A rapid screening assay for identifying mycobacteria targeted nanoparticle antibiotics.

Antibiotic resistance is a serious problem. Nanotechnology offers enormous potential in medicine, yet there is limited knowledge regarding the toxicity of nanoparticles (NP) for mycobacterial species that cause serious human diseases (e.g. tuberculosis (TB) and leprosy). Mycobacterial diseases are a major global health problem; TB caused by Mycobacterium tuberculosis (Mtb) kills up to 2 million people annually and there are over 200 000 leprosy cases each year caused by Mycobacterium leprae (M. leprae). Few drugs are effective against these mycobacteria and increasing antibiotic resistance exacerbates the problem. As such, alternative therapies are urgently needed but most current assays used to assess the effectiveness of therapeutics against mycobacteria are slow and expensive. This study aimed to develop a rapid, low-cost assay which can be used for screening the antimicrobial properties of compounds against pathogenic mycobacteria and to assess the toxicity of three NP (silver [Ag], copper oxide [Cu(II)O], and zinc oxide [ZnO]) against a green fluorescent protein reporter strain of Mycobacterium avium subspecies paratuberculosis, a slow growing, pathogenic mycobacterial species causing paratuberculosis in ruminants. Fluorescence was used to monitor mycobacterial growth over time, with NP concentrations of 6.25-100 µg/mL tested for up to 7 days, and a method of data analysis was designed to permit comparison between results. Mycobacterial sensitivity to the NP was found to be NP composition specific and toxicity could be ranked in the following order: Ag > Cu(II)O > ZnO.

The antibiotic resistance arrow of time: efflux pump induction is a general first step in the evolution of mycobacterial drug resistance.

We hypothesize that low-level efflux pump expression is the first step in the development of high-level drug resistance in mycobacteria. We performed 28-day azithromycin dose-effect and dose-scheduling studies in our hollow-fiber model of disseminated Mycobacterium avium-M. intracellulare complex. Both microbial kill and resistance emergence were most closely linked to the within-macrophage area under the concentration-time curve (AUC)/MIC ratio. Quantitative PCR revealed that subtherapeutic azithromycin exposures over 3 days led to a 56-fold increase in expression of MAV_3306, which encodes a putative ABC transporter, and MAV_1406, which encodes a putative major facilitator superfamily pump, in M. avium. By day 7, a subpopulation of M. avium with low-level resistance was encountered and exhibited the classic inverted U curve versus AUC/MIC ratios. The resistance was abolished by an efflux pump inhibitor. While the maximal microbial kill started to decrease after day 7, a population with high-level azithromycin resistance appeared at day 28. This resistance could not be reversed by efflux pump inhibitors. Orthologs of pumps encoded by MAV_3306 and MAV_1406 were identified in Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium abscessus, and Mycobacterium ulcerans. All had highly conserved protein secondary structures. We propose that induction of several efflux pumps is the first step in a general pathway to drug resistance that eventually leads to high-level chromosomal-mutation-related resistance in mycobacteria as ordered events in an "antibiotic resistance arrow of time."

The thioamides methimazole and thiourea inhibit growth of M. avium Subspecies paratuberculosis in culture.

BACKGROUND: Thyrotoxicosis is conceptualized as an "autoimmune" disease with no accepted infectious etiology. There are increasingly compelling data that another "autoimmune" affliction, Crohn disease, may be caused by Mycobacterium avium subspecies paratuberculosis (MAP). Like M. tb, MAP is systemic. We hypothesized that some cases of thyrotoxicosis may be initiated by a MAP infection. Because other thioamides treat tuberculosis, leprosy and M. avium complex, we hypothesized that a mode of action of some thioamide anti-thyrotoxicosis medications may include MAP growth inhibition. METHODS: The effect of the thioamides, thiourea, methimazole and 6-propo-2-thiouracil (6-PTU) were studied in radiometric Bactec culture, on ten strains of three mycobacterial species (six of MAP, two of M. avium and two of M. tb. complex). Data are presented as "cumulative growth index," (cGI) or "percent decrease in cumulative GI" (%-DeltacGI). PRINCIPAL FINDINGS: Methimazole was the most effective thioamide at inhibiting MAP growth. At 128microg/ml: MAP UCF-4; 65%-DeltacGI & MAP ATCC 19698; 90%-DeltacGI. Thiourea inhibited MAP "Ben" maximally; 70%-DeltacGI. Neither methimazole nor thiourea inhibited M. avium or M. tb. at the doses tested. 6-PTU has no inhibition on any strain studied, although a structurally analogous control, 5-PTU, was the most inhibitory thioamide tested. SIGNIFICANCE: We show inhibition of MAP growth by the thioamides, thiourea and methimazole in culture. These data are compatible with the hypothesis that these thioamides may have anti-prokaryotic in addition to their well-established eukaryotic actions in thyrotoxic individuals.

Genome and transcriptome scale portrait of sigma factors in Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (JD), a chronic gastroenteritis of ruminants and other animals, including primates. Many evidences suggested association of MAP to Crohn's disease, a chronic granulomatous gastrointestinal disease of humans with strong similarities with JD. The present study attempts to evaluate global gene regulation in MAP, which has not been addressed previously, despite the availability of MAP genome sequence. For this purpose, we investigated: (i) the presence of sigma factors and their relationship to sigma factors of other mycobacteria (M. avium subsp.avium, M. tuberculosis, M. bovis, M. leprae and M. smegmatis), and (ii) their expression during different growth conditions and in vitro infection of intestinal epithelial Caco2 cells. MAP genome contains 19 putative sigma factor, but only 12 belong to gene families common to other mycobacteria. Gene expression was evaluated with Real-Time PCR during growth in 7H9 medium and mycobactin J, in 7H9 medium plus mycobactin J and lisozyme, and during infection of Caco2 cells: very different expression patterns were observed and, on the whole, only 7 sigma factors were found to be expressed. sigJ was upregulated during the infection of Caco2 cells. Even if only few sigma factors were expressed in the three conditions tested, the overall high numbers of MAP sigma factors suggests a noteworthy flexibility of this pathogen. Thus, this first report on expression of MAP sigma factors opens the way to an extensive characterization of global gene regulation, as a key to understand strategies of survival and mechanisms of infections used by this organism.

Genes required for intrinsic multidrug resistance in Mycobacterium avium.

Genes required for intrinsic multidrug resistance by Mycobacterium avium were identified by screening a library of transposon insertion mutants for the inability to grow in the presence of ciprofloxacin, clarithromycin, and penicillin at subinhibitory concentrations. Two genes, pks12 and Maa2520, were disrupted in multiple drug-susceptible mutants. The pks12 gene (Maa1979), which may be cotranscribed with a downstream gene (Maa1980), is widely conserved in the actinomycetes. Its ortholog in Mycobacterium tuberculosis is a polyketide synthase required for the synthesis of dimycocerosyl phthiocerol, a major cell wall lipid. Mutants of M. avium with insertions into pks12 exhibited altered colony morphology and were drug susceptible, but they grew as well as the wild type did in vitro and intracellularly within THP-1 cells. A pks12 mutant of M. tuberculosis was moderately more susceptible to clarithromycin than was its parent strain; however, susceptibility to ciprofloxacin and penicillin was not altered. M. avium complex (MAC) and M. tuberculosis appear to have different genetic mechanisms for resisting the effects of these antibiotics, with pks12 playing a relatively more significant role in MAC. The second genetic locus identified in this study, Maa2520, is a conserved hypothetical gene with orthologs in M. tuberculosis and Mycobacterium leprae. It is immediately upstream of Maa2521, which may code for an exported protein. Mutants with insertions at this locus were susceptible to multiple antibiotics and slow growing in vitro and were unable to survive intracellularly within THP-1 cells. Like pks12 mutants, they exhibited increased Congo red binding, an indirect indication of cell wall modifications. Maa2520 and pks12 are the first genes to be linked by mutation to intrinsic drug resistance in MAC.

Effect of listeriolysin O-loaded erythrocytes on Mycobacterium avium replication within macrophages.

OBJECTIVE: To evaluate the efficacy of erythrocytes loaded with the haemolytic toxin listeriolysin O against Mycobacterium avium replication within human macrophages. METHODS: Recombinant listeriolysin O was loaded in human erythrocytes by a procedure of hypotonic dialysis and isotonic resealing. Loaded erythrocytes were modified to allow them to be recognized and taken up by human macrophages infected with M. avium. The antimycobacterial activity of the erythrocytes loaded with listeriolysin O was evaluated by supernatant and intracellular cfu counts on days 4 and 7 post-erythrocyte administration. RESULTS: Recombinant listeriolysin O was encapsulated in human erythrocytes to reach final concentrations ranging from 1 to 4 ng/mL of erythrocytes. Erythrocytes loaded with increasing quantities of recombinant protein were able to reduce (at most by 50%) M. avium replication in a dose-dependent fashion when administered to infected macrophages. CONCLUSIONS: Erythrocytes loaded with listeriolysin O are effective against M. avium replication within macrophages. We are confident that the strategy presented could be useful against mycobacteria other than M. avium (such as Mycobacterium tuberculosis and Mycobacterium leprae) by itself or as part of an antimycobacterial treatment.

Treatment of localized Mycobacterium avium complex infection with clofazimine and doxycycline in a cat.

Mycobacterium avium complex infection resulted in a granuloma that developed at the base of the left ear in a cat. The lesion caused vestibular dysfunction and facial palsy on the left side and protruded into the oral cavity on that side. The cat was treated successfully, with resolution of the lesion and elimination of the organism, by use of combined administration of clofazimine and doxycycline. Adverse effects of the clofazimine treatment included temporary reddish-orange discoloration of the cat's skin and mucous membranes.

Recent trends in rifamycin research.

Rifamycin is a clinically useful macrolide antibiotic produced by the gram positive bacterium Amycolatopsis mediterranei. This antibiotic is primarily used against Mycobacterium tuberculosis and Mycobacterium leprae, causative agents of tuberculosis and leprosy, respectively. In these bacteria, rifamycin treatment specifically inhibits the initiation of RNA synthesis by binding to beta-subunit of RNA polymerase. Apart from its activity against the bacteria, rifamycin has also been reported to inhibit reverse transcriptase (RT) of certain RNA viruses. Recently, rifamycin derivatives have been discovered that are effective against Mycobacterium avium, which is associated with the AIDS complex. Consequently, the importance of and demand for rifamycin has increased tremendously, the world over. In this article, recent trends in rifamycin research and accessibility of recombinant DNA techniques to increase rifamycin production are reviewed.

Comparative in vitro and in vivo activity of fleroxacin and ofloxacin against various mycobacteria.

In vitro antimicrobial activity of fleroxacin (6,8- difluoro-1-(2-fluoroethyl)-1, 4-dihydro-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid) and ofloxacin against representative pathogenic mycobacteria was evaluated by the agar dilution method, using 7H11 agar medium. Fleroxacin showed appreciable antimicrobial activity against Mycobacterium tuberculosis (MIC90 = 6.25 mg/l), M. kansasii (MIC90 = 3.13 mg/l), and M. fortuitum (MIC90 = 6.25 mg/l), whereas M. marinum, M. scrofulaceum, M. avium, M. intracellulare, and M. chelonae were highly resistant to the agent. The activity of fleroxacin was comparable to that of ofloxacin. Fleroxacin showed antimicrobial activity against M. intracellulare phagocytosed in murine peritoneal macrophages at a concentration of 10 mg/l in the culture medium, but its activity was considerably lower than that of ofloxacin. On the other hand, the therapeutic activity of fleroxacin against M. fortuitum infection induced in mice was higher than that of ofloxacin. Neither fleroxacin nor ofloxacin was efficacious against M. intracellulare infection. Fleroxacin significantly depressed the growth of M. leprae in the mouse footpad.

Methionine as methyl-group donor in the synthesis of Mycobacterium avium envelope lipids, and its inhibition by DL-ethionine, D-norleucine and DL-norleucine.

The radioactivity from 3H-methyl methionine was rapidly incorporated into the surface lipids of Mycobacterium avium. The transmethylation reaction was efficiently inhibited by DL-ethionine, D-norleucine and DL-norleucine. The structure of the outerlayer of the M. avium envelope was profoundly altered in the bacteria treated with DL-norleucine.

Rifabutin (ansamycin LM 427): a new rifamycin-S derivative for the treatment of mycobacterial diseases.

Rifabutin (ansamycin LM 427), a semisynthetic spiropiperidyl derivative of rifamycin S, shows good in vitro activity against most mycobacterial species, including Mycobacterium avium complex. In animal models, the drug is more active against both Mycobacterium tuberculosis and Mycobacterium leprae than in rifampin, and studies indicate that rifabutin is active against some rifampin-resistant strains of both species. The drug has a long half-life (16 hr) in humans and a marked tissue tropism, with tissue levels five- to 10-fold higher than that in the serum. In animals rifabutin is no more toxic than rifampin. A large experience from the compassionate use of rifabutin for life-threatening mycobacterial infections in humans, most commonly disseminated M. avium complex disease in patients with AIDS, has also indicated relative drug safety. Although some data suggest that rifabutin is effective, firm conclusions about drug efficacy await results from controlled clinical trials.

[Relationships between "Mycobacterium simiae" and the "M. avium-intracellulare-scrofulaceum" complex (author's transl)]. / Relations entre Mycobacterium simiae et le complexe M. avium-intracellulare-scofulaceum.

The 24 strains of Mycobacterium simiae described in this report were isolated from 12 black Africans, 6 from white Europeans, 5 from primates and 1 from a leprosy infected Armadillo. These strains form 3 groups having the similar morphologic and cultural properties as M. intracellulare. Two groups were similar with respect to pigmentation, urease activity and niacin production but differed serologically, the second group being of M. intracellulare serotype 18. The third group was less homogenous and was intermediate to M. simiae and M. intracellulare. Thus M. simiae belong to the M. avium-intracellulare-simiae-scrofulaceum (MAISS) complex. Two cases of well characterized pulmonary disease progressed like M. avium mycobacteriosis.