Inhaler Formulary Change in COPD and the Association with Exacerbations, Health Care Utilization, and Costs.

Rationale: Prescription formularies specify which medications are available to patients. Formularies change frequently, potentially forcing patients to switch medications for nonclinical indications (nonmedical switching). Nonmedical switching is known to impact disease control and adherence. The consequences of nonmedical switching have not been rigorously studied in COPD. Methods: We conducted a cohort study of Veterans with COPD on inhaler therapy in January 2016 when formoterol was removed from the Department of Veterans Affairs (VA) national formulary. A 2-point difference-in-differences analysis using multivariable negative binomial and generalized linear models was performed to estimate the association of the formulary change with patient outcomes in the 6 months before and after the change. Our primary outcome was the number of COPD exacerbations in 6 months, with secondary outcomes of total health care encounters and encounter-related costs in 6 months. Results: We identified 10,606 Veterans who met our inclusion criteria, of which 409 (3.9%) experienced nonmedical switching off formoterol. We did not identify a change in COPD exacerbations (-0.04 exacerbations; 95% confidence interval [CI] -0.12, 0.03) associated with the formulary change. In secondary outcome analysis, we did not observe a change in the number of health care encounters (-0.12 visits; 95% CI -1.00, 0.77) or encounter-related costs ($369; 95% CI -$1141, $1878). Conclusions: Among COPD patients on single inhaler therapy, nonmedical inhaler switches due to formulary discontinuation of formoterol were not associated with changes in COPD exacerbations, encounters, or encounter-related costs. Additional research is needed to confirm our findings in more severe disease and other settings.

Trends and Rural-Urban Differences in the Initial Prescription of Low-Value Inhaled Corticosteroids among U.S. Veterans with Chronic Obstructive Pulmonary Disease.

Rationale: Guidelines recommend inhaled corticosteroids (ICS) for patients with chronic obstructive pulmonary disease (COPD) and select indications, including asthma history, high exacerbation risk, or high serum eosinophils. ICS are commonly prescribed outside of these indications, despite evidence of harm. We defined a "low-value" ICS prescription as the receipt of an ICS without evidence of a guideline-recommended indication. ICS prescription patterns are not well characterized and could inform health system interventions to reduce low-value practices. Objectives: To evaluate the national trends in initial low-value ICS prescriptions in the U.S. Department of Veterans Affairs and to determine whether rural-urban differences in low-value ICS prescribing exist. Methods: We performed a cross-sectional study between January 4, 2010, and December 31, 2018, identifying veterans with COPD who were new users of inhaler therapy. We defined low-value ICS as prescriptions in patients with 1) no asthma, 2) low risk of future exacerbation (Global Initiative for Chronic Obstructive Lung Disease group A or B), and 3) serum eosinophils <300 cells/µl. We performed multivariable logistic regression to evaluate trends in low-value ICS prescription over time, adjusting for potential confounders. We performed fixed effects logistic regression to assess rural-urban prescribing patterns. Results: We identified a total of 131,009 veterans with COPD starting inhaler therapy, 57,472 (44%) of whom were prescribed low-value ICS as initial therapy. From 2010 to 2018, the probability of receiving low-value ICS as initial therapy increased by 0.42 percentage points per year (95% confidence interval, 0.31-0.53). Compared with urban residence, rural residence was associated with a 2.5-percentage-point (95% confidence interval, 1.9-3.1) higher probability of receiving low-value ICS as initial therapy. Conclusions: The prescription of low-value ICS as initial therapy is common and increasing slightly over time for both rural and urban veterans. Given the widespread and persistent nature of low-value ICS prescribing, health system leaders should consider system-wide approaches to address this low-value prescribing practice.

Low-Value Inhaled Corticosteroids in Chronic Obstructive Pulmonary Disease and the Association with Healthcare Utilization and Costs.

Rationale: Inhaled corticosteroids (ICS) are not first-line therapy for patients with chronic obstructive pulmonary disease (COPD) at low risk of exacerbations, but they are commonly prescribed despite evidence of harm. We consider ICS prescription in this population to be of "low value." The association of low-value ICS with subsequent healthcare utilization and costs is unknown. Understanding this relationship could inform efforts to reduce the delivery of low-value care. Objectives: To determine whether low-value ICS prescribing is associated with higher outpatient healthcare utilization and costs among patients with COPD who are at low risk of exacerbation. Methods: We performed a cohort study between January 1, 2010, and December 31, 2018, identifying a cohort of veterans with COPD who performed pulmonary function tests (PFTs) at 21 Veterans Affairs medical centers nationwide. Patients were defined as having low exacerbation risk if they experienced less than two outpatient exacerbations and no hospital admissions for COPD in the year before PFTs. Our primary exposure was the receipt of an ICS prescription in the 3 months before the date of PFTs. Our primary outcomes were outpatient utilization and outpatient costs in the 1 year after PFTs. For inference, we generated negative binomial models for utilization and generalized linear models for costs, adjusting for confounders. Results: We identified a total of 31,551 patients with COPD who were at low risk of exacerbation. Of these patients, 9,742 were prescribed low-value ICS (mean [standard deviation (SD)] age, 69 [9] yr), and 21,809 were not prescribed low-value ICS (mean [SD] age, 68 [9] yr). Compared with unexposed patients, those exposed to low-value ICS had 0.53 more encounters per patient per year (95% confidence interval CI, 0.23-0.83) and incurred $154.72 higher costs/patient/year (95% CI, $45.58-$263.86). Conclusions: Low-value ICS prescription was associated with higher subsequent outpatient healthcare utilization and costs. Potential mechanisms for the observed association are that 1) low-value ICS may be a marker of poor respiratory symptom control, 2) there is confounding by indication, or 3) low-value ICS results in increased drug costs or utilization. Health systems should identify low-value ICS prescriptions as a target to improve value-based care.

Association of Guideline-Recommended COPD Inhaler Regimens With Mortality, Respiratory Exacerbations, and Quality of Life: A Secondary Analysis of the Long-Term Oxygen Treatment Trial.

BACKGROUND: Although inhaled therapy reduces exacerbations among patients with COPD, the effectiveness of providing inhaled treatment per risk stratification models remains unclear. RESEARCH QUESTION: Are inhaled regimens that align with the 2017 Global Initiative for Chronic Obstructive Lung Disease (GOLD) strategy associated with clinically important outcomes? STUDY DESIGN AND METHODS: We conducted secondary analyses of Long-term Oxygen Treatment Trial (LOTT) data. The trial enrolled patients with COPD with moderate resting or exertional hypoxemia between 2009 and 2015. Our exposure was the patient-reported inhaled regimen at enrollment, categorized as either aligning with, undertreating, or potentially overtreating per the 2017 GOLD strategy. Our primary composite outcome was time to death or first hospitalization for COPD. Additional outcomes included individual components of the composite outcome and time to first exacerbation. We generated multivariable Cox proportional hazard models across strata of GOLD-predicted exacerbation risk (high vs low) to estimate between-group hazard ratios for time to event outcomes. We adjusted models a priori for potential confounders, clustered by site. RESULTS: The trial enrolled 738 patients (73.4% men; mean age, 68.8 years). Of the patients, 571 (77.4%) were low risk for future exacerbations. Of the patients, 233 (31.6%) reported regimens aligning with GOLD recommendations; most regimens (54.1%) potentially overtreated. During a 2.3-year median follow-up, 332 patients (44.9%) experienced the composite outcome. We found no difference in time to composite outcome or death among patients reporting regimens aligning with recommendations compared with undertreated patients. Among patients at low risk, potential overtreatment was associated with higher exacerbation risk (hazard ratio, 1.42; 95% CI, 1.09-1.87), whereas inhaled corticosteroid treatment was associated with 64% higher risk of pneumonia (incidence rate ratio, 1.64; 95% CI, 1.01-2.66). INTERPRETATION: Among patients with COPD with moderate hypoxemia, we found no difference in clinical outcomes between inhaled regimens aligning with the 2017 GOLD strategy compared with those that were undertreated. These findings suggest the need to reevaluate the effectiveness of risk stratification model-based inhaled treatment strategies.

MRP4 Modulation of the Guanylate Cyclase-C/cGMP Pathway: Effects on Linaclotide-Induced Electrolyte Secretion and cGMP Efflux.

MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.

Guanylate cyclase-C/cGMP: an emerging pathway in the regulation of visceral pain.

Activation of guanylate cyclase-C (GC-C) expressed predominantly on intestinal epithelial cells by guanylin, uroguanylin or the closely related GC-C agonist peptide, linaclotide, stimulates generation, and release of cyclic guanosine-3',5'-monophosphate (cGMP). Evidence that the visceral analgesic effects of linaclotide are mediated by a novel, GC-C-dependent peripheral sensory mechanism was first demonstrated in animal models of visceral pain. Subsequent studies with uroguanylin or linaclotide have confirmed the activation of a GC-C/cGMP pathway leading to increased submucosal cGMP mediated by cGMP efflux pumps, which modulates intestinal nociceptor function resulting in peripheral analgesia. These effects can be reproduced by the addition of exogenous cGMP and support a role for GC-C/cGMP signaling in the regulation of visceral sensation, a physiological function that has not previously been linked to the GC-C/cGMP pathway. Notably, targeting the GC-C/cGMP pathway for treatment of gastrointestinal pain and abdominal sensory symptoms has now been validated in the clinic. In 2012, linaclotide was approved in the United States and European Union for the treatment of adult patients with irritable bowel syndrome with constipation.

Gastrointestinal pain: unraveling a novel endogenous pathway through uroguanylin/guanylate cyclase-C/cGMP activation.

The natural hormone uroguanylin regulates intestinal fluid homeostasis and bowel function through activation of guanylate cyclase-C (GC-C), resulting in increased intracellular cyclic guanosine-3',5'-monophosphate (cGMP). We report the effects of uroguanylin-mediated activation of the GC-C/cGMP pathway in vitro on extracellular cGMP transport and in vivo in rat models of inflammation- and stress-induced visceral hypersensitivity. In vitro exposure of intestinal Caco-2 cells to uroguanylin stimulated bidirectional, active extracellular transport of cGMP into luminal and basolateral spaces. cGMP transport was significantly and concentration dependently decreased by probenecid, an inhibitor of cGMP efflux pumps. In ex vivo Ussing chamber assays, uroguanylin stimulated cGMP secretion from the basolateral side of rat colonic epithelium into the submucosal space. In a rat model of trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity, orally administered uroguanylin increased colonic thresholds required to elicit abdominal contractions in response to colorectal distension (CRD). Oral administration of cGMP mimicked the antihyperalgesic effects of uroguanylin, significantly decreasing TNBS- and restraint stress-induced visceromotor response to graded CRD in rats. The antihyperalgesic effects of cGMP were not associated with increased colonic spasmolytic activity, but were linked to significantly decreased firing rates of TNBS-sensitized colonic afferents in rats in response to mechanical stimuli. In conclusion, these data suggest that the continuous activation of the GC-C/cGMP pathway along the intestinal tract by the endogenous hormones guanylin and uroguanylin results in significant reduction of gastrointestinal pain. Extracellular cGMP produced on activation of GC-C is the primary mediator in this process via modulation of sensory afferent activity.

Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.

Linaclotide, a potent guanylate cyclase C agonist, is a therapeutic peptide approved in the United States for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation. We present for the first time the metabolism, degradation, and disposition of linaclotide in animals and humans. We examined the metabolic stability of linaclotide in conditions that mimic the gastrointestinal tract and characterized the metabolite MM-419447 (CCEYCCNPACTGC), which contributes to the pharmacologic effects of linaclotide. Systemic exposure to these active peptides is low in rats and humans, and the low systemic and portal vein concentrations of linaclotide and MM-419447 observed in the rat confirmed both peptides are minimally absorbed after oral administration. Linaclotide is stable in the acidic environment of the stomach and is converted to MM-419447 in the small intestine. The disulfide bonds of both peptides are reduced in the small intestine, where they are subsequently proteolyzed and degraded. After oral administration of linaclotide, <1% of the dose was excreted as active peptide in rat feces and a mean of 3-5% in human feces; in both cases MM-419447 was the predominant peptide recovered. MM-419447 exhibits high-affinity binding in vitro to T84 cells, resulting in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3',5'-monophosphate (cGMP). In rat models of gastrointestinal function, orally dosed MM-419447 significantly increased fluid secretion into small intestinal loops, increased intraluminal cGMP, and caused a dose-dependent acceleration in gastrointestinal transit. These results demonstrate the importance of the active metabolite in contributing to linaclotide's pharmacology.

Evaluation of an alternative posaconazole prophylaxis regimen in haematological malignancy patients receiving concomitant stress ulcer prophylaxis.

Posaconazole (PCZ) is approved for fungal prophylaxis in high-risk neutropenic patients. Unfortunately, PCZ oral absorption is affected by nutritional intake and drug interactions with proton pump inhibitors (PPIs) and possibly histamine-2 antagonists (H2As). Cancer patients frequently receive stress ulcer prophylaxis (SUP) with PPIs or H2As. Recommended PCZ steady-state concentrations (C(ss)) are difficult to achieve using the traditional dosing regimen of 200 mg thrice daily. Given the paucity of guidance on PCZ dosing in patients receiving SUP, this study evaluated attainment of targeted PCZ C(ss) (0.5 µg/mL and 0.7 µg/mL) with two different PCZ dosing regimens when SUP was given. Twenty patients received the traditional dosing and 34 patients received 400 mg twice daily. Median PCZ C(ss) levels were 0.37 µg/mL and 0.32 µg/mL with the traditional and 400 mg twice-daily regimens, respectively (P=0.809). When stratified by type of SUP, H2A patients had a median PCZ C(ss) of 0.39 µg/mL, whereas PPI patients had a median PCZ C(ss) of 0.32 µg/mL. Despite having more patients with PCZ C(ss) >0.5 µg/mL in the H2A group, a statistical significance was not found (P=0.368). Multiple logistic regression did show that increasing age [odds ratio (OR)=1.08, 95% confidence interval (CI) 1.03-1.19] and use of H2As over PPIs (OR=6.8, 95% CI 1.22-55.16] was associated with PCZ target attainment. These results suggest that PCZ target attainment is similar with either PCZ regimen but that there may be less of an interaction with H2As compared with PPIs.

A post-marketing evaluation of posaconazole plasma concentrations in neutropenic patients with haematological malignancy receiving posaconazole prophylaxis.

This is one of the first studies to report on therapeutic drug monitoring (TDM) of posaconazole (PCZ) for antifungal prophylaxis in patients with acute myelogenous leukaemia or myelodysplastic syndrome outside of the rigours of clinical licencing trials. A number of factors have been identified or proposed as causing poor oral absorption of PCZ. Putative PCZ concentrations have been recommended for TDM (0.5 µg/mL or 0.7 µg/mL). In this study, 19 (90.5%) of 21 patients failed to reach the higher putative target of 0.7 µg/mL, and 16 patients (76.2%) failed to reach the lower target of 0.5 µg/mL. Increasing the dose did not help four of six patients reach target concentrations. Three of the patients developed 'proven' or 'possible' fungal infections, all with PCZ concentrations <0.5 µg/mL. Use of acid-suppressing agents appears to explain some of the poor absorption. TDM of PCZ is warranted in patients receiving this orally administered agent.

Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.

Linaclotide is a first-in-class, orally administered 14-amino acid peptide that is in development for the treatment of irritable bowel syndrome with constipation and chronic constipation. We have characterized the solution structure of linaclotide, the in vitro binding and agonist activity to guanylate cyclase C receptors, the stability of linaclotide under conditions mimicking the gastric environment, oral bioavailability, and the pharmacodynamic effects in rat models of gastrointestinal transit and intestinal secretion. Nuclear magnetic resonance spectroscopy analysis determined that the molecular structure of linaclotide is stabilized by three intramolecular disulfide bridges. Linaclotide exhibited high affinity and pH-independent binding (K(i): 1.23-1.64 nM) to guanylate cyclase C receptors on human colon carcinoma T84 cells and concomitantly, linaclotide binding resulted in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3', 5'-monophosphate (cGMP) (EC50:99 nM). Linaclotide was stable after 3 h incubation in simulated gastric fluid (pH 1) and similarly, was completely resistant to hydrolysis by pepsin. Pharmacokinetic analysis of linaclotide showed very low oral bioavailability (0.1%). Orally administered linaclotide elicited a significant, dose-dependent increase in gastrointestinal transit rates in rats at doses of ≥5 µg/kg. Exposure of surgically ligated small intestinal loops to linaclotide induced a significant increase in fluid secretion, accompanied by a significant increase in intraluminal cGMP levels. These results suggest that the guanylate cyclase C agonist linaclotide elicits potent pharmacological responses locally in the gastrointestinal tract, and that orally administered guanylate cyclase C agonists may be capable of improving bowel habits in patients suffering from irritable bowel syndrome with constipation and chronic constipation.

Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.

AIMS: Linaclotide is an orally administered 14-amino acid peptide being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) and chronic constipation. We determined the stability of linaclotide in the intestine, measured the oral bioavailability, and investigated whether the pharmacodynamic effects elicited in rodent models of gastrointestinal function are mechanistically linked to the activation of intestinal guanylate cyclase C (GC-C). MAIN METHODS: Linaclotide binding to intestinal mucosal membranes was assessed in competitive binding assays. Stability and oral bioavailability of linaclotide were measured in small intestinal fluid and serum, respectively, and models of gastrointestinal function were conducted using wild type (wt) and GC-C null mice. KEY FINDINGS: Linaclotide inhibited in vitro [(125)I]-STa binding to intestinal mucosal membranes from wt mice in a concentration-dependent manner. In contrast, [(125)I]-STa binding to these membranes from GC-C null mice was significantly decreased. After incubation in vitro in jejunal fluid for 30 min, linaclotide was completely degraded. Pharmacokinetic analysis showed very low oral bioavailability (0.10%). In intestinal secretion and transit models, linaclotide exhibited significant pharmacological effects in wt, but not in GC-C null mice: induction of increased fluid secretion into surgically ligated jejunal loops was accompanied by the secretion of elevated levels of cyclic guanosine-3',5'-monophosphate and accelerated gastrointestinal transit. SIGNIFICANCE: Linaclotide is a potent and selective GC-C agonist that elicits pharmacological effects locally in the gastrointestinal tract. This pharmacological profile suggests that orally administered linaclotide may be capable of improving the abdominal symptoms and bowel habits of patients suffering from IBS-C and chronic constipation.

Inhibition of filamentation can be used to treat disseminated candidiasis.

Candida albicans remains the leading causative agent of invasive fungal infection. Although the importance of filamentation in C. albicans pathogenesis has been extensively investigated, in vivo studies to date have been unable to dissect the role of this developmental process in the establishment of infection versus the development of active disease as characterized by damage to the host leading to mortality. To address this issue, we genetically engineered a C. albicans tet-NRG1 strain in which filamentation and virulence can be modulated both in vitro and in vivo simply by the presence or absence of doxycycline (DOX): this strain enabled us, in a prior study, to demonstrate that yeast-form cells were able to infect the deep organs but caused no disease unless filamentation (induced by the addition of DOX) was allowed to occur. In the present study, we examined whether inhibiting filamentation (by withdrawing the DOX) at 24 or 48 h postinfection could serve as an effective therapeutic intervention against candidiasis. The results obtained indicate that DOX removal led to an alteration in the morphology of the infecting fungal cells and a dramatic increase in survival, but as with conventional antifungal drug therapy regimens, mortality rates increased markedly the longer this intervention was delayed. These observations reinforce the importance of invasive filamentous growth in causing the damage to the host and the lethality associated with active disease and suggest this process could be fruitfully targeted for the development of new antifungal agents.

A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function.

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.

Characterization of the Streptococcus pneumoniae NADH oxidase that is required for infection.

Streptococcus pneumoniae is an important human pathogen capable of causing serious infections. NADH oxidase, a factor necessary for infection, was previously identified as part of a signature-tagged mutagenesis screen of a S. pneumoniae clinical isolate, 0100993. The mutant, with a plasmid insertion disrupting the nox gene, was attenuated for virulence in a murine respiratory tract infection model. A complete refined nox deletion mutant was generated by allelic-replacement mutagenesis and found to be attenuated for virulence 10(5)-fold in the murine respiratory tract infection model and at least 10(4)-fold in a Mongolian gerbil otitis media infection model, confirming the importance of the NADH oxidase for both types of S. pneumoniae infection. NADH oxidase converts O(2) to H(2)O. If O(2) is not fully reduced, it can form superoxide anion (O2(-)) and hydrogen peroxide (H(2)O(2)), both of which can be toxic to cells. Bacterial cell extracts from the allelic-replacement mutant were found to lack NADH oxidase activity and the mutant was unable to grow exponentially under conditions of vigorous aeration. In contrast, the mutant displayed normal growth characteristics under conditions of limited aeration. The S. pneumoniae nox gene was cloned and expressed in E. coli. The purified His-tagged NADH oxidase was shown to oxidize NADH with a K:(m) of 32 microM, but was unable to oxidize NADPH. Oxidation of NADH was independent of exogenous FAD or FMN.

The bacA gene, which determines bacitracin susceptibility in Streptococcus pneumoniae and Staphylococcus aureus, is also required for virulence.

Homologues of Escherichia coli bacA, encoding extremely hydrophobic proteins, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Allelic replacement mutagenesis demonstrated that the gene is not essential for in vitro growth in either organism, and the mutants showed no significant changes in growth rate or morphology. The Staph. aureus bacA mutant showed slightly reduced virulence in a mouse model of infection and an eightfold increase in bacitracin susceptibility. However, a Strep. pneumoniae bacA mutant was highly attenuated in a mouse model of infection, and demonstrated an increase in susceptibility to bacitracin of up to 160000-fold. These observations are consistent with the previously proposed role of BacA protein as undecaprenol kinase.