The par locus of the Enterococcus faecalis plasmid pAD1 is an RNA-regulated addiction module encoding the peptide toxin Fst. Homology searches revealed that Fst belongs to a family of at least nine related peptides encoded on the chromosomes and plasmids of six different Grampositive bacterial species. Comparison of an alignment of these peptides with the results of a saturation mutagenesis analysis indicated regions of the peptides important for biological function. Examination of the genetic context of the fst genes revealed that all of these peptides are encoded within par-like loci with conserved features similar to pAD1 par. All four Ent. faecalis family members were demonstrated to produce the expected toxin-encoding and regulatory RNA products. The locus from the Ent. faecalis plasmid pAMS1 was demonstrated to function as an addiction module and Fst was shown to be toxic to Staphylococcus aureus, suggesting that a plasmid-encoded module in that species is performing the same function. Thus, the pAD1-encoded par locus appears to be the prototype of a family of related loci found in several Grampositive species. INTRODUCTIONToxin-antitoxin (TA) systems were first identified on bacterial plasmids, where they function as stability determinants by programming for death any daughter cells that fail to inherit a copy (for recent reviews see Gerdes & Wagner, 2007;Hayes, 2003). These systems, originally called post-segregation killing systems or addiction modules, encode a stable toxin and an unstable antitoxin. As long as the plasmid is properly inherited, antitoxin is continually replenished and the toxin remains inactive. If the plasmid is lost, the antitoxin is degraded and the toxin is free to exert its effect. Toxins have a variety of targets including gyrase (Bahassi et al., 1999;Jiang et al., 2002), ribosome-bound and free mRNA (Condon, 2006;Zhang et al., 2004), and the cell membrane (Gerdes et al., 1986). In most TA systems, both components are proteins, with the antitoxin targeted by a specific cellular protease (Gerdes et al., 2005). In two well-studied cases, the hok/sok system of Escherichia coli plasmid R1 (Gerdes et al., 1990) and the par system of Enterococcus faecalis plasmid pAD1 Greenfield et al., 2001, the antitoxin is a small regulatory RNA that represses the translation of the toxin. The RNA-regulated TA systems are sometimes referred to as type I TA loci while the proteinregulated systems are designated type II.Paradoxically, numerous TA systems have been identified on bacterial chromosomes and have been the subject of numerous recent reviews (Buts et al., 2005; EngelbergKulka et al., 2006;Fozo et al., 2008;Gerdes & Wagner, 2007;Gerdes et al., 2005;Van Melderen & Saavedra De Bast, 2009). Various roles have been either demonstrated or proposed for these systems, including stabilization of integrated mobile genetic elements, protection from plasmid-encoded addiction modules, programmed cell death, growth modulation during stress response, persistence, and developmental processes. It seems like...

Surgical site infection (SSI) remains a significant risk for any clean orthopedic surgical procedure. Complications resulting from an SSI often require a second surgery and lengthen patient recovery time. The efficacy of antimicrobial agents delivered to combat SSI is diminished by systemic toxicity, bacterial resistance, and patient compliance to dosing schedules. We submit that development of localized, controlled release formulations for antimicrobial compounds would improve the effectiveness of prophylactic surgical wound antibiotic treatment while decreasing systemic side effects. Our research group developed and characterized oligo(poly(ethylene glycol)fumarate) / sodium methacrylate (OPF/SMA) charged copolymers as biocompatible hydrogel matrices. Here, we report the engineering of this copolymer for use as an antibiotic delivery vehicle in surgical applications. We demonstrate that these hydrogels can be efficiently loaded with vancomycin (over 500 μg drug per mg hydrogel) and this loading mechanism is both time- and charge-dependent. Vancomycin release kinetics are shown to be dependent on copolymer negative charge. In the first 6 hours, we achieved as low as 33.7% release. In the first 24 hours, under 80% of total loaded drug was released. Further, vancomycin release from this system can be extended past four days. Finally, we show that the antimicrobial activity of released vancomycin is equivalent to stock vancomycin in inhibiting the growth of colonies of a clinically derived strain of methicillin-resistant Staphylococcus aureus. In summary, our work demonstrates that OPF/SMA hydrogels are appropriate candidates to deliver local antibiotic therapy for prophylaxis of surgical site infection.

dMicrobiological diagnosis is pivotal to the appropriate management and treatment of infective endocarditis. We evaluated PCRelectrospray ionization mass spectrometry (PCR/ESI-MS) for bacterial and candidal detection using 83 formalin-fixed paraffinembedded heart valves from subjects with endocarditis who had positive valve and/or blood cultures, 63 of whom had positive valvular Gram stains. PCR/ESI-MS yielded 55% positivity with concordant microbiology at the genus/species or organism group level (e.g., viridans group streptococci), 11% positivity with discordant microbiology, and 34% with no detection. PCR/ESI-MS detected all antimicrobial resistance encoded by mecA or vanA/B and identified a case of Tropheryma whipplei endocarditis not previously recognized.

Bacterial biofilms are difficult to treat using available antimicrobial agents, so new antibiofilm strategies are needed. We previously showed that 20, 200, and 2,000 A of electrical current reduced bacterial biofilms of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. Here, we tested continuous direct current at lower amperages, intermittent direct current, and combinations of surface materials (Teflon or titanium) and electrode compositions (stainless steel, graphite, titanium, or platinum) against S. aureus, S. epidermidis, and P. aeruginosa biofilms. In addition, we tested 200 or 2,000 A for 1 and 4 days against biofilms of 33 strains representing 13 species of microorganisms. The logarithmic reduction factor was used to measure treatment effects. Using continuous current delivery, the lowest active amperage was 2 A for 1, 4, or 7 days against P. aeruginosa and 5 A for 7 days against S. epidermidis and S. aureus biofilms. Delivery of 200 A for 4 h a day over 4 days reduced P. aeruginosa, S. aureus, and S. epidermidis biofilms on Teflon or titanium discs. A reduction of P. aeruginosa, S. aureus, and S. epidermidis biofilms was measured for 23 of 24 combinations of surface materials and electrode compositions tested. Four days of direct current delivery reduced biofilms of 25 of 33 strains studied. In conclusion, low-amperage current or 4 h a day of intermittent current delivered using a variety of electrode compositions reduced P. aeruginosa, S. aureus, and S. epidermidis biofilms on a variety of surface materials. The electricidal effect was observed against a majority of bacterial species studied.C hronic infections associated with medical devices such as joint replacements and other types of orthopedic instrumentation, prosthetic heart valves, pacemakers, implantable defibrillators, urinary catheters and stents, peritoneal dialysis catheters, intravascular catheters, cerebrospinal fluid shunts, breast implants, and vascular grafts and stents are common in today's medical practice. When these devices become infected, they must often be removed to successfully cure the associated infection. Device removal is associated with significant morbidity, cost, and, in some cases, mortality. Prosthetic joint removal, for example, may mean that a patient is left without a functional joint for months, along with the requirement for two surgeries (i.e., infected implant removal and eventual replacement) (1). Intrathoracic device infections may require major surgical procedures, involving repeat sternotomy. The removal of some devices, such as vascular graft bypasses, may be impossible, rendering associated infection incurable with current approaches.The pathogenesis of device-associated infections relates to the presence of microorganisms in biofilms. Existence within a biofilm represents a survival strategy for microorganisms, protecting them from environmental influences, the host immune system, and, unfortunately, therapeutic levels of conventional antimicrobial agents. Biofilms exhibit ...

The par stability determinant of Enterococcus faecalis plasmid pAD1 is the only antisense RNA-regulated addiction module identified to date in gram-positive bacteria. par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, that function as the toxin (Fst)-encoding and antitoxin components, respectively. Previous work showed that structures at the 5 end of RNA I are important in regulating its translation. The work presented here reveals that a stem-loop sequestering the Fst ribosome binding site is required for translational repression but a helix sequestering the 5 end of RNA I is not. Furthermore, disruption of the stem-loop prevented RNA II-mediated repression of Fst translation in vivo. Finally, although Fst-encoding wild-type RNA I is not toxic in Escherichia coli, mutations affecting stem-loop stability resulted in toxicity in this host, presumably due to increased translation.