Chemical Screening in an Estrogen Receptor Transactivation Assay With Metabolic Competence.

The U.S. EPA continues to utilize high-throughput screening data to evaluate potential biological effects of endocrine active substances without the use of animal testing. Determining the scope and need for in vitro metabolism in high-throughput assays requires the generation of larger data sets that assess the impact of xenobiotic transformations on toxicity-related endpoints. The objective of the current study was to screen a set of 768 ToxCast chemicals in the VM7Luc estrogen receptor transactivation assay (ERTA) using the Alginate Immobilization of Metabolic Enzymes hepatic metabolism method. Chemicals were screened with or without metabolism to identify estrogenic effects and metabolism-dependent changes in bioactivity. Based on estrogenic hit calls, 85 chemicals were active in both assay modes, 16 chemicals were only active without metabolism, and 27 chemicals were only active with metabolism. Using a novel metabolism curve shift method that evaluates the shift in concentration-response curves, 29 of these estrogenic chemicals were identified as bioactivated and 59 were bioinactivated. Human biotransformation routes and associated metabolites were predicted in silico across the chemicals to mechanistically characterize possible transformation-related ERTA effects. Overall, the study profiled novel chemicals associated with metabolism-dependent changes in ERTA bioactivity, and suggested routes of biotransformation and putative metabolites responsible for the observed estrogenic effects. The data demonstrate a range of metabolism-dependent effects across a diverse chemical library and highlight the need to evaluate the role of intrinsic xenobiotic metabolism for endocrine and other toxicity-related health effects.

Development of an In Vitro Human Thyroid Microtissue Model for Chemical Screening.

Thyroid hormones (TH) are essential for regulating a number of diverse physiological processes required for normal growth, development, and metabolism. The US EPA Endocrine Disruptor Screening Program (EDSP) has identified several molecular thyroid targets relevant to hormone synthesis dynamics that have been adapted to high-throughput screening (HTS) assays to rapidly evaluate the ToxCast/Tox21 chemical inventories for potential thyroid disrupting chemicals (TDCs). The uncertainty surrounding the specificity of active chemicals identified in these screens and the relevance to phenotypic effects on in vivo human TH synthesis are notable data gaps for hazard identification of TDCs. The objective of this study was to develop a medium-throughput organotypic screening assay comprised of reconstructed human thyroid microtissues to quantitatively evaluate the disruptive effects of chemicals on TH production and secretion. Primary human thyroid cells procured from qualified euthyroid donors were analyzed for retention of NK2 homeobox 1 (NKX2-1), Keratin 7 (KRT7), and Thyroglobulin (TG) protein expression by high-content image analysis to verify enrichment of follicular epithelial cells. A direct comparison of 2-dimensional (2D) and 3-dimensional (3D) 96-well culture formats was employed to characterize the morphology, differential gene expression, TG production, and TH synthesis over the course of 20 days. The results indicate that modeling human thyroid cells in the 3D format was sufficient to restore TH synthesis not observed in the 2D culture format. Inhibition of TH synthesis in an optimized 3D culture format was demonstrated with reference chemicals for key molecular targets within the thyroid gland. Implementation of the assay may prove useful for interpreting phenotypic effects of candidate TDCs identified by HTS efforts currently underway in the EDSP.

A novel rat model of foreign body osteomyelitis for evaluation of antimicrobial efficacy.

The most common organism-type causing orthopedic foreign body infection is the staphylococci, of which Staphylococcus aureus and Staphylococcus epidermidis are especially common. These organisms form biofilms on orthopedic foreign body surfaces, rendering such infections challenging and time consuming to treat. Our group evaluates novel therapeutics for orthopedic foreign body infection in animal models. A current limitation of most animal models is that that they only allow for the removal of one sample per animal, at the time of sacrifice. Herein, we describe a novel rat model of foreign body osteomyelitis that allows removal of foreign bodies at different time points, from the same infected animal. We demonstrate that this model can be used for both S. aureus and S. epidermidis orthopedic foreign body infection, with 3.56, 3.60 and 5.51 log10 cfu/cm2 S. aureus recovered at four, five and six weeks, respectively, after infection, and 2.08, 2.17 and 2.62 log10 cfu/cm2 S. epidermidis recovered at four, five and six weeks, respectively, after infection We evaluated the model with S. aureus infection treated with rifampin 25 mg/kg twice daily for 21 days. Using quantitative cultures, we were no longer able to detect bacteria as of the 14th day of treatment with bacteria becoming detectable again 7 days following die discontinuation of rifampin a period. This novel model allows monitoring of evolution of infection at the infection site in the same animal.

Effect of Direct Electrical Current on Bones Infected with Staphylococcus epidermidis.

We are developing electrical approaches to treat biofilm-associated orthopedic foreign-body infection. Although we have previously shown that such approaches have antibiofilm activity, the effects on bone have not been assessed. Herein, low-amperage 200 µA fixed direct current (DC) was compared with no current, in a rat femoral foreign-body infection model. In the infected group, a platinum implant seeded with S. epidermidis biofilm (105 CFU/cm2), plus 50 µL of a 109 CFU suspension of bacteria, were placed in the femoral medullary cavity of 71 rats. One week later, rats were assigned to one of four groups: infected with no current or DC, or uninfected with no current or DC. After 2 weeks, bones were removed and subjected to histopathology, micro-computed tomography (µCT), and strength testing. Histopathology showed no inflammation or bony changes/remodeling in the uninfected no current group, and some osteoid formation in the DC group; bones from the infected no current group had evidence of inflammation without bony changes/remodeling; along with inflammation, there was moderate osteoid present in the DC group. µCT showed more cortical bone volume and density, trabecular thickness, and cancellous bone volume in the DC group compared with the no current group, for both uninfected and infected bones (p < 0.05). There was no difference in torsional strength or stiffness between the no current versus DC groups, for both infected and uninfected bones (p > 0.05). © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Activity of fixed direct electrical current in experimental Staphylococcus aureus foreign-body osteomyelitis.

Fixed DC was compared to ceftriaxone, ceftriaxone with 200 µA fixed DC, or no treatment in a rat model of methicillin-susceptible Staphylococcus aureus foreign-body osteomyelitis. After 3 weeks, fewer bacteria were present in bones of the ceftriaxone group (5.71 log10cfu/g [P = 0.0004]) and the ceftriaxone/DC group (3.53 log10cfu/g [P = 0.0002]) than untreated controls (6.70 log10cfu/g). Fewer bacteria were present in the ceftriaxone/DC group than in the ceftriaxone-alone and DC-alone groups (P = 0.0012 and 0.0008, respectively). There were also fewer bacteria on the implanted wires in the groups treated with ceftriaxone (5.47 log10cfu/cm2) or ceftriaxone/DC (2.82 log10cfu/cm2) than in the untreated controls (6.44 log10cfu/cm2 [P = 0.0003 and 0.0002, respectively]). There were fewer bacteria in the ceftriaxone/DC rats than in the ceftriaxone-alone- and fixed DC-alone-treated rats (P = 0.0017 and 0.0016, respectively). Fixed DC with an antibiotic may be useful for treating foreign-body infections caused by S. aureus.

In Vitro Activity of Imipenem-Relebactam and Ceftolozane-Tazobactam against Resistant Gram-Negative Bacilli.

Understanding which antimicrobial agents are likely to be active against Gram-negative bacilli can guide selection of antimicrobials for empirical therapy as mechanistic rapid diagnostics are adopted. In this study, we determined the MICs of a novel ß-lactam-ß-lactamase inhibitor combination, imipenem-relebactam, along with ceftolozane-tazobactam, imipenem, ertapenem, meropenem, ceftriaxone, and cefepime, against 282 drug-resistant isolates of Gram-negative bacilli. For isolates harboring blaKPC (n = 110), the addition of relebactam to imipenem lowered the MIC50/MIC90 from 16/>128 µg/ml for imipenem alone to 0.25/1 µg/ml. For isolates harboring blaCTX-M (n = 48), the MIC50/MIC90 of ceftolozane-tazobactam were 0.5/16 µg/ml (83% susceptible). For isolates harboring blaCMY-2 (n = 17), the MIC50/MIC90 of ceftolozane-tazobactam were 4/8 µg/ml (47% susceptible). Imipenem-relebactam was active against most KPC-producing (but not NDM- or IMP-producing) Enterobacteriaceae and is an encouraging addition to the present antibiotic repertoire.

Rifampicin resistance in Staphylococcus epidermidis: molecular characterisation and fitness cost of rpoB mutations.

The molecular mechanisms and characteristics of rifampicin (RIF) resistance in Staphylococcus epidermidis are poorly characterised, even though S. epidermidis is one of the most common nosocomial pathogens associated with indwelling medical device-related infections. The aim of this study was to investigate the evolution of RIF resistance and to characterise the associated molecular mechanisms in S. epidermidis. RIF-resistant mutants from two RIF-susceptible S. epidermidis strains (RP62A and IDRL-8883) were selected through in vitro and in vivo exposure to RIF. A total of 16 colonies with an RP62A background and 63 colonies with an IDRL-8883 background were analysed for rpoB mutations. The fitness of RIF-susceptible and isogenic RIF-resistant strains was assessed using a paired competition assay and by comparing generation times. All mutations detected were in cluster I of rpoB. The following five amino acid substitutions were selected in vitro: Asp471→Asn; Asp471→Gly; Asp471→Val; Ser486→Tyr; and His481→Tyr. The following three amino acid substitutions were selected in vivo: His481→Tyr; Gln468→Lys; and Ser486→Phe. Asp471→Asn and Asp471→Gly changes were associated with susceptible minimal inhibitory concentrations (MICs). In vitro competition assays revealed that all RIF-resistant mutants other than Ser486→Tyr and Ser486→Phe had a relative fitness of <1.0. His481→Tyr mutations had their own specific fitness costs and effects on growth rate, irrespective of strain background. In conclusion, the current study presents molecular characterisations and fitness costs of several rpoB mutations in S. epidermidis.

Activity of Electrical Current in Experimental Propionibacterium acnes Foreign-Body Osteomyelitis.

Foreign-body-associated infections are often difficult to treat, given that the associated microorganisms are in a biofilm state. Previously, we showed that a low-amperage direct electrical current (DC) reduces Propionibacterium acnes biofilms formed on implant-associated materials in vitro In this study, low-amperage DC was compared to ceftriaxone treatment or no treatment in a novel rat femur model of foreign-body osteomyelitis. A platinum implant seeded with a P. acnes biofilm (107 CFU/cm2) and 109 CFU of planktonic P. acnes was placed in the femoral medullary cavity. One week later, rats were assigned to one of three treatment groups: no treatment, ceftriaxone treatment, or 200-µA-DC treatment. After 2 weeks of treatment, there were fewer bacteria in the bones of the ceftriaxone group (3.06 log10 CFU/g of bone [P = 0.0209]) and the 200-µA-DC group (0.5 log10 CFU/g [P = 0.0015]) than in those of the control group (6.58 log10 CFU/g). The DC-exposed animals exhibited fewer bacteria than the ceftriaxone-treated animals (P = 0.0330). There were fewer bacteria on the implanted wires in the groups treated with ceftriaxone (0.1 log10 CFU/cm2) or a 200-µA DC (0.1 log10 CFU/cm2) than in the control group (2.53 log10 CFU/cm2 [P, 0.0003 for both comparisons]). Low-amperage DC may be useful for treating, or aiding in the treatment of, foreign-body infections caused by P. acnes.

Rifampin-Based Combination Therapy Is Active in Foreign-Body Osteomyelitis after Prior Rifampin Monotherapy.

Staphylococcal prosthetic joint infections (PJIs) are associated with biofilm formation, making them difficult to treat; if managed with debridement and implant retention, rifampin-based therapy is usually employed. Rifampin resistance potentially challenges PJI treatment. In investigating the effects of rifampin monotherapy on methicillin-resistant Staphylococcus aureus (MRSA) foreign-body osteomyelitis in rats, we previously demonstrated that rifampin resistance was selected but that it disappeared 14 days following rifampin monotherapy (1) and that rifampin resistance occurred less frequently following two rounds than following one round of rifampin monotherapy (2). Here, we compared rifampin monotherapy followed by rifampin-vancomycin combination therapy to rifampin-vancomycin combination therapy alone in experimental MRSA foreign-body osteomyelitis. Animals treated with rifampin monotherapy followed by rifampin-vancomycin combination therapy had decreased quantities of bacteria 14 days following treatment completion (P = 0.034) compared to those in animals treated with combination therapy alone. Additionally, some isolates recovered from animals treated with combination therapy alone, although still susceptible to rifampin, had higher MIC, minimum biofilm-inhibitory concentration (MBIC), and minimum biofilm-bactericidal concentration (MBBC) values than those of the inoculating strain. This suggests that rifampin may remain a feasible treatment option in foreign-body-associated orthopedic infections following the selection of rifampin resistance.

Exposure of Bacterial Biofilms to Electrical Current Leads to Cell Death Mediated in Part by Reactive Oxygen Species.

Bacterial biofilms may form on indwelling medical devices such as prosthetic joints, heart valves and catheters, causing challenging-to-treat infections. We have previously described the 'electricidal effect', in which bacterial biofilms are decreased following exposure to direct electrical current. Herein, we sought to determine if the decreased bacterial quantities are due to detachment of biofilms or cell death and to investigate the role that reactive oxygen species (ROS) play in the observed effect. Using confocal and electron microscopy and flow cytometry, we found that direct current (DC) leads to cell death and changes in the architecture of biofilms formed by Gram-positive and Gram-negative bacteria. Reactive oxygen species (ROS) appear to play a role in DC-associated cell death, as there was an increase in ROS-production by Staphylococcus aureus and Staphylococcus epidermidis biofilms following exposure to DC. An increase in the production of ROS response enzymes catalase and superoxide dismutase (SOD) was observed for S. aureus, S. epidermidis and Pseudomonas aeruginosa biofilms following exposure to DC. Additionally, biofilms were protected from cell death when supplemented with antioxidants and oxidant scavengers, including catalase, mannitol and Tempol. Knocking out SOD (sodAB) in P. aeruginosa led to an enhanced DC effect. Microarray analysis of P. aeruginosa PAO1 showed transcriptional changes in genes related to the stress response and cell death. In conclusion, the electricidal effect results in death of bacteria in biofilms, mediated, at least in part, by production of ROS.

Direct Electrical Current Reduces Bacterial and Yeast Biofilm Formation.

New strategies are needed for prevention of biofilm formation. We have previously shown that 24 hr of 2,000 µA of direct current (DC) reduces Staphylococcus epidermidis biofilm formation in vitro. Herein, we examined the effect of a lower amount of DC exposure on S. epidermidis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Propionibacterium acnes, and Candida albicans biofilm formation. 12 hr of 500 µA DC decreased S. epidermidis, S. aureus, E. coli, and P. aeruginosa biofilm formation on Teflon discs by 2, 1, 1, and 2 log10 cfu/cm(2), respectively (p < 0.05). Reductions in S. epidermidis, S. aureus, and E. coli biofilm formation were observed with as few as 12 hr of 200 µA DC (2, 2 and 0.4 log10 cfu/cm(2), resp.); a 1 log10 cfu/cm(2) reduction in P. aeruginosa biofilm formation was observed at 36 hr. 24 hr of 500 µA DC decreased C. albicans biofilm formation on Teflon discs by 2 log10 cfu/cm(2). No reduction in P. acnes biofilm formation was observed. 1 and 2 log10 cfu/cm(2) reductions in E. coli and S. epidermidis biofilm formation on titanium discs, respectively, were observed with 12 hr of exposure to 500 µA. Electrical current is a potential strategy to reduce biofilm formation on medical biomaterials.

Controlled Delivery of Vancomycin via Charged Hydrogels.

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.

Evaluation of the Enterococcus faecalis Biofilm-Associated Virulence Factors AhrC and Eep in Rat Foreign Body Osteomyelitis and In Vitro Biofilm-Associated Antimicrobial Resistance.

Enterococcus faecalis can cause healthcare-associated biofilm infections, including those of orthopedic devices. Treatment of enterococcal prosthetic joint infection is difficult, in part, due to biofilm-associated antimicrobial resistance. We previously showed that the E. faecalis OG1RF genes ahrC and eep are in vitro biofilm determinants and virulence factors in animal models of endocarditis and catheter-associated urinary tract infection. In this study, we evaluated the role of these genes in a rat acute foreign body osteomyelitis model and in in vitro biofilm-associated antimicrobial resistance. Osteomyelitis was established for one week following the implantation of stainless steel orthopedic wires inoculated with E. faecalis strains OG1RF, ΩahrC, and ∆eep into the proximal tibiae of rats. The median bacterial loads recovered from bones and wires did not differ significantly between the strains at multiple inoculum concentrations. We hypothesize that factors present at the infection site that affect biofilm formation, such as the presence or absence of shear force, may account for the differences in attenuation in the various animal models we have used to study the ΩahrC and ∆eep strains. No differences among the three strains were observed in the planktonic and biofilm antimicrobial susceptibilities to ampicillin, vancomycin, daptomycin, linezolid, and tetracycline. These findings suggest that neither ahrC nor eep directly contribute to E. faecalis biofilm-associated antimicrobial resistance. Notably, the experimental evidence that the biofilm attachment mutant ΩahrC displays biofilm-associated antimicrobial resistance suggests that surface colonization alone is sufficient for E. faecalis cells to acquire the biofilm antimicrobial resistance phenotype.

Antibiofilm Activity of Low-Amperage Continuous and Intermittent Direct Electrical Current.

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 µA 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 µA 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 µA for 1, 4, or 7 days against P. aeruginosa and 5 µA for 7 days against S. epidermidis and S. aureus biofilms. Delivery of 200 µA 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.

Causes and Implications of the Disappearance of Rifampin Resistance in a Rat Model of Methicillin-Resistant Staphylococcus aureus Foreign Body Osteomyelitis.

Orthopedic foreign body-associated infections are often treated with rifampin-based combination antimicrobial therapy. We previously observed that rifampin-resistant and methicillin-resistant Staphylococcus aureus (MRSA) isolates were present 2 days after cessation of rifampin therapy in experimental foreign body osteomyelitis. Unexpectedly, only rifampin-susceptible isolates were detected 14 days after the completion of treatment. We studied two rifampin-resistant isolates recovered 2 days after treatment and one rifampin-susceptible isolate recovered 14 days after treatment. Growing these isolates alone in vitro or in vivo demonstrated no fitness defects; however, in mixed culture, rifampin-susceptible bacteria outcompeted rifampin-resistant bacteria. In vivo, two courses of rifampin treatment (25 mg/kg of body weight every 12 h for 21 days) yielded a greater decrease in bacterial quantity in the bones of treated animals 14 days following treatment than that in animals receiving a single course of treatment (P = 0.0398). In infections established with equal numbers of rifampin-resistant and rifampin-susceptible bacteria, one course of rifampin treatment did not affect bacterial quantities. Rifampin-resistant and rifampin-susceptible isolates were recovered both 2 days and 14 days following treatment completion; however, the proportion of animals with rifampin-resistant isolates was lower at 14 days than that at 2 days following treatment completion (P = 0.024). In untreated animals infected with equal numbers of rifampin-resistant and rifampin-susceptible bacteria for 4 weeks, rifampin-susceptible isolates were exclusively recovered, indicating the outcompetition of rifampin-resistant by rifampin-susceptible isolates. The data presented imply that although there is no apparent fitness defect in rifampin-resistant bacteria when grown alone, they are outcompeted by rifampin-susceptible bacteria when the two are present together. The findings also suggest that selected rifampin resistance may not persist in initially rifampin-susceptible infections following the discontinuation of rifampin.

PCR-electrospray ionization mass spectrometry for direct detection of pathogens and antimicrobial resistance from heart valves in patients with infective endocarditis.

Microbiological diagnosis is pivotal to the appropriate management and treatment of infective endocarditis. We evaluated PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) for bacterial and candidal detection using 83 formalin-fixed paraffin-embedded 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.

Characterization of the effects of an rpoC mutation that confers resistance to the Fst peptide toxin-antitoxin system toxin.

Overexpression of the Fst toxin in Enterococcus faecalis strain OG1X leads to defects in chromosome segregation, cell division and, eventually, membrane integrity. The M7 mutant derivative of OG1X is resistant to most of these effects but shows a slight growth defect in the absence of Fst. Full-genome sequencing revealed two differences between M7 and its OG1X parent. First, OG1X contains a frameshift mutation that inactivates the etaR response regulator gene, while M7 is a wild-type revertant for etaR. Second, the M7 mutant contains a missense mutation in the rpoC gene, which encodes the ß' subunit of RNA polymerase. Mutagenesis experiments revealed that the rpoC mutation was primarily responsible for the resistance phenotype. Microarray analysis revealed that a number of transporters were induced in OG1X when Fst was overexpressed. These transporters were not induced in M7 in response to Fst, and further experiments indicated that this had a direct protective effect on the mutant cells. Therefore, exposure of cells to Fst appears to have a cascading effect, first causing membrane stress and then potentiation of these effects by overexpression of certain transporters.

Identification and characterization of a family of toxin-antitoxin systems related to the Enterococcus faecalis plasmid pAD1 par addiction module.

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 Gram-positive 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 Gram-positive species.

Translational regulation by an intramolecular stem-loop is required for intermolecular RNA regulation of the par addiction module.

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.