Brilacidin, a novel antifungal agent against Cryptococcus neoformans.

Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans. BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae, BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans. BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii. There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans. We propose BRI as a new antifungal agent against cryptococcosis.

Membrane Phenotypic, Metabolic and Genotypic Adaptations of Streptococcus oralis Strains Destined to Rapidly Develop Stable, High-Level Daptomycin Resistance during Daptomycin Exposures.

The Streptococcus mitis-oralis subgroup of viridans group streptococci are important human pathogens. We previously showed that a substantial portion of S. mitis-oralis strains (>25%) are 'destined' to develop rapid, high-level, and stable daptomycin (DAP) resistance (DAP-R) during DAP exposures in vitro. Such DAP-R is often accompanied by perturbations in distinct membrane phenotypes and metabolic pathways. The current study evaluated two S. oralis bloodstream isolates, 73 and 205. Strain 73 developed stable, high-level DAP-R (minimum inhibitory concentration [MIC] > 256 µg/mL) within 2 days of in vitro DAP passage ("high level" DAP-R [HLDR]). In contrast, strain 205 evolved low-level and unstable DAP-R (MIC = 8 µg/mL) under the same exposure conditions in vitro ("non-HLDR"). Comparing the parental 73 vs. 73-D2 (HLDR) strain-pair, we observed the 73-D2 had the following major differences: (i) altered cell membrane (CM) phospholipid profiles, featuring the disappearance of phosphatidylglycerol (PG) and cardiolipin (CL), with accumulation of the PG-CL pathway precursor, phosphatidic acid (PA); (ii) enhanced CM fluidity; (iii) increased DAP surface binding; (iv) reduced growth rates; (v) decreased glucose utilization and lactate accumulation; and (vi) increased enzymatic activity within the glycolytic (i.e., lactate dehydrogenase [LDH]) and lipid biosynthetic (glycerol-3-phosphate dehydrogenase [GPDH]) pathways. In contrast, the 205 (non-HLDR) strain-pair did not show these same phenotypic or metabolic changes over the 2-day DAP exposure. WGS analyses confirmed the presence of mutations in genes involved in the above glycolytic and phospholipid biosynthetic pathways in the 73-D2 passage variant. These data suggest that S. oralis strains which are 'destined' to rapidly develop HLDR do so via a conserved cadre of genotypic, membrane phenotypic, and metabolic adaptations.

Combinations of Daptomycin plus Ceftriaxone, but Not Ascending Daptomycin Dose-Regimens, Are Effective in Experimental Endocarditis Caused by Streptococcus mitis-oralis Strains: Target Tissue Clearances and Prevention of Emergence of Daptomycin-Resistance.

The Streptococcus mitis-oralis subgroup of the viridans group streptococci (VGS) are the most common cause of infective endocarditis (IE) in many parts of the world. These organisms are frequently resistant in vitro to standard ß-lactams (e.g., penicillin; ceftriaxone [CRO]), and have the notable capacity for rapidly developing high-level and durable daptomycin resistance (DAP-R) during exposures in vitro, ex vivo, and in vivo. In this study, we used 2 prototypic DAP-susceptible (DAP-S) S. mitis-oralis strains (351; and SF100), which both evolved stable, high-level DAP-R in vitro within 1 to 3 days of DAP passage (5 to 20 µg/mL DAP). Of note, the combination of DAP + CRO prevented this rapid emergence of DAP-R in both strains during in vitro passage. The experimental rabbit IE model was then employed to quantify both the clearance of these strains from multiple target tissues, as well as the emergence of DAP-R in vivo under the following treatment conditions: (i) ascending DAP-alone dose-strategies encompassing human standard-dose and high-dose-regimens; and (ii) combinations of DAP + CRO on these same metrics. Ascending DAP-alone dose-regimens (4 to 18 mg/kg/d) were relatively ineffective at either reducing target organ bioburdens or preventing emergence of DAP-R in vivo. In contrast, the combination of DAP (4 or 8 mg/kg/d) + CRO was effective at clearing both strains from multiple target tissues (often with sterilization of bio-burdens in such organs), as well as preventing the emergence of DAP-R. In patients with serious S. mitis-oralis infections such as IE, especially caused by strains exhibiting intrinsic ß-lactam resistance, initial therapy with combinations of DAP + CRO may be warranted.

Impact of Bicarbonate on PBP2a Production, Maturation, and Functionality in Methicillin-Resistant Staphylococcus aureus (MRSA).

Certain methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit ß-lactam-susceptibility in vitro, ex vivo and in vivo in the presence of NaHCO3 (NaHCO3-responsive MRSA). Herein, we investigate the impact of NaHCO3 on factors required for PBP2a functionality. Prototype NaHCO3-responsive and -nonresponsive MRSA strains (as defined in vitro) were assessed for the impact of NaHCO3 on: expression of genes involved in PBP2a production-maturation pathways (mecA, blaZ, pbp4, vraSR, prsA, sigB, and floA); membrane PBP2a and PrsA protein content; and membrane carotenoid content. Following NaHCO3 exposure in NaHCO3-responsive (vs - nonresponsive) MRSA, there was significantly reduced expression of: i) mecA and blaZ; ii) the vraSR-prsA gene axis; and iii) pbp4 Carotenoid production was reduced, while floA expression was increased by NaHCO3 exposure in all MRSA strains. This work underscores the distinct regulatory impact of NaHCO3 on a cadre of genes encoding factors required for maintenance of the MRSA phenotype through PBP2a functionality and maturation.

Activity of the Lactate Dehydrogenase Inhibitor Oxamic Acid against the Fermentative Bacterium Streptococcus mitis/oralis: Bactericidal Effects and Prevention of Daptomycin Resistance In Vitro and in an Ex Vivo Model.

Streptococcus mitis/oralis is a fermentative bacterium that relies on lactate dehydrogenase to balance its redox poise and keep glycolysis active. Metabolomic analysis of an in vitro-derived daptomycin-resistant (DAP-R) S. mitis/oralis strain (351-D10) revealed differences in glucose catabolism relative to its DAP-susceptible (DAP-S) parental strain, 351. Metabolic changes associated with the transition to this DAP-R phenotype suggested that inhibiting glycolysis could alter DAP susceptibility. In addition, the strong reliance of S. mitis/oralis on glycolysis for energy and biosynthetic intermediates suggested that inhibiting glycolysis would adversely affect growth and biomass accumulation. To test these hypotheses, we used the lactate dehydrogenase inhibitor oxamic acid (OXA) to assess its efficacy against DAP-S S. mitis/oralis strain 351 during DAP exposures in vitro and ex vivo. As expected, OXA was growth inhibitory to S. mitis/oralis in a dose-dependent manner in vitro; however, it did not alter in vitro DAP susceptibility profiles. In contrast, OXA did prevent the emergence of DAP-R in an ex vivo model of simulated endocardial vegetations. These data suggest that metabolic inhibitors directed against this fermentative bacterium with limited metabolic capabilities could enhance killing and potentially forestall the emergence of DAP resistance.

Interstitial cystitis/bladder pain syndrome (IC/BPS): Single-center 20 year experience and treatment results in India.

OBJECTIVES: Interstitial cystitis/bladder pain syndrome (IC/BPS) is an enigmatic disease that is difficult to treat. Even among physicians, the prevalent belief is that patients do not improve over time. In this study, we retrospectively reviewed our experience and treatment results for patients diagnosed with IC/BPS at our clinic in India over the past 20 years. MATERIALS AND METHODS: Three hundred and eighty IC/BPS patients diagnosed between January 2001 and December 2020 were included. Patients underwent cystoscopy and hydrodistension and were treated with oral drugs, intravesical instillations, and surgery as needed. From January 2021 to June 2021, all patients were contacted by telephone. RESULTS: The study had 380 participants, but only 231 could be contacted for analysis. Follow-up averaged 6.37 years and the median was 14 years. Eighteen percent showed no improvement, 2% showed a slight improvement on Global Response Assessment (GRA) questionnaire and were considered nonresponders (NR). Yet, 67% reported notable improvements, and 13% moderate improvements, all of which make up 80% responders (R). In 11 patients who were operated on for ileocystoplasty, 9 showed significant improvements. In addition, three patients developed Urothelial Malignancy. Pregnancy did not affect the disease in any way. CONCLUSIONS: Long-term results have been encouraging for IC/BPS patients. Unfortunately, Hunner's lesions patients need a more intensive treatment regimen. Re-evaluation with cystoscopy is recommended in NR. In spite of good results of surgery in our series, it is best to perform surgery only as a last resort.

Synergy Mechanisms of Daptomycin-Fosfomycin Combinations in Daptomycin-Susceptible and -Resistant Methicillin-Resistant Staphylococcus aureus: In Vitro, Ex Vivo, and In Vivo Metrics.

Increased usage of daptomycin (DAP) for methicillin-resistant Staphylococcus aureus (MRSA) infections has led to emergence of DAP-resistant (DAP-R) strains, resulting in treatment failures. DAP-fosfomycin (Fosfo) combinations are synergistically active against MRSA, although the mechanism(s) of this interaction is not fully understood. The current study explored four unique but likely interrelated activities of DAP-Fosfo combinations: (i) synergistic killing, (ii) prevention of evolution of DAP-R, (iii) resensitization of already DAP-R subpopulations to a DAP-susceptible (DAP-S) phenotype, and (iv) perturbations of specific cell envelope phenotypes known to correlate with DAP-R in MRSA. Using an isogenic DAP-S (CB1483)/DAP-R (CB185) clinical MRSA strain pair, we demonstrated that combinations of DAP plus Fosfo (DAP+Fosfo) (i) enhanced killing of both strains in vitro and ex vivo, (ii) increased target tissue clearances of the DAP-R strain in an in vivo model of experimental infective endocarditis (IE), (iii) prevented emergence of DAP-R in the DAP-S parental strain both in vitro and ex vivo, and (iv) resensitized the DAP-R strain to a DAP-S phenotype ex vivo. Phenotypically, following exposure to sub-MIC Fosfo, the DAP-S/DAP-R strain pair exhibited distinct modifications in (i) net positive surface charge (P < 0.05), (ii) quantity (P < 0.0001) and localization of cell membrane cardiolipin (CL), (iii) DAP surface binding, and (iv) membrane fluidity (P < 0.05). Furthermore, preconditioning this strain pair to DAP with or without Fosfo (DAP+/-Fosfo) sensitized these organisms to killing by the human host defense peptide LL37. These data underscore the notion that DAP-Fosfo combinations can impact MRSA clearances within multiple microenvironments, likely based on specific phenotypic adaptations.

Cell Membrane Adaptations Mediate ß-Lactam-Induced Resensitization of Daptomycin-Resistant (DAP-R) Staphylococcus aureus In Vitro.

The reversal of daptomycin resistance in MRSA to a daptomycin-susceptible phenotype following prolonged passage in selected ß-lactams occurs coincident with the accumulation of multiple point mutations in the mprF gene. MprF regulates surface charge by modulating the content and translocation of the positively charged cell membrane phospholipid, lysyl-phosphatidylglycerol (LPG). The precise cell membrane adaptations accompanying such ß-lactam-induced mprF perturbations are unknown. This study examined key cell membrane metrics relevant to antimicrobial resistance among three daptomycin-resistant MRSA clinical strains, which became daptomycin-susceptible following prolonged exposure to cloxacillin ('daptomycin-resensitized'). The causal role of such secondary mprF mutations in mediating daptomycin resensitization was confirmed through allelic exchange strategies. The daptomycin-resensitized strains derived either post-cloxacillin passage or via allelic exchange (vs. their respective daptomycin-resistant strains) showed the following cell membrane changes: (i) enhanced BODIPY-DAP binding; (ii) significant reductions in LPG content, accompanied by significant increases in phosphatidylglycerol content (p < 0.05); (iii) no significant changes in positive cell surface charge; (iv) decreased cell membrane fluidity (p < 0.05); (v) enhanced carotenoid content (p < 0.05); and (vi) lower branched chain fatty acid profiles (antiso- vs. iso-), resulting in increases in saturated fatty acid composition (p < 0.05). Overall, the cell membrane characteristics of the daptomycin-resensitized strains resembled those of parental daptomycin-susceptible strains. Daptomycin resensitization with selected ß-lactams results in both definable genetic changes (i.e., mprF mutations) and a number of key cell membrane phenotype modifications, which likely facilitate daptomycin activity.

ß-Lactam-Induced Cell Envelope Adaptations, Not Solely Enhanced Daptomycin Binding, Underlie Daptomycin-ß-Lactam Synergy in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious clinical threat due to innate virulence properties, high infection rates, and the ability to develop resistance to multiple antibiotics, including the lipopeptide daptomycin (DAP). The acquisition of DAP resistance (DAP-R) in MRSA has been linked with several characteristic alterations in the cell envelope. Clinical treatment of DAP-R MRSA infections has generally involved DAP-plus-ß-lactam combinations, although definable synergy of such combinations varies in a strain-dependent as well as a ß-lactam-dependent manner. We investigated distinct ß-lactam-induced cell envelope adaptations of nine clinically derived DAP-susceptible (DAP-S)/DAP-R strain pairs following in vitro exposure to a panel of six standard ß-lactams (nafcillin, meropenem, cloxacillin, ceftriaxone, cefaclor, or cefoxitin), which differ in their penicillin-binding protein (PBP)-targeting profiles. In general, in both DAP-S and DAP-R strains, exposure to these ß-lactams led to (i) a decreased positive surface charge; (ii) decreased cell membrane (CM) fluidity; (iii) increased content and delocalization of anionic phospholipids (i.e., cardiolipin), with delocalization being more pronounced in DAP-R strains; and (iv) increased DAP binding in DAP-S (but not DAP-R) strains. Collectively, these results suggest that ß-lactam-induced alterations in at least three major cell envelope phenotypes (surface charge, membrane fluidity, and cardiolipin content) could underlie improved DAP activity, not mediated solely by an increase in DAP binding. (Note that for ease of presentation, we utilize the terminology "DAP-R" instead of "DAP nonsusceptibility.").

Mechanistic Fingerprinting Reveals Kinetic Signatures of Resistance to Daptomycin and Host Defense Peptides in Streptococcus mitis-oralis.

Streptococcus mitis-oralis (S. mitis-oralis) infections are increasingly prevalent in specific populations, including neutropenic cancer and endocarditis patients. S. mitis-oralis strains have a propensity to evolve rapid, high-level and durable resistance to daptomycin (DAP-R) in vitro and in vivo, although the mechanism(s) involved remain incompletely defined. We examined mechanisms of DAP-R versus cross-resistance to cationic host defense peptides (HDPs), using an isogenic S. mitis-oralis strain-pair: (i) DAP-susceptible (DAP-S) parental 351-WT (DAP MIC = 0.5 µg/mL), and its (ii) DAP-R variant 351-D10 (DAP MIC > 256 µg/mL). DAP binding was quantified by flow cytometry, in-parallel with temporal (1-4 h) killing by either DAP or comparative prototypic cationic HDPs (hNP-1; LL-37). Multicolor flow cytometry was used to determine kinetic cell responses associated with resistance or susceptibility to these molecules. While overall DAP binding was similar between strains, a significant subpopulation of 351-D10 cells hyper-accumulated DAP (>2-4-fold vs. 351-WT). Further, both DAP and hNP-1 induced cell membrane (CM) hyper-polarization in 351-WT, corresponding to significantly greater temporal DAP-killing (vs. 351-D10). No strain-specific differences in CM permeabilization, lipid turnover or regulated cell death were observed post-exposure to DAP, hNP-1 or LL-37. Thus, the adaptive energetics of the CM appear coupled to the outcomes of interactions of S. mitis-oralis with DAP and selected HDPs. In contrast, altered CM permeabilization, proposed as a major mechanism of action of both DAP and HDPs, did not differentiate DAP-S vs. DAP-R phenotypes in this S. mitis-oralis strain-pair.

Strain-Specific Adaptations of Streptococcus mitis-oralis to Serial In Vitro Passage in Daptomycin (DAP): Genotypic and Phenotypic Characteristics.

Viridans group streptococci (VGS), especially the Streptococcus mitis-oralis subgroup, are pivotal pathogens in a variety of invasive endovascular infections, including "toxic shock" in neutropenic cancer patients and infective endocarditis (IE). Previously, we showed that the serial in vitro passage of S. mitis-oralis strains in sublethal daptomycin (DAP) resulted in rapid, high-level and stable DAP-resistance (DAP-R), which is accompanied by distinct changes in several genotypic and phenotypic signatures: (1) the disappearance of two key membrane phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL); (2) increased membrane fluidity; (3) increased positive surface charge; (4) single nucleotide polymorphisms (SNPs) in two loci involved in CL biosynthesis (pgsA; cdsA); and (5) DAP hyperaccumulation. The current study examined these same metrics following in vitro serial DAP passages of a separate well-characterized S. mitis-oralis bloodstream isolate (SF100). Although some metrics seen in prior DAP post-passage strains were recapitulated with SF100 (e.g., pgsA SNPs, enhanced membrane fluidity), we observed the following major differences (comparing the parental versus post-passage variant): (1) no change in PG content; (2) reduced, but not absent, CL, with enhancement in phosphatidic acid (PA) content; (3) an unusual pattern of CL localization; (4) significantly decreased positive surface charge; (5) no difference in DAP accumulation; and (6) no cdsA SNPs. Thus, S. mitis-oralis strains are not "pre-programmed" phenotypically and/or genotypically to adapt in an identical manner during the evolution of the DAP-R.

Prolonged Exposure to ß-Lactam Antibiotics Reestablishes Susceptibility of Daptomycin-Nonsusceptible Staphylococcus aureus to Daptomycin.

Daptomycin-nonsusceptible (DAP-NS) Staphylococcus aureus often exhibits gain-in-function mutations in the mprF gene (involved in positive surface charge maintenance). Standard ß-lactams, although relatively inactive against methicillin-resistant S. aureus (MRSA), may prevent the emergence of mprF mutations and DAP-NS. We determined if ß-lactams might also impact DAP-NS isolates already possessing an mprF mutation to revert them to DAP-susceptible (DAP-S) phenotypes and, if so, whether this is associated with specific penicillin-binding protein (PBP) targeting. This study included 25 DAP-S/DAP-NS isogenic, clinically derived MRSA bloodstream isolates. MICs were performed for DAP, nafcillin (NAF; PBP-promiscuous), cloxacillin (LOX; PBP-1), ceftriaxone (CRO; PBP-2), and cefoxitin (FOX; PBP-4). Three DAP-NS isolates were selected for a 28-day serial passage in subinhibitory ß-lactams. DAP MICs and time-kill assays, host defense peptide (LL-37) susceptibilities, and whole-genome sequencing were performed to associate genetic changes with key phenotypic profiles. Pronounced decreases in baseline MICs were observed for NAF and LOX (but not for CRO or FOX) among DAP-NS versus DAP-S isolates ("seesaw" effect). Prolonged (28-d) ß-lactam passage of three DAP-NS isolates significantly reduced DAP MICs. LOX was most impactful (∼16-fold decrease in DAP MIC; 2 to 0.125 mg/liter). In these DAP-NS isolates with preexisting mprF polymorphisms, accumulation of additional mprF mutations occurred with prolonged LOX exposures. This was associated with enhanced LL-37 killing activity and reduced surface charge (both mprF-dependent phenotypes). ß-lactams that either promiscuously or specifically target PBP-1 have significant DAP "resensitizing" effects against DAP-NS S. aureus strains. This may relate to the acquisition of multiple mprF single nucleotide polymorphism (SNPs), which, in turn, affect cell envelope function and metabolism.

Metabolic changes associated with adaptive resistance to daptomycin in Streptococcus mitis-oralis.

BACKGROUND: Viridans group streptococci of the Streptococcus mitis-oralis subgroup are important endovascular pathogens. They can rapidly develop high-level and durable non-susceptibility to daptomycin both in vitro and in vivo upon exposure to daptomycin. Two consistent genetic adaptations associated with this phenotype (i.e., mutations in cdsA and pgsA) lead to the depletion of the phospholipids, phosphatidylglycerol and cardiolipin, from the bacterial membrane. Such alterations in phospholipid biosynthesis will modify carbon flow and change the bacterial metabolic status. To determine the metabolic differences between daptomycin-susceptible and non-susceptible bacteria, the physiology and metabolomes of S. mitis-oralis strains 351 (daptomycin-susceptible) and 351-D10 (daptomycin non-susceptible) were analyzed. S. mitis-oralis strain 351-D10 was made daptomycin non-susceptible through serial passage in the presence of daptomycin. RESULTS: Daptomycin non-susceptible S. mitis-oralis had significant alterations in glucose catabolism and a re-balancing of the redox status through amino acid biosynthesis relative to daptomycin susceptible S. mitis-oralis. These changes were accompanied by a reduced capacity to generate biomass, creating a fitness cost in exchange for daptomycin non-susceptibility. CONCLUSIONS: S. mitis-oralis metabolism is altered in daptomycin non-susceptible bacteria relative to the daptomycin susceptible parent strain. As demonstrated in Staphylococcus aureus, inhibiting the metabolic changes that facilitate the transition from a daptomycin susceptible state to a non-susceptible one, inhibits daptomycin non-susceptibility. By preventing these metabolic adaptations in S. mitis-oralis, it should be possible to deter the formation of daptomycin non-susceptibility.

Why parents consent to their children's participation in genetic research: A study of parental decision making.

Parents need to be asked to provide informed consent on behalf of their child for participation in genetic research. Decision making for such parents is difficult because ethical challenges in paediatric genetic research studies are different from similar adult studies. This paper focuses on interviews conducted with parents who were asked to consent to their children's participation (or not) in a genetic research study of intellectual disability and/or autism.

Proteomic and Membrane Lipid Correlates of Reduced Host Defense Peptide.

We previously described a transposon mutant in Staphylococcus aureus strain SH1000 that exhibited reduced susceptibility to cationic thrombin-induced platelet microbicidal proteins (tPMPs). The transposon insertion site was mapped to the gene snoD, the staphylococcal nuo orthologue. Hence, further studies have been performed to understand how this mutation impacts susceptibility to tPMP, by comparing proteomics profiling and membrane lipid analyses of the parent vs. mutant strains. Surprisingly, the mutant showed differential regulation of only a single protein when cultivated aerobically (FadB), and only a small number of proteins under anaerobic growth conditions (AdhE, DapE, Ddh, Ald1, IlvA1, AgrA, Rot, SA2366, and SA2367). Corresponding to FadB impact on lipid remodeling, membrane fatty acid analyses showed that the snoD mutant contained more short chain anteiso-, but fewer short chain iso-branched chain fatty acids under both aerobic and anaerobic conditions vs. the parental strain. Based upon these proteomic and membrane compositional data, a hypothetical "network" model was developed to explain the impact of the snoD mutation upon tPMP susceptibility.

Metabolic interventions for the prevention and treatment of daptomycin non-susceptibility in Staphylococcus aureus.

BACKGROUND: A major developing problem in the treatment of Staphylococcus aureus infections is the emergence of resistance during treatment with daptomycin. Previous metabolomic analyses of isogenic S. aureus strains prior to and after evolution into a daptomycin non-susceptible (DapNS) state provided important metabolic information about this transition (e.g. perturbations of the tricarboxylic acid cycle). OBJECTIVES: To assess the significance of these metabolic changes, in vitro susceptibility to daptomycin was determined in daptomycin-susceptible (DapS) and DapNSS. aureus strains cultivated with metabolic inhibitors targeting these changes. METHODS: Only inhibitors that are approved for use in humans were chosen (i.e. fosfomycin, valproate, trimetazidine and 6-mercaptopurine) to assess the importance of metabolic pathways for daptomycin non-susceptibility. The ability of these inhibitors to forestall the emergence of DapNS strains was also assessed. RESULTS: The combination of daptomycin and fosfomycin synergistically killed both DapS and DapNS strains in vitro and enhanced the in vivo outcome against a DapNS strain in experimental endocarditis. Interestingly, fosfomycin acts on the peptidoglycan biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA); however, it also had a significant effect on the enzymatic activity of enolase, an essential enzyme in S. aureus. While fosfomycin acted synergistically with daptomycin, it failed to prevent the in vitro evolution of daptomycin non-susceptibility. In contrast, trimetazidine, an anti-angina drug that stimulates glucose oxidation, abolished the ability of DapSS. aureus strains to transition to a DapNS state. CONCLUSIONS: These data reveal that metabolic adaptations associated with DapNS strains can be targeted to prevent the emergence of and/or reverse pre-existing resistance to daptomycin.

Intravesical tacrolimus in treatment of intractable interstitial cystitis/bladder pain syndrome - A pilot study.

AIMS: Interstitial cystitis/Bladder pain syndrome (IC/BPS) is a chronic condition with limited effectiveness of current treatments without any cure. Cyclosporine A is effective in intractable cases of BPS/IC. Tacrolimus has same mechanism of action. The purpose of this pilot study was to find if tacrolimus instilled in bladder is effective in treating BPS/IC without side effects. METHODS: From February 2013 till Dec. 2017 tacrolimus dissolved in DMSO/sterile water was instilled in bladder of 24 patients of intractable BPS/IC. Patients received one to six cycles of therapy at interval of 14 days. Base line complete blood count, blood glucose, renal and liver function test were done and repeated after every three instillations. Serum tacrolimus level was also measured in 10 patients. Primary study endpoint was Global Response Assessment (GRA) score. RESULTS: 13 out of 24 patients showed improvement in a follow up extending from 6 to 63 month. Except for post- instillation flare in symptoms no side effects were observed in the patients during follow-up. Blood levels of tacrolimus reach same safe level irrespective of using either DMSO or water for preparing the solution. CONCLUSIONS: Intravesical tacrolimus dissolved in DMSO/water has been found effective in 54% patients of intractable BPS/IC without significant side effects in this pilot study. For the first time we have discovered that though tacrolimus is believed to be insoluble in water it gets absorbed by bladder urothelium when a solution of tacrolimus in water is instilled in urinary bladder. It should be offered to the patients before offering surgery.

Daptomycin Dose-Ranging Evaluation with Single-Dose versus Multidose Ceftriaxone Combinations against Streptococcus mitis/oralis in an Ex Vivo Simulated Endocarditis Vegetation Model.

The viridans group streptococci (VGS) are a heterogeneous group of organisms which are important components of the normal human oral flora. Among the VGS, the Streptococcus mitis/oralis subgroup is one of the most common causes of infective endocarditis (IE). Daptomycin (DAP) is a potential alternative therapeutic option for invasive S. mitis infections, given high rates of ß-lactam resistance and vancomycin tolerance in such strains. However, the ability of these strains to rapidly evolve high-level and durable DAP resistance (DAP-R) is problematic. Recent data suggest that combination DAP-ß-lactam therapy circumvents this issue. Human-simulated dose-escalating DAP-alone dose regimens (6, 8, 10, or 12 mg/kg/day times 4 days) versus DAP (6 mg/kg/day) plus ceftriaxone (CRO) (2 g once daily times 4 days or 0.5 g, single dose) were assessed against two prototypical DAP-susceptible (DAP-S) S. mitis/oralis strains (SF100 and 351), as measured by a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs). No DAP-alone regimen was effective, with regrowth of high-level DAP-R isolates observed for both strains over 96-h exposures. Combinations of DAP-CRO with either single- or multidose regimens yielded significant reductions in log10 CFU/g amounts within SEVs for both strains (∼6 log10 CFU/g) within 24 h. In addition, no DAP-R strains were detected in either DAP-CRO combination regimens over the 96-h exposure. In contrast to prior in vitro studies, no perturbations in two key cardiolipin biosynthetic genes (cdsA and pgsA) were identified in DAP-R SEV isolates emerging from strain 351, despite defective phospholipid production. The combination of DAP-CRO warrants further investigation for treatment of IE due to S. mitis/oralis.

Comparative Efficacies of Linezolid vs. Tedizolid in an Experimental Murine Model of Vancomycin-Resistant Enterococcal (VRE) Bacteremia.

Tedizolid (TZD) is an oxazolidinone derivative which demonstrates bacteriostatic activity through inhibition of protein synthesis. We compared the efficacies of TZD and an earlier-generation oxazolidinone, linezolid (LZD), in an experimental murine model of bacteremia caused by two VRE strains (one each E. faecium and E. faecalis). LZD exhibited significantly better efficacy in terms of reduced VRE blood and target tissue densities than TZD in this model.

Mutations in cdsA and pgsA Correlate with Daptomycin Resistance in Streptococcus mitis and S. oralis.

We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.