Ceftobiprole Medocaril Is an Effective Post-Exposure Treatment in the Fischer 344 Rat Model of Pneumonic Tularemia.

Francisella tularensis subspecies tularensis is a category-A biothreat agent that can cause lethal tularemia. Ceftobiprole medocaril is being explored as a medical countermeasure for the treatment of pneumonic tularemia. The efficacy of ceftobiprole medocaril against inhalational tularemia was evaluated in the Fischer 344 rat model of infection. The dose was expected to be effective against F. tularensis isolates with ceftobiprole minimum inhibitory concentrations ≤0.5 µg/mL. Animals treated with ceftobiprole medocaril exhibited a 92% survival rate 31 days post-challenge, identical to the survival of levofloxacin-treated rats. By comparison, rats receiving placebo experienced 100% mortality. Terminally collected blood, liver, lung, and spleen samples confirmed disseminated F. tularensis infections in most animals that died prior to completing treatments (placebo animals and a rat treated with ceftobiprole medocaril), although levels of bacteria detected in the placebo samples were significantly elevated compared to the ceftobiprole-medocaril-treated group geometric mean. Furthermore, no evidence of infection was detected in any rat that completed ceftobiprole medocaril or levofloxacin treatment and survived to the end of the post-treatment observation period. Overall, survival rates, body weights, and bacterial burdens consistently demonstrated that treatment with ceftobiprole medocaril is efficacious against otherwise fatal cases of pneumonic tularemia in the rat model.

Ceftobiprole activity against Gram-positive and Gram-negative pathogens causing bone and joint infections in the United States from 2016 to 2020.

Bone and joint infections (BJIs) present significant treatment challenges. Ceftobiprole, a broad-spectrum cephalosporin with activity against methicillin-resistant Staphylococcus aureus, is approved in many European and other countries for the treatment of adults with community- and hospital-acquired pneumonia, excluding ventilator-associated pneumonia. In this study, the in vitro activity of ceftobiprole and comparators was evaluated against clinical isolates collected from BJIs in the USA from 2016 to 2020. Gram-positive pathogens made up 70.6% of all BJI isolates and included S. aureus (47.4% of all isolates), ß-hemolytic streptococci, coagulase-negative staphylococci, and Enterococcus faecalis. Ceftobiprole was highly active against S. aureus (MIC50/90 values, 0.5/1 mg/L; 99.6% susceptible using the European Committee on Antimicrobial Susceptibility Testing susceptibility breakpoint of ≤2 mg/L for the treatment of pneumonia patients) and exhibited potent activity against the other Gram-positive cocci and the predominant BJI Gram-negative groups. These results support the further evaluation of ceftobiprole for this potential indication.

A Phase 3, Randomized, Investigator-blinded Trial Comparing Ceftobiprole With a Standard-of-care Cephalosporin, With or Without Vancomycin, for the Treatment of Pneumonia in Pediatric Patients.

BACKGROUND: The advanced-generation, broad-spectrum, intravenous (IV) cephalosporin, ceftobiprole, is an effective and well-tolerated treatment for adults with hospital-acquired pneumonia (HAP) or community-acquired pneumonia (CAP), but its effects in pediatric patients have not been established. METHODS: In this multicenter, investigator-blinded, active-controlled, phase 3 study, patients 3 months to <18 years old with HAP or CAP requiring hospitalization were randomized (2:1) to ceftobiprole versus standard-of-care (SoC) IV cephalosporin treatments (ceftazidime or ceftriaxone), with or without vancomycin. After at least 3 days' IV treatment, patients demonstrating clinical improvement could be switched to an oral antibiotic, to complete a minimum of 7 days' treatment. RESULTS: Overall, 138 patients were randomized to ceftobiprole (n = 94) or a SoC cephalosporin (n = 44). Median time to oral switch was 6.0 days in the ceftobiprole group and 8.0 days in the SoC cephalosporin group. While on IV therapy, adverse events and treatment-related adverse events were reported by 20.2% and 8.5% of ceftobiprole-treated patients and 18.2% and 0% of SoC cephalosporin-treated patients. Early clinical response rates at day 4 in the intention-to-treat population were 95.7% and 93.2% (between-group difference, 2.6%; 95% confidence interval, -5.5% to 14.7%) in the ceftobiprole and comparator groups, and clinical cure rates at the test-of-cure visit were 90.4% and 97.7% (between-group difference, -7.3%; 95% confidence interval, -15.7% to 3.6%), respectively. CONCLUSIONS: Ceftobiprole was well tolerated and, in this small phase 3 study, demonstrated similar efficacy to SoC cephalosporins in pediatric patients with HAP or CAP requiring hospitalization.

Ceftobiprole Compared With Vancomycin Plus Aztreonam in the Treatment of Acute Bacterial Skin and Skin Structure Infections: Results of a Phase 3, Randomized, Double-blind Trial (TARGET).

BACKGROUND: The development of novel broad-spectrum antibiotics, with efficacy against both gram-positive and gram-negative bacteria, has the potential to enhance treatment options for acute bacterial skin and skin structure infections (ABSSSIs). Ceftobiprole is an advanced-generation intravenous cephalosporin with broad in vitro activity against gram-positive (including methicillin-resistant Staphylococcus aureus) and gram-negative pathogens. METHODS: TARGET was a randomized, double-blind, active-controlled, parallel-group, multicenter, phase 3 noninferiority study that compared ceftobiprole with vancomycin plus aztreonam. The Food and Drug Administration-defined primary efficacy endpoint was early clinical response 48-72 hours after treatment initiation in the intent-to-treat (ITT) population and the European Medicines Agency-defined primary endpoint was investigator-assessed clinical success at the test-of-cure (TOC) visit. Noninferiority was defined as the lower limit of the 95% CI for the difference in success rates (ceftobiprole minus vancomycin/aztreonam) >-10%. Safety was assessed through adverse event and laboratory data collection. RESULTS: In total, 679 patients were randomized to ceftobiprole (n = 335) or vancomycin/aztreonam (n = 344). Early clinical success rates were 91.3% and 88.1% in the ceftobiprole and vancomycin/aztreonam groups, respectively, and noninferiority was demonstrated (adjusted difference: 3.3%; 95% CI: -1.2, 7.8). Investigator-assessed clinical success at the TOC visit was similar between the 2 groups, and noninferiority was demonstrated for both the ITT (90.1% vs 89.0%) and clinically evaluable (97.9% vs 95.2%) populations. Both treatment groups displayed similar microbiological success and safety profiles. CONCLUSIONS: TARGET demonstrated that ceftobiprole is noninferior to vancomycin/aztreonam in the treatment of ABSSSIs, in terms of early clinical response and investigator-assessed clinical success at the TOC visit. CLINICAL TRIALS REGISTRATION: NCT03137173.

Ceftobiprole Activity against Bacteria from Skin and Skin Structure Infections in the United States from 2016 through 2018.

Ceftobiprole medocaril is an advanced-generation cephalosporin prodrug that has qualified infectious disease product status granted by the US FDA and is currently being evaluated in phase 3 clinical trials in patients with acute bacterial skin and skin structure infections (ABSSSIs) and in patients with Staphylococcus aureus bacteremia. In this study, the activity of ceftobiprole and comparators was evaluated against more than 7,300 clinical isolates collected in the United States from 2016 through 2018 from patients with skin and skin structure infections. The major species/pathogen groups were S. aureus (53%), Enterobacterales (23%), Pseudomonas aeruginosa (7%), beta-hemolytic streptococci (6%), Enterococcus spp. (4%), and coagulase-negative staphylococci (2%). Ceftobiprole was highly active against S. aureus (MIC50/90, 0.5/1 mg/liter; 99.7% susceptible by EUCAST criteria; 42% methicillin-resistant S. aureus [MRSA]). Ceftobiprole also exhibited potent activity against other Gram-positive cocci. The overall susceptibility of Enterobacterales to ceftobiprole was 84.8% (>99.0% susceptible for isolate subsets that exhibited a non-extended-spectrum ß-lactamase [ESBL] phenotype). A total of 74.4% of P. aeruginosa, 100% of beta-hemolytic streptococci and coagulase-negative staphylococci, and 99.6% of Enterococcus faecalis isolates were inhibited by ceftobiprole at ≤4 mg/liter. As expected, ceftobiprole was largely inactive against Enterobacterales that contained ESBL genes and Enterococcus faecium Overall, ceftobiprole was highly active against most clinical isolates from the major Gram-positive and Gram-negative skin and skin structure pathogen groups collected at U.S. medical centers participating in the SENTRY Antimicrobial Surveillance Program during 2016 to 2018. The broad-spectrum activity of ceftobiprole, including potent activity against MRSA, supports its further evaluation for a potential ABSSSI indication.

Ceftobiprole activity when tested against contemporary bacteria causing bloodstream infections in the United States (2016-2017).

Ceftobiprole medocaril, the prodrug of ceftobiprole, is an advanced-generation cephalosporin that is approved in many European and non-European countries for the treatment of adults with hospital-acquired pneumonia (excluding ventilator-associated pneumonia) and community-acquired pneumonia and is currently being evaluated in a global phase 3 clinical trial of patients with Staphylococcus aureus bacteremia. This study investigated the in vitro activity of ceftobiprole and comparators against a total of 5466 gram-positive and -negative isolates from bloodstream infections (BSIs) that were collected in the United States during 2016 and 2017 as part of the SENTRY Antimicrobial Surveillance Program. Ceftobiprole was highly active (isolates were >99% susceptible) against S. aureus (including methicillin-resistant S. aureus), coagulase-negative staphylococci, Enterococcus faecalis, streptococci, and non-extended-spectrum ß-lactamase (non-ESBL) phenotype Enterobacteriaceae. As expected, lower activities were observed against Enterococcus faecium, ESBL-phenotype Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii. These results support further clinical evaluation of ceftobiprole for the treatment of BSIs caused by susceptible organisms.

Ceftobiprole Activity against Gram-Positive and -Negative Pathogens Collected from the United States in 2006 and 2016.

Ceftobiprole is an advanced cephalosporin with potent activity against Gram-positive and Gram-negative bacteria that has been approved in many European and non-European countries to treat community- and hospital-acquired pneumonia (excluding ventilator-associated pneumonia). This study reports on the activity of ceftobiprole against a large set of clinical isolates obtained from hospitalized patients in the United States in 2016 that caused serious infections, including pneumonia, bacteremia, and skin and skin structure infections. To assess any potential temporal changes in ceftobiprole activity, the 2016 results were compared to corresponding MIC data from a 2006 U.S. survey that included key target pathogens. Ceftobiprole exhibited potent activity against Staphylococcus aureus (including methicillin-resistant S. aureus isolates, which were 99.3% susceptible), coagulase-negative staphylococci (100% susceptible), Enterococcus faecalis (100% susceptible), Streptococcus pneumoniae (99.7% susceptible), and other tested streptococci. Similarly, ceftobiprole was highly active against Enterobacteriaceae isolates that did not exhibit an extended-spectrum ß-lactamase (ESBL) phenotype, including Escherichia coli (99.8% susceptible) and Klebsiella pneumoniae (99.6% susceptible). A total of 99.6% of all Haemophilus influenzae and Moraxella catarrhalis isolates were inhibited at ≤1 mg/liter ceftobiprole, and 72.7% of the Pseudomonas aeruginosa isolates were susceptible to ceftobiprole. With the exception of decreased cephalosporin susceptibility among Enterobacteriaceae isolates, which correlates with an increased prevalence of ESBL-producing isolates, ceftobiprole had similar activities against the isolate sets collected in 2006 and 2016. Therefore, ceftobiprole remains highly active when tested in vitro against a large number of current Gram-positive or Gram-negative pathogens that cause serious infections.

Susceptibility to ceftobiprole of respiratory-tract pathogens collected in the United Kingdom and Ireland during 2014-2015.

PURPOSE: Lower respiratory tract infections (LRTIs) can cause significant morbidity and mortality and are becoming increasingly difficult to treat because of the growing prevalence of resistance to conventional antimicrobial agents. This study aimed to assess the current in vitro susceptibility of respiratory tract pathogens collected from the UK and Ireland to ceftobiprole, an advanced-generation cephalosporin, as compared with other antibiotics. METHODS: Pathogens isolated from patients with LRTIs were analyzed as part of the British Society for Antimicrobial Chemotherapy Antimicrobial Resistance Surveillance Programme during 2014-2015. Antibiotic susceptibility was evaluated using European Committee on Antimicrobial Susceptibility Testing breakpoints, including the ceftobiprole pharmacokinetic/pharmacodynamic non-species-specific breakpoint when species-specific breakpoints were not available. RESULTS: One thousand one hundred and sixty-eight isolates from community-onset LRTIs and 1,264 isolates from hospital-onset LRTIs were analyzed. The ceftobiprole susceptibility rate was 99.8% (428/429) for Streptococcus pneumoniae, 100% (502/502) for Haemophilus influenzae, and 99.6% (236/237) for Moraxella catarrhalis. All Staphylococcus aureus isolates, including methicillin-susceptible S. aureus (MSSA; N=181) and methicillin-resistant S. aureus (MRSA; N=35), were susceptible to ceftobiprole. Overall, ceftobiprole susceptibility was observed in 88.1% (215/244) of Escherichia coli isolates, 83.4% (156/187) of Klebsiella pneumoniae isolates and 86.7% (98/113) of Enterobacter spp. isolates. CONCLUSION: Ceftobiprole had in vitro activity against all S. aureus (both MSSA and MRSA) isolates, and almost all S. pneumoniae isolates, as well as against Gram-negative bacteria associated with community-onset or hospital-onset LRTIs. Based on this analysis, ceftobiprole is a good treatment option when broad-spectrum antibiotic coverage is needed for LRTIs.

Evaluation of the bactericidal activity of Telavancin against Staphylococcus aureus using revised testing guidelines.

The in vitro broth microdilution testing method for telavancin, a lipoglycopeptide active against S. aureus, was revised in 2014 to include polysorbate-80 in the test media. This study evaluates the bactericidal activity of telavancin against S. aureus in media containing polysorbate-80 by in vitro time-kill analysis alongside relevant comparators.

In Vitro Activity of Telavancin Against Clinically Important Gram-Positive Pathogens from 69 U.S. Medical Centers (2015): Potency Analysis by U.S. Census Divisions.

A total of 8,072 gram-positive isolates collected from 69 medical centers among all 9 U.S. Census Divisions during the 2015 SENTRY Antimicrobial Surveillance Program were included. Telavancin had minimal inhibitory concentration (MIC)50 and MIC90 values of 0.03/0.06 µg/mL, respectively, against methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). Similar activity was also observed among coagulase-negative staphylococci (MIC50/90, 0.03/0.06 µg/ml; 100.0% inhibited by ≤0.12 µg/ml). Telavancin was active against vancomycin-susceptible Enterococcus faecalis (MIC50/90, 0.12/0.12 µg/ml) and Enterococcus faecium (MIC50/90, 0.03/0.06 µg/ml), but was less active against vancomycin-resistant E. faecium (MIC90, >2 µg/ml) and E. faecalis (MIC90, >2 µg/ml). Streptococci showed modal MIC results of 0.008 µg/ml (Streptococcus pneumoniae) to 0.015 µg/ml (ß-hemolytic streptococci and viridans group streptococci). Potency variations for telavancin within Census Divisions were not observed. Telavancin remains potent in vitro against indicated pathogens recovered from U.S. medical centers (2015).

Telavancin activity in vitro tested against a worldwide collection of Gram-positive clinical isolates (2014).

OBJECTIVES: Telavancin activity was measured against Gram-positive pathogens collected worldwide during 2014 using the revised broth microdilution testing method. The results were compared with previous reports. METHODS: A total of 10552 isolates from 86 sites located in 32 countries were included as part of the Telavancin International Surveillance Program for the Americas, Europe and Asia-Pacific. RESULTS: Telavancin had MIC50 and MIC90 values of 0.03µg/mL and 0.06µg/mL, respectively, against Staphylococcus aureus, regardless of methicillin susceptibility, and inhibited all S. aureus isolates at the CLSI breakpoint (≤0.12µg/mL). Telavancin was eight-fold more active than daptomycin (MIC50/90, 0.25/0.5µg/mL) and 16-32-fold more active than vancomycin (MIC50/90, 1/1µg/mL) and linezolid (MIC50/90, 1/1µg/mL) against methicillin-resistant S. aureus (MRSA). Telavancin was also active against coagulase-negative staphylococci isolates (MIC50/90, 0.06/0.06µg/mL). Vancomycin-susceptible Enterococcus faecalis isolates (n=718) were all inhibited by telavancin (MIC50/90, 0.12/0.12µg/mL) at ≤0.25µg/mL (CLSI breakpoint), except for a single isolate (MIC, 0.5µg/mL). All Enterococcus faecium and E. faecalis isolates with telavancin MICs of ≥0.5µg/mL and ≥1µg/mL, respectively, had a VanA phenotype. Based on the MIC90 values, telavancin was at least eight-fold more potent than comparators against vancomycin-susceptible enterococci. With the exception of Streptococcus agalactiae (MIC50, 0.03µg/mL), all tested species of streptococci exhibited telavancin MIC50 values of ≤0.015µg/mL. CONCLUSIONS: These in vitro results from 2014 highlight the potent in vitro activity of telavancin and demonstrate that its activity was virtually unchanged compared with the corresponding 2013 telavancin surveillance results.

Activity of telavancin against Gram-positive pathogens isolated from bone and joint infections in North American, Latin American, European and Asia-Pacific nations.

Telavancin was tested against a worldwide collection of Gram-positive pathogens (967) isolated from bone and joint infections (BJI). Most BJI isolates were from the United States (US) (49.9%) followed by Europe (26.4%), Latin America (LATAM; 14.4%), and Asia-Pacific (APAC; 9.3%). Organisms were tested by broth microdilution susceptibility methods. S. aureus (66.4%; range of 48.9% in APAC to 71.2% in LATAM) was the most common pathogen and had a 35.7% methicillin resistance (MRSA) rate and telavancin MIC50/90 of 0.03/0.06µg/mL (100% susceptible). MRSA isolates that were daptomycin resistant (0.2%) were telavancin susceptible. CoNS (12.1% of BJI) had telavancin MIC50/90 at 0.06/0.06µg/mL, and 13.7% were teicoplanin resistant. Enterococci had telavancin MIC50/90 at 0.12/0.25µg/mL, but telavancin inhibited vancomycin-susceptible isolates at ≤0.25µg/mL. All streptococci were telavancin susceptible (MIC90, 0.03-0.06µg/mL). The in vitro results presented here warrant further investigations to access the role of telavancin for BJI/osteomyelitis treatment caused by Gram-positive cocci.

Assessment of telavancin minimal inhibitory concentrations by revised broth microdilution method in phase 3 complicated skin and skin-structure infection clinical trial isolates.

The broth microdilution (BMD) MIC testing method for telavancin was recently revised BMD (rBMD) to improve accuracy and reproducibility. Staphylococcus aureus isolates from telavancin phase 3 complicated skin and skin-structure infection (cSSSI) studies were tested using the rBMD method. Retesting of 1132 isolates produced MICs ranging from ≤0.015 to 0.12µg/mL that were 8-fold lower than the original method. All isolates tested remained susceptible to telavancin at the revised susceptibility breakpoint of 0.12µg/mL. The clinical cure and microbiological eradication rates were 90% (368/409) and 89% (366/409) for telavancin-treated patients, and were similar for patients with methicillin-susceptible and -resistant S. aureus isolates and S. aureus isolates with elevated vancomycin MICs (≥1µg/mL). The data presented here are aimed to update the literature and better inform clinicians and clinical microbiologists about the revised telavancin MICs, as well as the corresponding clinical and microbiological cure rates for cSSSI patients.

Assessment of Minimum Inhibitory Concentrations of Telavancin by Revised Broth Microdilution Method in Phase 3 Hospital-Acquired Pneumonia/Ventilator-Associated Pneumonia Clinical Isolates.

INTRODUCTION: The broth microdilution method (BMD) for testing telavancin minimum inhibitory concentrations (MICs) was revised (rBMD) in 2014 to improve the accuracy, precision, and reproducibility of the testing method. The aim of this study was to determine the effect of the revised method on telavancin MIC values for Staphylococcus aureus (S. aureus) clinical isolates obtained from hospital-acquired pneumonia (HAP) patients. METHODS: Isolates from patients who participated in the phase 3 Assessment of Telavancin for Treatment of HAP Studies were retested using the rBMD method. RESULTS: Retesting of 647 isolates produced a range of telavancin MIC values from 0.015 µg/mL to 0.12 µg/mL with MIC50/90 values of 0.06/0.06 µg/mL for the total pool of samples. For methicillin-resistant S. aureus (MRSA), MIC50/90 values were 0.06/0.12 µg/mL. These values are up to 4-fold lower than MIC50/90 values obtained using the original method. These results were used in part to justify lowering the telavancin breakpoints. All tested isolates remained susceptible to telavancin at the revised susceptibility breakpoint of ≤0.12 µg/mL. Overall, the clinical cure rate for microbiologically evaluable telavancin-treated patients was 78% for S. aureus, 76% for patients with MRSA, and 79% for patients with isolates with reduced susceptibility to vancomycin (MIC ≥1 µg/mL). CONCLUSION: Results from the rBMD method support the in vitro potency of telavancin against S. aureus. TRIAL REGISTRATION: ATTAIN (NCT00107952 and NCT00124020). FUNDING: Theravance Biopharma Antibiotics, Inc.

Telavancin activity tested against a collection of Staphylococcus aureus isolates causing pneumonia in hospitalized patients in the United States (2013-2014).

Telavancin is approved in the United States and Europe for the treatment of hospital-acquired bacterial pneumonia (HABP) and ventilator-associated bacterial pneumonia caused by Staphylococcus aureus. This study provides a current in vitro activity analysis for telavancin and comparators against 1353 United States S. aureus causing pneumonia in hospitalized patients during 2013-2014. Telavancin was found to exhibit potent activity (MIC90, 0.06µg/mL; 16-fold more active than vancomycin or linezolid) against all S. aureus isolates, including the HABP subset, multidrug-resistant isolates, and those less susceptible to vancomycin.

A review of telavancin activity in in vitro biofilms and animal models of biofilm-associated infections.

Tissue- and device-associated biofilm infections are important medical problems. These infections are difficult to treat due to a high-level of tolerance to antibiotics. Telavancin has been studied in several in vitro biofilm models and has demonstrated efficacy against staphylococcal and enterococcal-associated biofilm infections, including those formed by methicillin-resistant Staphylococcus aureus. Telavancin was effective against the difficult-to-treat vancomycin- and glycopeptide-intermediate strains of S. aureus in these models. Furthermore, the efficacy of telavancin has been evaluated in several biofilm-related in vivo models, including osteomyelitis, endocarditis and device-associated infections in rabbits. Overall, telavancin exhibited similar or greater efficacy than vancomycin and other comparators in these animal models and maintained activity against vancomycin-intermediate and daptomycin nonsusceptible strains of S. aureus.

A novel C-terminal mutation resulting in constitutive activation of the Listeria monocytogenes central virulence regulatory factor PrfA.

The environmental bacterium Listeria monocytogenes survives and replicates in a variety of diverse ecological niches that range from the soil to the cytosol of infected mammalian cells. The ability of L. monocytogenes to replicate within an infected host requires the expression of a number of secreted bacterial gene products whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells; however, the mechanism by which this activation occurs remains unknown. Here we describe a novel C-terminal mutation that results in the high-level constitutive activation of PrfA and yet, in contrast with other described prfA* activation mutations, only modestly increases PrfA DNA binding affinity. L. monocytogenes strains containing the prfA P219S mutation exhibited high levels of PrfA-dependent virulence gene expression, were hyperinvasive in tissue culture models of infection, were fully motile and were hypervirulent in mice. In contrast with PrfA G145S and other mutationally activated PrfA proteins, the PrfA P219S protein readily formed homodimers and did not exhibit a dramatic increase in its DNA-binding affinity for target promoters. Interestingly, the prfA P219S mutation is located adjacent to the prfA K220 residue that has been previously reported to contribute to PrfA DNA binding activity. prfA P219S therefore appears to constitutively activate PrfA via a novel mechanism which minimally affects PrfA DNA binding in vitro.

Probing the role of protein surface charge in the activation of PrfA, the central regulator of Listeria monocytogenes pathogenesis.

Listeria monocytogenes is a food-borne intracellular bacterial pathogen capable of causing serious human disease. L. monocytogenes survival within mammalian cells depends upon the synthesis of a number of secreted virulence factors whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells where it induces the expression of gene products required for bacterial spread to adjacent cells. Activation of PrfA appears to occur via the binding of a small molecule cofactor whose identity remains unknown. Electrostatic modeling of the predicted PrfA cofactor binding pocket revealed a highly positively charged region with two lysine residues, K64 and K122, located at the edge of the pocket and another (K130) located deep within the interior. Mutational analysis of these residues indicated that K64 and K122 contribute to intracellular activation of PrfA, whereas a K130 substitution abolished protein activity. The requirement of K64 and K122 for intracellular PrfA activation could be bypassed via the introduction of the prfA G145S mutation that constitutively activates PrfA in the absence of cofactor binding. Our data indicate that the positive charge of the PrfA binding pocket contributes to intracellular activation of PrfA, presumably by facilitating binding of an anionic cofactor.