is associated with alterations of the CW and properties of the CM. However, the genes linked to these changes in enterococci appear to be different from those described in S. aureus.

Studies of the structure and function of a fibrinogen binding MSCRAMM(RTM) from Staphylococcus epidermidis

Coagulase-negative staphylococci (CNS) are recognized as important pathogens and are particularly associated with foreign body infections. S. epidermidis accounts for approximately 75% of the infections caused by CNS. Three genes, sdrF, sdrG, and sdrH, were identified by screening a S. epidermidis genomic library with a probe encompassing the serine-aspartate dipeptide repeat-encoding region (region R) of clfA from S. aureus. SdrG has significant amino acid identity to ClfA, ClfB and other surface proteins of S. aureus. SdrG is also similar to a protein (Fbe) recently described by Nilsson, et al. (Infection and Immunity, 1998, 66:2666–73) from S. epidermidis. The N-terminal domain (A region) of SdrG was expressed as a his-tag fusion protein in E. coli. In an ELISA, this protein, rSdrG(50-597) was shown to bind specifically to fibrinogen (Fg). Western ligand blot analysis showed that SdrG binds the Bβ chain of Fg. To further characterize the rSdrG(50-597)-Fg interaction, truncates of the Fg Bβ chain were made and expressed as recombinant proteins in E. coli. SdrG was shown to bind the full-length Bβ chain (1462), as well as the N-terminal three-quarters (1-341), the N-terminal one-half (1-220) and the N-terminal one-quarter (1-95) Bβ chain constructs. rSdrG(50-597) failed to bind to the recombinant truncates that lacked the N-terminal 25 amino acid residues of this polypeptide suggesting that SdrG recognizes a site within this region of the Bβ chain. Inhibition ELISAs have shown that peptide mimetics, including β1–25, and β6–20, encompassing this 25 residue region can inhibit binding of rSdrG(50-597) to Fg coated wells. Using fluorescence polarization we were able to determine an equilibrium constant (KD) for the interaction of rSdrG(50-597) with the Fg Bβ chain peptide β1–25. The labeled peptide was shown to bind to rSdrG(50-597) with a KD of 0.14 ± 0.01μM. Because rSdrG(50-597) recognizes a site in the Fg Bβ chain close to the thrombin cleavage site, we investigated the possibility of the rSdrG(50-597) site either overlapping or lying close to this cleavage site. An ELISA showed that rSdrG(50-597) binding to thrombin-treated Fg was significantly reduced. In a clot inhibition assay rSdrG(50-597) was able to inhibit fibrin clot formation in a concentration dependent manner. Furthermore, rSdrG(50-597) was able to inhibit clot formation by preventing the release of fibrinopeptide B as determined by HPLC. To further define the interaction between rSdrG(50-597) and peptide β6–20, we utilized an alanine amino acid replacement strategy. The residues in β6–20 that appear to be important in rSdrG(50-597) binding to Fg, were confirmed by the rSdrG(273-597)-β6–20 co-crystal structure that was recently solved by our collaborators at University of Alabama-Birmingham. Additionally, rSdrG(50-597) was not able to bind to Fg from different animal species, rather it bound specifically to human Fg in an ELISA. This suggests that the sequence variation between Fg Bβ chains of different species, specifically with in the N-terminal 25 residues, affects the ability of rSdrG(50-597) binding to Fg, and this may explain why S. epidermidis is primarily a human pathogen. ^