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 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.

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 (AU)

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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.

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.

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.

Effective therapy with the neuroleptic thioridazine as an adjunct to second line of defence drugs, and the potential that thioridazine offers for new patents that cover a variety of "new uses".

New and active infections of tuberculosis continue to increase globally. Although antibiotic susceptible infections can be readily cured with isoniazide and rifampicin, infections resistant to these two antibiotics, named Multi-Drug Resistant TB (MDR TB), are problematic for therapy, extol high costs in terms of human suffering and finances, and when these MDR infections progress to Extensive Drug Resistant TB (XDR TB) status, they are not only difficult to treat, they produce high levels of mortality regardless of therapeutic modality employed. The neuroleptic thioridazine (TZ) has been shown to have wide spectrum in vitro and ex vivo activities against antibiotic susceptible, MDR and XDR strains, and has been successfully used for curing mice of active tuberculosis produced by antibiotic susceptible and MDR strains, and has cured 10 out of 12 XDR TB patients when used in combination with three antibiotics to which the XDR TB patients were non-responsive. Mycobacterium tuberculosis TZ has been recommended for "Compassionate Therapy" of MDR/XDR TB infections whose prognoses are significantly serious and anticipated to result in mortality. This review of TZ activity and its potential to cure MDR/XDR TB supports the contention that this neuroleptic offers patenting opportunities for "New Use". The motivation for patents therefore is expected to rapidly bring TZ to the forefront for therapy of MDR/XDR TB and therefore, the striving for new patents is expected to contribute to the prevention of new infections of antibiotic resistant tuberculosis.

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.

Thioridazine protects the mouse from a virulent infection by Salmonella enterica serovar Typhimurium 74.

When administered to mice at doses of 100microg/mouse and 200microg/mouse, thioridazine (TDZ) significantly protected animals from the lethality produced by a virulent strain of Salmonella enterica serovar Typhimurium and reduced the number of bacteria retrieved from the spleen, liver and heart blood. The protection conferred by TDZ against a virulent Salmonella infection is hypothesised to be due to a reduction in the 55kDa virulence protein of the outer membrane of the organism, as this protein is almost totally absent when the organism is exposed to the phenothiazine. It is further hypothesised that the reduction in the 55kDa virulence factor renders the organism susceptible to the action of hydrolytic enzymes of the neutrophil phagolysosome, whereas in the absence of exposure to TDZ intracellular ingestion and localisation of the phagocytosed bacterium does not result in killing owing to rapid induction of the two-step PmrA/B regulon that results in the eventual synthesis and insertion of lipid A into the nascent lipopolysaccharide layer of the outer membrane.

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.

[Pigs as an infection source for methicillin resistant Staphylococcus aureus infections in humans]. / Svin som smittekilde til infektioner med methicillinresistente Staphylococcus aureus hos mennesker.

Recent Dutch studies indicate that methicillin-resistant Staphylococcus aureus (MRSA) sequence type 398 is widely distributed in pigs and may give rise to infection in humans. In this study we present the first two Danish cases of MRSA infection, which in all probability were acquired from occupational contact with pigs. One infection presented as a severe surgical wound infection, following knee surgery, the other as a superficial ear lobe infection. Both MRSA strains were multiresistant, sequence type 398, Spa-type t034, and Panton-Valentine leukocidin-encoding gene negative.

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.

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.

Mycobacterium alsiense, a novel, slowly growing species isolated from two patients with pulmonary disease.

A previously undescribed, slowly growing Mycobacterium species was isolated from pulmonary specimens of two patients, one from Denmark and one from Italy. The isolates showed unique 16S rRNA internal transcribed spacers and hsp65 sequences: the 16S rRNA was most closely related to Mycobacterium szulgai and Mycobacterium malmoense.

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.

Reversal of resistance in microorganisms by help of non-antibiotics.

Intracellular efflux pumps have been largely the research focus in multidrug-resistant (MDR) Gram-positive and Gram-negative microorganisms and parasites including cancers. However, drug efflux mechanisms other than pumps per se have been observed, supported by the effects of isomeric, non-antibiotic depressant (DPR), phenothiazines and thixenes, and antidepressant (ADPR) phenylpiperidine neurotropic drugs, alone or in combination with classical antimicrobials on MDR Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes and Streptococcus pneumoniae. Of the non-antibiotics we investigated, the DPR l-thioridazine, trans-clopenthixol and isomers of phenylpiperidines NNC 20-4962 (isomer of femoxetine) and NNC 20-7052 (isomer of paroxetine) were potent antimicrobials with the least neurotropic activity, pointing to a possible general isomeric structure-activity relationship. These compounds may be regarded as new efflux inhibitors. Moreover, these isomers have considerably reduced, in some cases absent, neurotropism and reduced mammalian toxicity. This may alleviate concerns about adverse effects and therapeutic safety for infected patients in life-threatening situations where the non-antibiotic dosage would be in the lower, non-chronic dosage ranges generally prescribed for individuals with mild mental health problems. The results point to the prokaryotic and eukaryotic microorganisms' phospholipid/protein domain involvement of the cationic, amphiphilic, non-antibiotic DPR and ADPR, with the phospholipids being the initial sites attracting and concentrating the neurotropes to induce a form of suspended animation, followed by gross changes of cell wall and membrane structure, and thereby potentiating their destructive or immobilizing effects on various as yet only hinted at resistance and efflux mechanisms. Combination of appropriate isomeric non-antibiotic DPR and ADPR of low neurotropism and toxicity with conventional and classical antimicrobials promises early, new therapeutic strategies salutary against microbial resistance, resistance development, pathogenicity and virulence.

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.

"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).