Differential binding of human and murine IgGs to catalytic and cell wall binding domains of Staphylococcus aureus peptidoglycan hydrolases.

Staphylococcus aureus is an opportunistic pathogen causing high morbidity and mortality. Since multi-drug resistant S. aureus lineages are nowadays omnipresent, alternative tools for preventive or therapeutic interventions, like immunotherapy, are urgently needed. However, there are currently no vaccines against S. aureus. Surface-exposed and secreted proteins are regarded as potential targets for immunization against S. aureus infections. Yet, many potential staphylococcal antigens of this category do not elicit protective immune responses. To obtain a better understanding of this problem, we compared the binding of serum IgGs from healthy human volunteers, highly S. aureus-colonized patients with the genetic blistering disease epidermolysis bullosa (EB), or immunized mice to the purified S. aureus peptidoglycan hydrolases Sle1, Aly and LytM and their different domains. The results show that the most abundant serum IgGs from humans and immunized mice target the cell wall-binding domain of Sle1, and the catalytic domains of Aly and LytM. Interestingly, in a murine infection model, these particular IgGs were not protective against S. aureus bacteremia. In contrast, relatively less abundant IgGs against the catalytic domain of Sle1 and the N-terminal domains of Aly and LytM were almost exclusively detected in sera from EB patients and healthy volunteers. These latter IgGs may contribute to the protection against staphylococcal infections, as previous studies suggest that serum IgGs protect EB patients against severe S. aureus infection. Together, these observations focus attention on the use of particular protein domains for vaccination to direct potentially protective immune responses towards the most promising epitopes within staphylococcal antigens.

Therapeutic Efficacy of Novel Antimicrobial Peptide AA139-Nanomedicines in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

Antimicrobial peptides (AMPs) have seen limited clinical use as antimicrobial agents, largely due to issues relating to toxicity, short biological half-life, and lack of efficacy against Gram-negative bacteria. However, the development of novel AMP-nanomedicines, i.e., AMPs entrapped in nanoparticles, has the potential to ameliorate these clinical problems. The authors investigated two novel nanomedicines based on AA139, an AMP currently in development for the treatment of multidrug-resistant Gram-negative infections. AA139 was entrapped in polymeric nanoparticles (PNPs) or lipid-core micelles (MCLs). The antimicrobial activity of AA139-PNP and AA139-MCL was determined in vitro The biodistribution and limiting doses of AA139-nanomedicines were determined in uninfected rats via endotracheal aerosolization. The early bacterial killing activity of the AA139-nanomedicines in infected lungs was assessed in a rat model of pneumonia-septicemia caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae In this model, the therapeutic efficacy was determined by once-daily (q24h) administration over 10 days. Both AA139-nanomedicines showed equivalent in vitro antimicrobial activities (similar to free AA139). In uninfected rats, they exhibited longer residence times in the lungs than free AA139 (∼20% longer for AA139-PNP and ∼80% longer for AA139-MCL), as well as reduced toxicity, enabling a higher limiting dose. In rats with pneumonia-septicemia, both AA139-nanomedicines showed significantly improved therapeutic efficacy in terms of an extended rat survival time, although survival of all rats was not achieved. These results demonstrate potential advantages that can be achieved using AMP-nanomedicines. AA139-PNP and AA139-MCL may be promising novel therapeutic agents for the treatment of patients suffering from multidrug-resistant Gram-negative pneumonia-septicemia.

Successful High-Dosage Monotherapy of Tigecycline in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

Background: Recent scientific reports on the use of high dose tigecycline monotherapy as a "drug of last resort" warrant further research into the use of this regimen for the treatment of severe multidrug-resistant, Gram-negative bacterial infections. In the current study, the therapeutic efficacy of tigecycline monotherapy was investigated and compared to meropenem monotherapy in a newly developed rat model of fatal lobar pneumonia-septicemia. Methods: A Klebsiella pneumoniae producing extended-spectrum ß-lactamase (ESBL) and an isogenic variant producing K. pneumoniae carbapenemase (KPC) were used in the study. Both strains were tested for their in vitro antibiotic susceptibility and used to induce pneumonia-septicemia in rats, which was characterized using disease progression parameters. Therapy with tigecycline or meropenem was initiated at the moment that rats suffered from progressive infection and was administered 12-hourly over 10 days. The pharmacokinetics of meropenem were determined in infected rats. Results: In rats with ESBL pneumonia-septicemia, the minimum dosage of meropenem achieving survival of all rats was 25 mg/kg/day. However, in rats with KPC pneumonia-septicemia, this meropenem dosage was unsuccessful. In contrast, all rats with KPC pneumonia-septicemia were successfully cured by administration of high-dose tigecycline monotherapy of 25 mg/kg/day (i.e., the minimum tigecycline dosage achieving 100% survival of rats with ESBL pneumonia-septicemia in a previous study). Conclusions: The current study supports recent literature recommending high-dose tigecycline as a last resort regimen for the treatment of severe multidrug-resistant bacterial infections. The use of ESBL- and KPC-producing K. pneumoniae strains in the current rat model of pneumonia-septicemia enables further investigation, helping provide supporting data for follow-up clinical trials in patients suffering from severe multidrug-resistant bacterial respiratory infections.

Cefpirome Treatment Results in Limited Selection of Stable Derepressed Enterobacter cloacae Mutants in the Intestinal Flora of Rats Treated for an Experimental Klebsiella pneumoniae Pulmonary Infection.

Background: Fourth-generation cephalosporins have been developed to improve their potency, that is, low minimal inhibitory concentrations (MICs) and to prevent resistance selection of derepressed AmpC-producing mutants in comparison to third-generation cephalosporins as ceftazidime. Objectives: We investigated the role of the administered cefpirome dose on the efficacy of treatment of a Klebsiella pneumoniae lung infection as well as in the selection of resistant Enterobacter cloacae isolates in the intestines of rats treated for a K. pneumoniae lung infection. Materials and Methods: Rats with K. pneumoniae lung infection received therapy with cefpirome doses of 0.4 to 50 mg/kg/day b.i.d. for 18 days. Resistance selection in intestinal E. cloacae was monitored during 43 days. Mutants were checked for ß-lactamase activity, mutations in their structural ampC gene, ampD gene, and omp39-40 gene. Results: A 45% and 100% rat survival rate was obtained by administration of 3.1 and 12.5 mg/kg b.i.d. of cefpirome. A significant correlation was demonstrated in the reduction of the susceptible E. cloacae isolates with %fT>MIC at days 7, 14, 22, and 29. Cefpirome E. cloacae mutants, with increased cefpirome MICs, were obtained in only four rats. Conclusions: The treatment with cefpirome resulted in less selection of derepressed mutants in comparison to ceftazidime as shown by their low number per gram of feces and in a limited number of animals.

Antimicrobial activity of two novel antimicrobial peptides AA139 and SET-M33 against clinically and genotypically diverse Klebsiella pneumoniae isolates with differing antibiotic resistance profiles.

Colistin is an antimicrobial peptide (AMP) used as a drug of last resort, although plasmid-mediated colistin resistance (MCR) has been reported. AA139 and SET-M33 are novel AMPs currently in development for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. As many AMPs have a similar mode of action to colistin, potentially leading to cross-resistance, the antimicrobial activity of AA139 and SET-M33 was investigated against a collection of 50 clinically and genotypically diverse Klebsiella pneumoniae isolates with differing antibiotic resistance profiles, including colistin-resistant strains. The collection was genotypically characterised and susceptibility to clinically relevant antibiotics was determined. Susceptibility to AA139 and SET-M33 did not differ among the collection despite differences in underlying mechanisms of resistance or susceptibility to colistin. For three colistin-susceptible and three colistin-resistant strains with distinct MDR profiles as well as an additional MCR-producing strain, the bactericidal activity of AA139, SET-M33 and colistin during 24 h of exposure was examined. Following 24 h of exposure to AA139, SET-M33 or colistin, the seven strains were tested for changes in susceptibility to the respective AMPs. AA139 and SET-M33 showed a concentration-dependent bactericidal effect irrespective of bacterial susceptibility to colistin. Exposure to low colistin concentrations resulted in the development of colistin resistance in colistin-susceptible strains, whereas susceptibility to AA139 and SET-M33 following exposure to the respective AMPs was maintained. The two novel AMPs remained effective against colistin-resistant strains and may be promising novel drugs for the treatment of clinically and genotypically diverse MDR K. pneumoniae infections, including infections associated with colistin-resistant bacteria.

Chitinase Induction Prior to Caspofungin Treatment of Experimental Invasive Aspergillosis in Neutropenic Rats Does Not Enhance Survival.

Host chitinases, chitotriosidase and acidic mammalian chitinase (AMCase), improved the antifungal activity of caspofungin (CAS) against Aspergillus fumigatus in vitro These chitinases are not constitutively expressed in the lung. Here, we investigated whether chitosan derivatives were able to induce chitinase activity in the lungs of neutropenic rats and, if so, whether these chitinases were able to prolong survival of rats with invasive pulmonary aspergillosis (IPA) or of rats with IPA and treated with CAS. An oligosaccharide-lactate chitosan (OLC) derivative was instilled in the left lung of neutropenic rats to induce chitotriosidase and AMCase activities. Rats instilled with OLC or with phosphate-buffered saline (PBS) were subsequently infected with A. fumigatus and then treated with suboptimal doses of CAS. Survival, histopathology, and galactomannan indexes were determined. Instillation of OLC resulted in chitotriosidase and AMCase activities. However, instillation of OLC did not prolong rat survival when rats were subsequently challenged with A. fumigatus In 5 of 7 rats instilled with OLC, the fungal foci in the lungs were smaller than those in rats instilled with PBS. Instillation of OLC did not significantly enhance the survival of neutropenic rats challenged with A. fumigatus and treated with a suboptimal dosage of CAS. Chitotriosidase and AMCase activities can be induced with OLC, but the presence of active chitinases in the lung did not prevent the development of IPA or significantly enhance the therapeutic outcome of CAS treatment.

Differential epitope recognition in the immunodominant staphylococcal antigen A of Staphylococcus aureus by mouse versus human IgG antibodies.

The immunodominant staphylococcal antigen A (IsaA) is a potential target for active or passive immunization against the important human pathogen Staphylococcus aureus. Consistent with this view, monoclonal antibodies against IsaA were previously shown to be protective against S. aureus infections in mouse models. Further, patients with the genetic blistering disease epidermolysis bullosa (EB) displayed high IsaA-specific IgG levels that could potentially be protective. Yet, mice actively immunized with IsaA were not protected against S. aureus infection. The present study was aimed at explaining these differences in IsaA-specific immune responses. By epitope mapping, we show that the protective human monoclonal antibody (humAb) 1D9 recognizes a conserved 62-residue N-terminal domain of IsaA. The same region of IsaA is recognized by IgGs in EB patient sera. Further, we show by immunofluorescence microscopy that this N-terminal IsaA domain is exposed on the S. aureus cell surface. In contrast to the humAb 1D9 and IgGs from EB patients, the non-protective IgGs from mice immunized with IsaA were shown to predominantly bind the C-terminal domain of IsaA. Altogether, these observations focus attention on the N-terminal region of IsaA as a potential target for future immunization against S. aureus.

Addition of 17-(allylamino)-17-demethoxygeldanamycin to a suboptimal caspofungin treatment regimen in neutropenic rats with invasive pulmonary aspergillosis delays the time to death but does not enhance the overall therapeutic efficacy.

Caspofungin (CAS) which is used as salvage therapy in patients with invasive pulmonary aspergillosis (IPA) inhibits the 1,3-ß-D-glucan synthesis in Aspergillus fumigatus. Inhibiting 1,3-ß-D-glucan synthesis induces a stress response and in an invertebrate model it was demonstrated that inhibiting this response with geldamycin enhanced the therapeutic efficacy of CAS. Since geldamycin itself is toxic to mammalians, the therapeutic efficacy of combining geldamycin with CAS was not studied in rodent models. Therefore in this study we investigated if the geldamycin derivate 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) was able to enhance the therapeutic efficacy of CAS in vitro and in our IPA model in transiently neutropenic rats. In vitro we confirmed the earlier demonstrated synergy between 17-AAG and CAS in ten A. fumigatus isolates. In vivo we treated A. fumigatus infected neutropenic rats with a sub-optimal dose of 0.75 mg/kg/day CAS and 1 mg/kg/day 17-AAG for ten days. Survival was monitored for 21 days after fungal inoculation. It appeared that the addition 17-AAG delayed death but did not improve overall survival of rats with IPA. Increasing the doses of 17-AAG was not possible due to hepatic toxicity. This study underlines the need to develop less toxic and more fungal specific geldamycin derivatives and the need to test such drugs not only in invertebrate models but also in mammalian models.

Assessment of treatment response by colony forming units, time to culture positivity and the molecular bacterial load assay compared in a mouse tuberculosis model.

The aim of the study is to compare counting of colony forming units (CFU), the time to positivity (TTP) assay and the molecular bacterial load (MBL) assay, and explore whether the last assays can detect a subpopulation which is unable to grown on solid media. CFU counting, TTP and the MBL assay were used to determine the mycobacterial load in matched lung samples of a murine tuberculosis model. Mice were treated for 24 weeks with 4 treatment arms: isoniazid (H) - rifampicin (R) - pyrazinamide (Z), HRZ-Streptomycin (S), HRZ - ethambutol (E) or ZES. Inverse relationships were observed when comparing TPP with CFU or MBL. Positive associations were observed when comparing CFU with MBL. Description of the net elimination of bacteria was performed for CFU vs. time, MBL vs. time and 1/TTP vs. time and fitted by nonlinear regression. CFU vs. time and 1/TTP vs. time showed bi-phasic declines with the exception of HRZE. A similar rank order, based on the alpha slope, was found comparing CFU vs. time and TTP vs. time, respectively HRZE, HRZ, HRZS and ZES. In contrast, MBL vs. time showed a mono-phasic decline with a flat gradient of elimination and a different rank order respectively, ZES, HRZ, HRZE and HRZS. The correlations found between methods reflects the ability of each to discern the general mycobacterial load. Based on the description of net elimination, we conclude that the MBL assay can detect a subpopulation of Mycobacterium tuberculosis which is not detected by the CFU or TTP assays.

The role of the time-kill kinetics assay as part of a preclinical modeling framework for assessing the activity of anti-tuberculosis drugs.

Novel treatment strategies for tuberculosis are urgently needed. Many different preclinical models assessing anti-tuberculosis drug activity are available, but it is yet unclear which combination of models is most predictive of clinical treatment efficacy. The aim of this study was to determine the role of our in vitro time kill-kinetics assay as an asset to a predictive preclinical modeling framework assessing anti-tuberculosis drug activity. The concentration- and time-dependent mycobacterial killing capacities of six anti-tuberculosis drugs were determined during exposure as single drugs or in dual, triple and quadruple combinations towards a Mycobacterium tuberculosis Beijing genotype strain and drug resistance was assessed. Streptomycin, rifampicin and isoniazid were most active against fast-growing M. tuberculosis. Isoniazid with rifampicin or high dose ethambutol were the only synergistic drug combinations. The addition of rifampicin or streptomycin to isoniazid prevented isoniazid resistance. In vitro ranking showed agreement with early bactericidal activity in tuberculosis patients for some but not all anti-tuberculosis drugs. The time-kill kinetics assay provides important information on the mycobacterial killing dynamics of anti-tuberculosis drugs during the early phase of drug exposure. As such, this assay is a valuable component of the preclinical modeling framework.

Investigations into the killing activity of an antimicrobial peptide active against extensively antibiotic-resistant K. pneumon iae and P. aeruginosa.

SET-M33 is a multimeric antimicrobial peptide active against Gram-negative bacteria in vitro and in vivo. Insights into its killing mechanism could elucidate correlations with selectivity. SET-M33 showed concentration-dependent bactericidal activity against colistin-susceptible and resistant isolates of P. aeruginosa and K. pneumoniae. Scanning and transmission microscopy studies showed that SET-M33 generated cell blisters, blebs, membrane stacks and deep craters in K. pneumoniae and P. aeruginosa cells. NMR analysis and CD spectra in the presence of sodium dodecyl sulfate micelles showed a transition from an unstructured state to a stable α-helix, driving the peptide to arrange itself on the surface of micelles. SET-M33 kills Gram-negative bacteria after an initial interaction with bacterial LPS. The molecule becomes then embedded in the outer membrane surface, thereby impairing cell function. This activity of SET-M33, in contrast to other similar antimicrobial peptides such as colistin, does not generate resistant mutants after 24h of exposure, non-specific interactions or toxicity against eukaryotic cell membranes, suggesting that SET-M33 is a promising new option for the treatment of Gram-negative antibiotic-resistant infections.

Immunotherapy Added to Antibiotic Treatment Reduces Relapse of Disease in a Mouse Model of Tuberculosis.

Immune-modulating drugs that target myeloid-derived suppressor cells or stimulate natural killer T cells have been shown to reduce mycobacterial loads in tuberculosis (TB). We aimed to determine if a combination of these drugs as adjunct immunotherapy to conventional antibiotic treatment could also increase therapeutic efficacy against TB. In our model of pulmonary TB in mice, we applied treatment with isoniazid, rifampicin, and pyrazinamide for 13 weeks alone or combined with immunotherapy consisting of all-trans retinoic acid, 1,25(OH)2-vitamin D3, and α-galactosylceramide. Outcome parameters were mycobacterial load during treatment (therapeutic activity) and 13 weeks after termination of treatment (therapeutic efficacy). Moreover, cellular changes were analyzed using flow cytometry and cytokine expression was assessed at the mRNA and protein levels. Addition of immunotherapy was associated with lower mycobacterial loads after 5 weeks of treatment and significantly reduced relapse of disease after a shortened 13-week treatment course compared with antibiotic treatment alone. This was accompanied by reduced accumulation of immature myeloid cells in the lungs at the end of treatment and increased TNF-α protein levels throughout the treatment period. We demonstrate, in a mouse model of pulmonary TB, that immunotherapy consisting of three clinically approved drugs can improve the therapeutic efficacy of standard antibiotic treatment.

Tigecycline Potentiates Clarithromycin Activity against Mycobacterium avium In Vitro.

Thein vitroactivities of clarithromycin and tigecycline alone and in combination againstMycobacterium aviumwere assessed. The activity of clarithromycin was time dependent, highly variable, and often resulted in clarithromycin resistance. Tigecycline showed concentration-dependent activity, and mycobacterial killing could only be achieved at high concentrations. Tigecycline enhanced clarithromycin activity againstM. aviumand prevented clarithromycin resistance. Whether there is clinical usefulness of tigecycline in the treatment ofM. aviuminfections needs further study.

Colistin as a potentiator of anti-TB drug activity against Mycobacterium tuberculosis.

OBJECTIVES: The mycobacterial cell wall is an effective permeability barrier that limits intracellular concentrations of anti-TB drugs and hampers the success of treatment. We hypothesized that colistin might enhance the efficacy of anti-TB drugs by increasing mycobacterial cell wall permeability. In this study, we investigated the additional effect of colistin on the activity of anti-TB drugs against Mycobacterium tuberculosis in vitro. METHODS: The concentration-dependent and time-dependent killing activity of isoniazid, rifampicin or amikacin alone or in combination with colistin against M. tuberculosis H37Rv was determined. Mycobacterial populations with both high and low metabolic activity were studied, and these were characterized by increasing or steady levels of ATP, respectively. RESULTS: With exposure to a single drug, striking differences in anti-TB drug activity were observed when the two mycobacterial populations were compared. The addition of colistin to isoniazid and amikacin resulted in sterilization of the mycobacterial load, but only in the M. tuberculosis population with high metabolic activity. The emergence of isoniazid and amikacin resistance was completely prevented by the addition of colistin. CONCLUSIONS: The results of this study emphasize the importance of investigating mycobacterial populations with both high and low metabolic activity when evaluating the efficacy of anti-TB drugs in vitro. This is the first study showing that colistin potentiates the activity of isoniazid and amikacin against M. tuberculosis and prevents the emergence of resistance to anti-TB drugs. These results form the basis for further studies on the applicability of colistin as a potentiator of anti-TB drugs.

Mild Staphylococcus aureus Skin Infection Improves the Course of Subsequent Endogenous S. aureus Bacteremia in Mice.

Staphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and evaluate protection in relation to anti-staphylococcal antibody levels. Skin infections once or twice by a clinical S. aureus isolate (isolate P) or S. aureus strain 8325-4 were induced in mice free of S. aureus and anti-staphylococcal antibodies. Five weeks later, immunoglobulin G (IgG) levels in blood against 25 S. aureus antigens were determined, and LD50 or LD100 bacteremia caused by S. aureus isolate P was induced. S. aureus skin infections led to elevated levels of anti-staphylococcal IgG in blood. One skin infection improved the course of subsequent severe endogenous bacteremia only. A second skin infection further improved animal survival rate, which was associated with increased pre-bacteremia IgG levels against Efb, IsaA, LukD, LukE, Nuc, PrsA and WTA. In conclusion, S. aureus isolate P skin infection in mice reduces the severity of subsequent endogenous S. aureus bacteremia only. Although cellular immune effects cannot be rules out, anti-staphylococcal IgG against specified antigens may contribute to this effect.

SILA-421 activity in vitro against rifampicin-susceptible and rifampicin-resistant Mycobacterium tuberculosis, and in vivo in a murine tuberculosis model.

Due to the emergence of multidrug-resistant and extensively drug-resistant tuberculosis (TB), there is an urgent need for new TB drugs or for compounds that improve the efficacy of currently used drugs. In this study, time-kill kinetics of SILA-421 as a single drug and in combination with isoniazid (INH), rifampicin (RIF), moxifloxacin (MXF) or amikacin (AMK) against Mycobacterium tuberculosis were assessed. Therapeutic efficacy in vivo in a mouse TB model was also studied. Further in vitro analysis was performed with a RIF-susceptible and RIF-resistant strains of M. tuberculosis. When used as a single drug, SILA-421 in vitro showed concentration-dependent and time-dependent bactericidal activity. SILA-421 also enhanced the activity of INH and RIF, resulting in synergy in the case of INH. Emergence of INH resistance following exposure to INH can be prevented by the addition SILA-421. SILA-421 had no additional value in combination with MXF or AMK. Furthermore, SILA-421 enhanced the activity of RIF towards a RIF-resistant strain and resulted in complete elimination of RIF-resistant mycobacteria. Unfortunately, in mice with TB induced by a Beijing genotype strain, addition of SILA-421 to an isoniazid-rifampicin-pyrazinamide regimen for 13 weeks did not result in enhanced therapeutic efficacy.

Active immunization with an octa-valent Staphylococcus aureus antigen mixture in models of S. aureus bacteremia and skin infection in mice.

Proteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl) and four phenol-soluble modulins α (PSMα) are invariantly produced by this pathogen. Therefore the present study was aimed at investigating whether these proteins can be used for active immunization against S. aureus infection in mouse models of bacteremia and skin infection. To this end, recombinant His-tagged fusions of IsaA, LytM, Nuc and pro-Atl were isolated from Lactococcus lactis or Escherichia coli, while the PSMα1-4 peptides were chemically synthesized. Importantly, patients colonized by S. aureus showed significant immunoglobulin G (IgG) responses against all eight antigens. BALB/cBYJ mice were immunized subcutaneously with a mixture of the antigens at day one (5 µg each), and boosted twice (25 µg of each antigen) with 28 days interval. This resulted in high IgG responses against all antigens although the response against pro-Atl was around one log lower compared to the other antigens. Compared to placebo-immunized mice, immunization with the octa-valent antigen mixture did not reduce the S. aureus isolate P load in blood, lungs, spleen, liver, and kidneys in a bacteremia model in which the animals were challenged for 14 days with a primary load of 3 × 10(5) CFU. Discomfort scores and animal survival rates over 14 days did not differ between immunized mice and placebo-immunized mice upon bacteremia with S. aureus USA300 (6 × 10(5) CFU). In addition, this immunization did not reduce the S. aureus isolate P load in mice with skin infection. These results show that the target antigens are immunogenic in both humans and mice, but in the used animal models do not result in protection against S. aureus infection.

A human monoclonal antibody targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus aureus bacteremia.

Due to substantial therapy failure and the emergence of antibiotic-resistant Staphylococcus aureus strains, alternatives for antibiotic treatment of S. aureus infections are urgently needed. Passive immunization using S. aureus-specific monoclonal antibodies (mAb) could be such an alternative to prevent and treat severe S. aureus infections. The invariantly expressed immunodominant staphylococcal antigen A (IsaA) is a promising target for passive immunization. Here we report the development of the human anti-IsaA IgG1 mAb 1D9, which was shown to bind to all 26 S. aureus isolates tested. These included both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively). Immune complexes consisting of IsaA and 1D9 stimulated human as well as murine neutrophils to generate an oxidative burst. In a murine bacteremia model, the prophylactic treatment with a single dose of 5 mg/kg 1D9 improved the survival of mice challenged with S. aureus isolate P (MSSA) significantly, while therapeutic treatment with the same dose did not influence animal survival. Neither prophylactic nor therapeutic treatment with 5 mg/kg 1D9 resulted in improved survival of mice with S. aureus USA300 (MRSA) bacteremia. Importantly, our studies show that healthy S. aureus carriers elicit an immune response which is sufficient to generate protective mAbs against invariant staphylococcal surface antigens. Human mAb 1D9, possibly conjugated to for example another antibody, antibiotics, cytokines or chemokines, may be valuable to fight S. aureus infections in patients.

Enhancement of in vitro activity of tuberculosis drugs by addition of thioridazine is not reflected by improved in vivo therapeutic efficacy.

OBJECTIVES: Assessment of the activity of thioridazine towards Mycobacterium tuberculosis (Mtb), in vitro and in vivo as a single drug and in combination with tuberculosis (TB) drugs. METHODS: The in vitro activity of thioridazine as single drug or in combination with TB drugs was assessed in terms of MIC and by use of the time-kill kinetics assay. Various Mtb strains among which the Beijing genotype strain BE-1585 were included. In vivo, mice with TB induced by BE-1585 were treated with a TB drug regimen with thioridazine during 13 weeks. Therapeutic efficacy was assessed by the change in mycobacterial load in the lung, spleen and liver during treatment and 13 weeks post-treatment. RESULTS: In vitro, thioridazine showed a concentration-dependent and time-dependent bactericidal activity towards both actively-replicating and slowly-replicating Mtb. Thioridazine at high concentrations could enhance the activity of isoniazid and rifampicin, and in case of isoniazid resulted in elimination of mycobacteria and prevention of isoniazid-resistant mutants. Thioridazine had no added value in combination with moxifloxacin or amikacin. In mice with TB, thioridazine was poorly tolerated, limiting the maximum tolerated dose (MTD). The addition of thioridazine at the MTD to an isoniazid-rifampicin-pyrazinamide regimen for 13 weeks did not result in enhanced therapeutic efficacy. CONCLUSIONS: Thioridazine is bactericidal towards Mtb in vitro, irrespective the mycobacterial growth rate and results in enhanced activity of the standard regimen. The in vitro activity of thioridazine in potentiating isoniazid and rifampicin is not reflected by improved therapeutic efficacy in a murine TB-model.

Enhancement of in vitro activity of tuberculosis drugs by addition of thioridazine is not reflected by improved in vivo therapeutic efficacy.

OBJECTIVES: Assessment of the activity of thioridazine towards Mycobacterium tuberculosis (Mtb), in vitro and in vivo as a single drug and in combination with tuberculosis (TB) drugs. METHODS: The in vitro activity of thioridazine as single drug or in combination with TB drugs was assessed in terms of MIC and by use of the time-kill kinetics assay. Various Mtb strains among which the Beijing genotype strain BE-1585 were included. In vivo, mice with TB induced by BE-1585 were treated with a TB drug regimen with thioridazine during 13 weeks. Therapeutic efficacy was assessed by the change in mycobacterial load in the lung, spleen and liver during treatment and 13 weeks post-treatment. RESULTS: In vitro, thioridazine showed a concentration-dependent and time-dependent bactericidal activity towards both actively-replicating and slowly-replicating Mtb. Thioridazine at high concentrations could enhance the activity of isoniazid and rifampicin, and in case of isoniazid resulted in elimination of mycobacteria and prevention of isoniazid-resistant mutants. Thioridazine had no added value in combination with moxifloxacin or amikacin. In mice with TB, thioridazine was poorly tolerated, limiting the maximum tolerated dose (MTD). The addition of thioridazine at the MTD to an isoniazid-rifampicin-pyrazinamide regimen for 13 weeks did not result in enhanced therapeutic efficacy. CONCLUSIONS: Thioridazine is bactericidal towards Mtb in vitro, irrespective the mycobacterial growth rate and results in enhanced activity of the standard regimen. The in vitro activity of thioridazine in potentiating isoniazid and rifampicin is not reflected by improved therapeutic efficacy in a murine TB-model.