Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.Staphylococcus aureus is a gram-positive bacterium that is notable for the frequency and severity of infections that it causes in hospitalized patients. These infections range from localized skin infections to bacteremia and septic shock. In the past 20 years there has been a dramatic increase in the incidence of nosocomial staphylococcal infections; this increase parallels the increased use of intravascular devices and invasive procedures. S. aureus has been identified as one of the three most frequent nosocomial pathogens and is responsible for approximately 25% of the 2 million nosocomial infections reported in the United States each year (38, 39). A second trend has been the increase in the incidence of methicillin-resistant S. aureus, largely due to selective antibiotic pressure. Resistant strains were initially identified in tertiary care hospitals but have been increasingly reported among infections in the community (25, 30). Resistance to methicillin is often accompanied by resistance to other antibiotics; a CDC survey showed that the proportion of methicillin-resistant isolates which were susceptible only to vancomycin rose from 22.8% to 56. 2% from 1987 to 1997 (18). More recently, S. aureus strains with intermediate susceptibility or resistance to vancomycin have been reported (11,24,36). Infections caused by multidrug-resistant S. aureus limit therapeutic options, and they may be associated with higher mortality and higher costs than infections caused by susceptible staphylococci. There is clearly a need for new treatment and prevention strategies.In an immunological survey of S. aureus su...

Escherichia coli UB1005 and two mutants of this strain (DC2 and DC3) have been used to assess indirectly the relative ability of various β-lactam antibiotics to penetrate the outer layers of E. coli . Benzylpenicillin, ampicillin, methicillin, cloxacillin, cephaloridine, cephalothin, and cephalexin have been examined. The results confirm those obtained with other methods and show that, among the compounds studied here, cephalosporins seem to penetrate more readily than penicillins.

The actinomycetes traditionally represent one of the most important sources for the discovery of new metabolites with biological activity; and many of these are described as being produced by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We present a strain characterization system based on the metabolic potential of microbial strains by targeting these biosynthetic genes. After an initial evaluation of the existing bias derived from the PCR detection in a well defined biosynthetic systems, we developed a new fingerprinting approach based on the restriction analysis of these PKS and NRPS amplified sequences. This method was applied to study the distribution of PKS and NRPS biosynthetic systems in a collection of wild-type actinomycetes isolated from tropical soil samples that were evaluated for the production of antimicrobial activities. We discuss the application of this tool as an alternative characterization approach for actinomycetes and we comment on the relationship observed between the presence of PKS-I, PKS-II and NRPS sequences and the antimicrobial activities observed in some of the microbial groups tested.

A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.

We previously reported the development of a human monoclonal antibody (CS-D7, IgG1) with specificity and affinity for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17), not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. The mAb was examined to determine if it blocked heme binding to IsdB in vitro. The mAb was not found to have heme blocking activity, nor did it prevent bacterial growth under in vivo conditions, in an implanted growth chamber. To assess the role of the mAb Fc a point mutation was introduced at aa 297 (CS-D7·N297A). This point mutation removes Fc effector functions. In vitro analysis of the mutein confirmed that it lacked measurable binding to FcγR, and that it did not fix complement. The mutein had dramatically reduced in vitro opsonic OP activity compared to CS-D7. Nonetheless, the mutein conferred protection equivalent to the wild type mAb in the murine sepsis model. Both wild type and mutein mAbs were efficacious in FcγR deletion mice (including both FcγRII−/− mice and FcγRIII−/− mice), indicating that these receptors were not essential for mAb mediated protection in vivo. Protection mediated by CS-D7 was lost in Balb/c mice depleted of C3 with cobra venom factor (CFV), was lost in mice depleted of superoxide dismutase (SOD) in P47phox deletion mice, and as previously reported, was absent in SCID mice (Joshi et al., 2012). Enhanced clearance of S. aureus in the liver of CS-D7 treated mice and enhanced production of IFN-γ, but not of IL17, may play a role in the mechanism of protection mediated by the mAb. CS-D7 apparently mediates survival in challenged mice through a mechanism involving complement, phagocytes, and lymphocytes, but which does not depend on interaction with FcγR, or on blocking heme uptake.

Three natural populations of actinomycetes were investigated by PCR for the presence of type I 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA), a gene associated with isoprenoid biosynthesis. The populations were obtained from an agricultural site (69 isolates), a coastal salt marsh (220 isolates), and a desert soil (96 isolates). A set (34) of standard actinomycete reference strains were also investigated. The target gene was only detected in 5 of the 419 actinomycetes screened, which represented 4 from the coastal salt marsh and one reference strain. The isolates that contained the gene were taxonomically diverse (4 Streptomyces spp. and 1 Nocardia sp.). These results suggest that type I HMG CoA containing pathways are rare in actinomycetes and their distribution within actinomycetes populations is not random.

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