Mycobacterium alsense sp. nov., a scotochromogenic slow grower isolated from clinical respiratory specimens.

The name 'Mycobacterium alsiense', although reported in 2007, has not been validly published. Polyphasic characterization of three available strains of this species led us to the conclusion that they represent a distinct species within the genus Mycobacterium. The proposed novel species grows slowly and presents pale yellow-pigmented colonies. Differentiation from other mycobacteria is not feasible on the basis of biochemical and cultural features alone while genetic analysis, extended to eight housekeeping genes and one spacer region, reveals its clear distinction from all other mycobacteria. Mycobacterium asiaticum is the most closely related species on the basis of 16S rRNA gene sequences (similarity 99.3 %); the average nucleotide identity between the genomes of the two species is 80.72 %, clearly below the suggested cut-off (95-96 %). The name Mycobacterium alsense sp. nov. is proposed here for the novel species and replaces the name 'M. alsiense', ex Richter et al. 2007, given at the time of isolation of the first strain. The type strain is TB 1906T ( = DSM 45230T = CCUG 56586T).

Phenothiazines, bacterial efflux pumps and targeting the macrophage for enhanced killing of intracellular XDRTB.

Phenothiazines have their primary effects on the plasma membrane of prokaryotes and eukaryotes. Among the components of the prokaryotic plasma membrane affected are efflux pumps, their energy sources, energy providing enzymes such as ATPases, and genes that regulate and code for permeability aspects of the bacterium. The responses of multi-drug (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis to the neuroleptic phenothiazine thioridazine are reviewed. The information collated suggests that this phenothiazine has the potential to cure XDR and MDR tuberculosis infections, a potential that has been recently demonstrated by its ability to cure 10 patients who presented with XDR TB infections. The mechanism by which this phenothiazine produces the desired effects within the infected macrophage is also discussed.

Characterization of intrinsic efflux activity of Enterococcus faecalis ATCC29212 by a semi-automated ethidium bromide method.

Enterococcus faecalis is recognized as a multidrug-resistant nosocomial pathogen. The phenotypic basis for this is largely uncharacterized. The intrinsic efflux system of the antibiotic-susceptible E. faecalis ATCC29212 strain was studied using a semi-automated method that assesses accumulation and efflux of the universal efflux pump substrate ethidium bromide (EB). The results show that the intrinsic efflux system of this Enterococcus strain is controlled by energy derived from the catabolism of glucose and the proton concentration of the medium. At pH 5, agents that inhibit efflux pumps in Gram-positive organisms and the proton gradient un-coupler CCCP do not increase accumulation nor inhibit efflux of EB. In contrast, at pH 8, where the proton concentration is 1,000-fold lower, these agents increase accumulation and efflux of EB. These results are relevant to infections produced by E. faecalis and subsequent antibiotic therapy with antibiotics to which the organism is known to be intrinsically resistant.

Potential role of non-antibiotics (helper compounds) in the treatment of multidrug-resistant Gram-negative infections: mechanisms for their direct and indirect activities.

Multidrug resistance in Gram-negative bacteria is now known to be primarily caused by overexpression of efflux pumps that extrude unrelated antibiotics from the periplasm or cytoplasm of the bacterium prior to their reaching their intended target. This review focuses on a variety of agents that have been shown to be efflux pump inhibitors (EPIs) and which, if used as 'helper compounds' in combination with antibiotics to which the organism is initially resistant, may produce the required cure. Although not all of the EPIs may serve a helper role owing to their toxicity, they may nevertheless serve as lead compounds.

Prolonged exposure of methicillin-resistant Staphylococcus aureus (MRSA) COL strain to increasing concentrations of oxacillin results in a multidrug-resistant phenotype.

Our previous studies demonstrated that exposure of a bacterium to increasing concentrations of an antibiotic would increase resistance to that antibiotic as a consequence of activating efflux pumps. This study utilises the same approach; however, it employs the methicillin-resistant Staphylococcus aureus (MRSA) COL strain, which is highly resistant to oxacillin (OXA). MRSA COL was adapted to 3200 mg/L of OXA. Changes in resistance to other antibiotics were evaluated and efflux pump activity during the adaptation process was determined. MRSA COL was exposed to stepwise two-fold increases of OXA. At the end of each step, minimum inhibitory concentration determination for erythromycin (ERY) and other antibiotics was conducted. Reserpine (RES) was employed to evaluate whether resistance to ERY was dependent on efflux pump activity. Efflux pump activity was also evaluated using the ethidium bromide (EB) assay. DNA typing of the products of each culture step was conducted to assess purity. Serial exposure of MRSA COL to increasing concentrations of OXA resulted in increased resistance to ERY, which could be eliminated with RES. Evaluation of efflux pump activity by the EB method indicated increased efflux activity. Resistance to ERY was accompanied by resistance to kanamycin, amikacin, ofloxacin, norfloxacin, ciprofloxacin and rifampicin. This is the first time that a multidrug-resistant phenotype has been experimentally produced as a consequence of exposure of the organism to an antibiotic to which it is initially highly resistant.

Potential management of resistant microbial infections with a novel non-antibiotic: the anti-inflammatory drug diclofenac sodium.

Diclofenac sodium (Dc), an anti-inflammatory agent, has remarkable inhibitory action both against drug-sensitive and drug-resistant clinical isolates of various Gram-positive and Gram-negative bacteria. The aim of this study was to determine the ability of Dc to protect mice from a virulent Salmonella infection. Dc injected at 1.5 microg/g and 3.0 microg/g mouse body weight significantly protected animals from the lethality of Salmonella infection. As was the case for the in vitro interaction, Dc in combination with streptomycin was even more effective. The non-antibiotic drug Dc has potential for the management of problematic antibiotic-resistant bacterial infections.

The curative activity of thioridazine on mice infected with Mycobacterium tuberculosis.

BACKGROUND: The aim of the study was to evaluate the effectiveness of thioridazine (TZ) at different dose levels on mice that had been infected intraperitoneally (i.p.) with a high dose of the Mycobacterium tuberculosis ATCC H37Rv strain. SUBJECTS AND METHODS: Groups of five female BALB/C mice were infected i.p. with 10(6) colony forming units/mL. After thirty days, treatment with TZ was initiated, except for the control group. Mice were treated with TZ at equivalent concentrations to that used in the humans (1200 mg/day), ranging from 0.05 to 0.5 mg/day. RESULTS: The results demonstrated that a daily dose of 0.5 mg/day of TZ reduced the number of colony forming units retrieved from the lungs of infected mice within one month. CONCLUSION: By the end of 300 days of therapy, although mycobacteria were still retained their presence, in comparison to that of the control was 8 orders of magnitude lower.

"Non-Antibiotics": alternative therapy for the management of MDRTB and MRSA in economically disadvantaged countries.

The antibiotic resistance is now common place throughout the globe. Two highly problematic antibiotic resistant infections are those produced by multi-drug resistant Mycobacterium tuberculosis (MDRTB) and methicillin resistant Staphylococcus aureus (MRSA). Although vancomycin is useful for therapy of MRSA, there is now evidence that resistance to this antibiotic is taking place. Intracellular infections of MRSA are very difficult to manage and are recurrent especially when invasive prosthetic devices are employed. This mini-review provides cogent evidence that both intracellular MDRTB and intracellular MRSA can be killed by concentrations of the non-antibiotic phenothiazine, Thioridazine, at concentrations in the medium that are below those present in the plasma of patients treated with this agent. Although thioridazine has been claimed to cause arrhythmias and even sudden death, the frequencies of these episodes are rare and when present, they are related to the patients underlying cardiac status as opposed to the direct effect of the agent itself. The authors do not suggest that thioridazine be used indiscriminately for MDRTB or intracellular infections produced by MRSA. However, there are circumstances where there are no alternative forms of therapy and the patient faces an unfavourable prognosis. For these highly selective and controlled situations, the use of thioridazine in the manner employed for the therapy of psychosis is recommended (compassionate therapy).

The in vitro activity of phenothiazines against Mycobacterium avium: potential of thioridazine for therapy of the co-infected AIDS patient.

Patients presenting with Acquired Immune Deficiency Syndrome (AIDS) are predisposed to co-infection with Mycobacterium avium. The management of such patients is problematic due to underlying immuno-incompetence and the high resistance of M. avium to most non-toxic compounds. Therefore, the need for effective agents is obvious. Because phenothiazines, especially the relatively mild thioridazine, have significant activity against Mycobacterium tuberculosis, we investigated the in vitro activity of chlorpromazine, thioridazine, promazine, promethazine and desipramine against a reference and clinical strains of M. avium. The results obtained show that whereas all of the phenothiazines employed in this study had an minimum inhibitory concentration (MIC) against the strains studied that ranged from ca. 10 to > 50 mg/L, as was previously shown for M. tuberculosis, thioridazine was the most active of the group against M. avium.

In vitro activity of phenothiazine derivatives in Enterococcus faecalis and Enterococcus faecium.

The antimicrobial activity of the phenothiazine derivatives thioridazine and prochlorperazine have been evaluated with 11 Enterococcus faecalis strains and 9 Enterococcus faecium strains, originating from human infections and animal faecal flora. We found that all E. faecalis and E. faecium strains, regardless of their susceptibility to commonly used antibiotics, were inhibited by thioridazine at a concentration of 16-32 microg/ml and by prochlorperazine at a concentration of 32-64 microg/ml. Combinations of the antibiotics vancomycin or ampicillin and thioridazine and prochlorperazine at subinhibitory concentrations, could render vancomycin- or ampicillin-resistant bacteria sensitive to each of the antibiotics. Verapamil and reserpine, inhibitors of P-glycoprotein-mediated multidrug resistance, did not reduce resistance. Our results outline modification of resistance in enterococci induced by phenothiazine derivatives unrelated to P-glycoprotein-mediated multidrug resistance.

Phenothiazines as a solution for multidrug resistant tuberculosis: From the origin to present.

Historically, multiplicity of actions in synthetic compounds is a rule rather than exception. The science of non-antibiotics evolved in this background. From the antimalarial and antitrypanosomial dye methylene blue, chemically similar compounds, the phenothiazines, were developed. The phenothiazines were first recognised for their antipsychotic properties, but soon after their antimicrobial functions came to be known and then such compounds were designated as non-antibiotics. The emergence of highly drug-resistant bacteria had initiated an urgent need to search for novel affordable compounds. Several phenothiazines awakened the interest among scientists to determine their antimycobacterial activity. Chlorpromazine, trifluoperazine, methdilazine and thioridazine were found to have distinct antitubercular action. Thioridazine took the lead as researchers repeatedly claimed its potentiality. Although thioridazine is known for its central nervous system and cardiotoxic side-effects, extensive and repeated in vitro and in vivo studies by several research groups revealed that a very small dose of thioridazine is required to kill tubercle bacilli inside macrophages in the lungs, where the bacteria try to remain and multiply silently. Such a small dose is devoid of its adverse side-effects. Recent studies have shown that the (-) thioridazine is a more active antimicrobial agent and devoid of the toxic side effects normally encountered. This review describes the possibilities of bringing down thioridazine and its (-) form to be combined with other antitubercular drugs to treat infections by drug-resistant strains of Mycobacterium tuberculosis and try to eradicate this deadly disease.

Quinoline and cyanine dyes--putative anti-MRSA drugs.

One way in which drug-resistant bacteria may be attacked is to screen new series of candidate compounds. Quaternary quinoline compounds and dyes were studied by Carl Browning (1887-1972) and Julius Cohen (1859-1935). A remarkable part of Browning and Cohen's work was the early development of structure-activity relationships for their series of compounds. Thus cationic species were found generally to be more effective antibacterials than neutrals or anionics, and the testing of partial or deconstructed active molecules was also carried out. Much of this work underpinned the fuller understanding of e.g. aminoacridine action developed by Adrien Albert (1907-1989), himself also a collaborator of Browning. Analysis of the activity of a range of compounds developed by Browning and Cohen suggests that these might again be examined as topical antimicrobials in the fight against methicillin-resistant S. aureus (MRSA) and other resistant bacteria.

Nizatidine and omeprazole enhance the effect of metronidazole on Helicobacter pylori in vitro.

Treatment failures are common in patients infected with metronidazole-resistant Helicobacter pylori in the gastric mucosa when triple therapy including metronidazole is used. In patients with treatment failure and metronidazole-resistant H. pylori, a higher eradication rate for H. pylori was found after secondary treatment with bismuth/ranitidine in combination with antibiotics including metronidazole, compared with the same antibiotics combined with a standard dose of omeprazole. This agrees with our previous finding that bismuth was able to reduce the susceptibility of H. pylori to metronidazole. In this study, we have found that nizatidine, an H(2)-receptor antagonist, is also able to reduce the susceptibility of H. pylori to metronidazole in vitro, despite having no direct inhibitory effect on the growth of H. pylori. This agrees with earlier findings that compounds having the ability to reverse antibiotic resistance do not necessarily have an antibiotic or chemotherapeutic effect in the sense of growth inhibition. Therefore, it was decided to investigate the effect of nizatidine and omeprazole on the oxidative respiratory chain, as it is known that metronidazole is able to inhibit the activity of fumarate reductase of H. pylori. This enzyme is a key enzyme in the alternative respiratory chain under anaerobic conditions. Nizatidine was, in these preliminary experiments, found to inhibit fumarate reductase in a dose-dependent way, like metronidazole, whereas omeprazole had almost no effect on fumarate reductase. No other significant effects on the enzymes of the respiratory chain were found. The synergistic effect of nizatidine on metronidazole resistant H. pylori strains could be explained by the effect on fumarate reductase, whereas the effect of omeprazole is different and could be an inhibition of a proton pump in H. pylori. Reversal of antimicrobial resistance with the help of different non-antibiotics seems to be possible by using quite different compounds, and is therefore to be explained by different molecular mechanisms.

Phenylpiperidine selective serotonin reuptake inhibitors interfere with multidrug efflux pump activity in Staphylococcus aureus.

Structural variants of phenylpiperidine selective serotonin reuptake inhibitors (P-SSRIs) inhibited the function of two unique Staphylococcus aureus multidrug efflux pumps. The most active compound was the paroxetine isomer NNC 20-7052, which had an IC(50) for ethidium, acriflavine, and pyronin Y efflux of 9, 53, and 18% of its MIC, respectively, against the NorA pump. The unbalanced effect of NNC 20-7052 on the efflux of different substrates suggests the possibility that P-SSRIs function by a physical interaction with NorA. Under the conditions employed pump inhibition partially extended to the resistance-nodulation-division (RND) pump AcrAB-TolC, but not to the Pseudomonas aeruginosa RND pumps MexAB-OprM or MexCD-OprJ.

Phenothiazines alter resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) to oxacillin in vitro.

Mechanisms of antibiotic resistance of bacteria include efflux pumps which extrude the antibiotic prior to reaching its target. Phenothiazines inhibit the activity of some efflux pumps thereby altering the susceptibility of bacteria. This study demonstrated that chlorpromazine and thioridazine reduce the susceptibility of methicillin-resistant strains (MRSA) but not that of methicillin-susceptible Staphylococcus aureus (MSSA) strains to oxacillin (MIC of oxacillin reduced from >500 to 10 mg/l). Reserpine, an inhibitor of antibiotic efflux pumps also reduced the resistance of MRSA strains to oxacillin suggesting the presence of an efflux pump that contributes to antibiotic resistance of MRSA strains.

Clinical concentrations of thioridazine enhance the killing of intracellular methicillin-resistant Staphylococcus aureus: an in vivo, ex vivo and electron microscopy study.

Chlorpromazine (CPZ) is concentrated by human macrophages where it kills intracellular mycobacteria when the concentration outside the macrophage is sub-clinical. We have previously demonstrated that thioridazine (TZ), a much milder phenothiazine, has similar activity and kills intracellular methicillin-susceptible S. aureus at sub-clinical concentrations. We have extended this latter study to include methicillin-resistant S. aureus (MRSA) and show that TZ kills intracellular MRSA at clinically relevant concentrations. The ultrastructure of MRSA exposed to in vitro concentrations of TZ just below its MIC and that of MRSA phagocytosed by macrophages previously exposed to a clinically relevant concentration of TZ was also studied. TZ inhibits the replication of phagocytosed MRSA, affecting the structure of the cell envelope, resulting in lysis of the bacterium 6 hours post-phagocytosis. These ultrastructural changes are identical to those produced in vitro by a TZ concentration that is just below the MIC. Because macrophage intracellular MRSA is not killed by the macrophage and its intracellular location protects it from antibiotics that are unable to reach that site, recurrent infections which result may be successfully managed with the use of TZ.

Role of Phenothiazines and Structurally Similar Compounds of Plant Origin in the Fight against Infections by Drug Resistant Bacteria.

Phenothiazines have their primary effects on the plasma membranes of prokaryotes and eukaryotes. Among the components of the prokaryotic plasma membrane affected are efflux pumps, their energy sources and energy providing enzymes, such as ATPase, and genes that regulate and code for the permeability aspect of a bacterium. The response of multidrug and extensively drug resistant tuberculosis to phenothiazines shows an alternative therapy for the treatment of these dreaded diseases, which are claiming more and more lives every year throughout the world. Many phenothiazines have shown synergistic activity with several antibiotics thereby lowering the doses of antibiotics administered to patients suffering from specific bacterial infections. Trimeprazine is synergistic with trimethoprim. Flupenthixol (Fp) has been found to be synergistic with penicillin and chlorpromazine (CPZ); in addition, some antibiotics are also synergistic. Along with the antibacterial action described in this review, many phenothiazines possess plasmid curing activities, which render the bacterial carrier of the plasmid sensitive to antibiotics. Thus, simultaneous applications of a phenothiazine like TZ would not only act as an additional antibacterial agent but also would help to eliminate drug resistant plasmid from the infectious bacterial cells.

A comparative analysis of in vitro and in vivo efficacies of the enantiomers of thioridazine and its racemate.

A long list of chemotherapeutical drugs used in the treatment of the peripheral and the central nervous systems possess anti-microbial activity. Some of these neurotropic compounds are chiral, with the one stereo isomeric form exaggerating reduced neurotropism. This is the case for the levorotatory form of thioridazine. The phenothiazine thioridazine is an interesting compound, characterized by exhibiting a significant growth inhibiting activity on a wide array of micro-organisms. Thioridazine is characterized by another challenging feature, because the compound is concentrated in certain human tissue cells. The present study describes a comparative study of the two enantiomers as well as the racemic form of thioridazine. The study exploits the stereochemical aspect and the in vitro and in vivo potential of these compounds, with a focus on the effects on gram negative organism Salmonella enterica serover Typhimurium. In summary, the results of this study yielded a significant antibacterial activity of all forms of thioridazine, indicating the levorotatory (-)-form to be superior in terms of both its in vitro and in vivo efficacies.

Effect of thioridazine stereoisomers on the drug accumulation of mouse lymphoma and human prostate cancer cell lines in vitro.

BACKGROUND: Cancer cells become refractory to chemotherapy as a consequence of their overexpression of multidrug transporters. MATERIALS AND METHODS: The anticancer and multidrug resistance (MDR) reversal effects of the racemic form and the two enantiomers of thoridazine were investigated on a mouse T-lymphoma cell line over-expressing the ATP-binding cassette, subfamily-B (MDR/TAP), member 1 (ABCB1) transporter (also known as P-glycoprotein) and on human PC3 prostate cancer cell line by 3-(4.5-dimethylthiazolyl-2)-2.5-diphenyl tetrazolium bromide (MTT) assay. The modulation of ABCB1 transporter activity was studied by rhodamine123 accumulation, the apoptosis-inducing effect was investigated using fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide. RESULTS: The thioridazine racemic and (+) and (-) enantiomers were similarly effective. Drug accumulation by MDR mouse T-lymphoma cells was moderately modified in the presence of thioridazine derivatives. Thioridazine induced apoptosis of the MDR cancer cell line, but there was no significant apoptotic effect on the PC3 cell line. CONCLUSION: Apparently, the chirality of thioridazine has no importance in the inhibition of MDR phenotype of cancer cells.