Discerning novel drug targets for treating Mycobacterium avium ss. paratuberculosis-associated autoimmune disorders: an in silico approach.

Mycobacterium avium subspecies paratuberculosis (MAP) exhibits 'molecular mimicry' with the human host resulting in several autoimmune diseases such as multiple sclerosis, type 1 diabetes mellitus (T1DM), Hashimoto's thyroiditis, Crohn's disease (CD), etc. The conventional therapy for autoimmune diseases includes immunosuppressants or immunomodulators that treat the symptoms rather than the etiology and/or causative mechanism(s). Eliminating MAP-the etiopathological agent might be a better strategy to treat MAP-associated autoimmune diseases. In this case study, we conducted a systematic in silico analysis to identify the metabolic chokepoints of MAP's mimicry proteins and their interacting partners. The probable inhibitors of chokepoint proteins were identified using DrugBank. DrugBank molecules were stringently screened and molecular interactions were analyzed by molecular docking and 'off-target' binding. Thus, we identified 18 metabolic chokepoints of MAP mimicry proteins and 13 DrugBank molecules that could inhibit three chokepoint proteins viz. katG, rpoB and narH. On the basis of molecular interaction between drug and target proteins finally eight DrugBank molecules, viz. DB00609, DB00951, DB00615, DB01220, DB08638, DB08226, DB08266 and DB07349 were selected and are proposed for treatment of three MAP-associated autoimmune diseases namely, T1DM, CD and multiple sclerosis. Because these molecules are either approved by the Food and Drug Administration or these are experimental drugs that can be easily incorporated in clinical studies or tested in vitro. The proposed strategy may be used to repurpose drugs to treat autoimmune diseases induced by other pathogens.

Bovine NK-lysin-derived peptides have bactericidal effects against Mycobacterium avium subspecies paratuberculosis.

Infection with Mycobacterium avium subspecies paratuberculosis (MAP) is complex, but little is known about the role that natural killer (NK) cells play. In the present study, four bovine NK-lysin peptides were synthesized to evaluate their bactericidal activity against MAP. The results demonstrated that bNK-lysin peptides were directly bactericidal against MAP, with bNK1 and bNK2A being more potent than bNK2B and bNK2C. Mechanistically, transmission electron microscopy revealed that the incubation of MAP with bNK2A resulted in extensive damage to cell membranes and cytosolic content leakage. Furthermore, the addition of bNK2A linked with a cell-penetrating peptide resulted in increased MAP killing in a macrophage model.

Effectiveness of copper ions against Mycobacterium avium subsp. paratuberculosis and bacterial communities in naturally contaminated raw cow's milk.

AIM: The focus of the present study was to evaluate the copper ions treatment on the viability of Mycobacterium avium subsp. paratuberculosis (MAP) and other bacterial communities in cow's milk. METHODS AND RESULTS: A copper ions treatment was evaluated in naturally contaminated cow's milk to assay MAP load and/or viability, and relative abundance of other bacterial communities. In addition, physical-chemical analyses of the milk were also performed. All analyses were carried out before and after a copper ions treatment. After copper ions treatment, pH and copper concentration markedly increased in milk; the numbers of viable MAP significantly decreased. The relative abundance of the four target phyla decreased, with the phyla Bacteroidetes and Firmicutes surviving treatment in higher proportions (4 and 2·1% of original populations, respectively). A progressively higher percentage of dead bacterial cells after 5 and 20 min copper ions treatments was found (12 and 35%, respectively). CONCLUSION: With the exception of some MAP-tolerant strains, we have once again demonstrated that copper ions have a significant inactivating effect on MAP as well as certain other bacterial communities found in naturally contaminated cow's milk. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed a significant inactivation of both MAP and other bacteria by copper ions in raw cow's milk, information that could be useful as a tool for MAP control.

Synthetic cathelicidin LL-37 reduces Mycobacterium avium subsp. paratuberculosis internalization and pro-inflammatory cytokines in macrophages.

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic diarrheic intestinal infections in domestic and wild ruminants (paratuberculosis or Johne's disease) for which there is no effective treatment. Critical in the pathogenesis of MAP infection is the invasion and survival into macrophages, immune cells with ability to carry on phagocytosis of microbes. In a search for effective therapeutics, our objective was to determine whether human cathelicidin LL-37, a small peptide secreted by leuckocytes and epithelial cells, enhances the macrophage ability to clear MAP infection. In murine (J774A.1) macrophages, MAP was quickly internalized, as determined by confocal microscopy using green fluorescence protein expressing MAPs. Macrophages infected with MAP had increased transcriptional gene expression of pro-inflammatory TNF-α, IFN-γ, and IL-1ß cytokines and the leukocyte chemoattractant IL-8. Pretreatment of macrophages with synthetic LL-37 reduced MAP load and diminished the transcriptional expression of TNF-α and IFN-γ whereas increased IL-8. Synthetic LL-37 also reduced the gene expression of Toll-like receptor (TLR)-2, key for mycobacterial invasion into macrophages. We concluded that cathelicidin LL-37 enhances MAP clearance into macrophages and suppressed production of tissue-damaging inflammatory cytokines. This cathelicidin peptide could represent a foundational molecule to develop therapeutics for controlling MAP infection.

Fate of Mycobacterium avium subsp. paratuberculosis and changes in bacterial diversity populations in dairy slurry after chemical treatments.

AIMS: A major drawback of using dairy slurry as fertilizer is that it may contains pathogens such as Mycobacterium avium subsp. paratuberculosis (MAP), and it could represent a risk to animal and public health. Thus, the aim of this study was to evaluate the fate of MAP and bacterial communities in dairy slurry after chemical treatments. METHODS AND RESULTS: Cattle slurry, naturally contaminated with MAP, was collected from a dairy herd and divided into 32 glass bottles which were assigned to eight different treatments (control, 3·0% CaO, 0·5% NaOH; 0·087%, 0·11% and 0·14% H2 SO4 ; and 1·0 and 2·5% KMnO4 ). Treated dairy slurry samples were evaluated at 0, 1, 3, 7, 15, 30 and 60-days following treatment application for viable MAP and dairy slurry pH, and in addition temperature in this material was monitored continuously. Bacterial counts were estimated at each sampling time. A Bayesian zero-inflated Poisson mixed model was fitted to assess the effect of each treatment on the count of MAP cells. Model results indicated that only the 3·0% CaO treatment had a statistically important negative effect on MAP counts during the study period. For most treatments, MAP was undetectable immediately after chemical treatment but re-appeared over time, in some replicates at low concentrations. However, in those cases MAP counts were not statistically different than the control treatment. Regarding the fate of the other bacterial populations, the Firmicutes phylum was the dominant population in the un-treated slurry while Clostridia class members were among the most prevalent bacteria after the application of most chemical treatments. CONCLUSION: Only 3% CaO treatment had a statistically important negative effect on MAP viability in cattle slurry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence of MAP partial control in dairy slurry. This information should be considered as a best management practice to reduce MAP and other pathogens for slurry management on dairy farms.

The inhibitory effect of nisin on Mycobacterium avium ssp. paratuberculosis and its effect on mycobacterial cell wall.

Infection with Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) is a widespread problem in the United States and worldwide, and it constitutes a significant health problem for dairy animals with a potential effect on human health. Mycobacterium paratuberculosis is easily transmitted through consumption of contaminated milk; therefore, finding safe methods to reduce the mycobacterial load in milk and other dairy products is important to the dairy industry. The main objective of the current study was to investigate the effect of natural products, such as bacteriocins designated as "generally regarded as safe" (GRAS), on the survival of M. paratuberculosis in milk. Commercially synthesized bacteriocin (nisin) was used to examine its effect on the survival of laboratory and field isolates of M. paratuberculosis and in contaminated milk. Surprisingly, nisin had a higher minimum inhibitory concentration (MIC) against the laboratory strain (M. paratuberculosis K10), at 500 U/mL, than against field isolates (e.g., M. paratuberculosis 4B and JTC 1281), at 15 U/mL. In milk, growth of M. paratuberculosis was inhibited after treatment with levels of nisin that are permissible in human food at 4°C and 37°C. Using both fluorescent and scanning electron microscopy, we were able to identify defects in the bacterial cell walls of treated cultures. Our analysis indicated that nisin reduced membrane integrity by forming pores in the mycobacterial cell wall, thereby decreasing survival of M. paratuberculosis. Thus, nisin treatment of milk could be implemented as a control measure to reduce M. paratuberculosis secreted in milk from infected herds. Nisin could also be used to reduce M. paratuberculosis in colostrum given to calves from infected animals, improving biosecurity control in dairy herds affected by Johne's disease.

In vitro inactivation of Mycobacterium avium subsp. paratuberculosis (MAP) by use of copper ions.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a contagious infectious disease that affects domestic and wild ruminants causing chronic inflammation of the intestine. MAP has proven to be very resistant to both physical and chemical processes, making it difficult to control this pathogen. Based on the recognized antimicrobial properties of copper, the objective of this study was to evaluate the effectiveness of copper ions to reduce MAP numbers and/or MAP viability in a fluid matrix. Besides, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli were used as controls of the effectiveness of copper ions. MAP-spiked PBS was subjected to copper ions treatment at 24 V for 5 min and the PBS suspensions were sampled before and after treatment. MAP viability and quantification were determined using three complementary techniques: a phage amplification assay, MGIT culture and qPCR. RESULTS: Moderate numbers (103 CFU ml-1) of the two control bacteria were completely eliminated by treatment with copper ions. For MAP, copper ions treatment reduced both the viability and numbers of this pathogen. Phage assay information quickly showed that copper ions (24 V for 5 min) resulted in a significant reduction in viable MAP. MGIT culture results over time showed statistically significant differences in time-to-detection (TTD) values between PRE and POST treatment. MAP genome equivalent estimates for PBS suspensions indicated that MAP numbers were lower in samples POST-treatment with copper ions than PRE-treatment. CONCLUSIONS: The use of copper ions resulted in a significant reduction of MAP in a liquid matrix, although some MAP survival on some occasions was observed.

Development of an Interspecies Nested Dose-Response Model for Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic inflammation of the intestines in humans, ruminants, and other species. It is the causative agent of Johne's disease in cattle, and has been implicated as the causative agent of Crohn's disease in humans. To date, no quantitative microbial risk assessment (QMRA) for MAP utilizing a dose-response function exists. The objective of this study is to develop a nested dose-response model for infection from oral exposure to MAP utilizing data from the peer-reviewed literature. Four studies amenable to dose-response modeling were identified in the literature search and optimized to the one-parameter exponential or two-parameter beta-Poisson dose-response models. A nesting analysis was performed on all permutations of the candidate data sets to determine the acceptability of pooling data sets across host species. Three of four data sets exhibited goodness of fit to at least one model. All three data sets exhibited good fit to the beta-Poisson model, and one data set exhibited goodness of fit, and best fit, to the exponential model. Two data sets were successfully nested using the beta-Poisson model with parameters α = 0.0978 and N50 = 2.70 × 10(2) CFU. These data sets were derived from sheep and red deer host species, indicating successful interspecies nesting, and demonstrate the highly infective nature of MAP. The nested dose-response model described should be used for future QMRA research regarding oral exposure to MAP.

Synthesis and Biological Evaluation of N-Alkoxyphenyl-3-hydroxynaphthalene-2-carboxanilides.

A series of fifteen new N-alkoxyphenylanilides of 3-hydroxynaphthalene-2-carboxylic acid was prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Staphylococcus aureus, three methicillin-resistant S. aureus strains, Mycobacterium tuberculosis H37Ra and M. avium subsp. paratuberculosis. Some of the tested compounds showed antibacterial and antimycobacterial activity against the tested strains comparable with or higher than that of the standards ampicillin or rifampicin. 3-Hydroxy-N-(2-propoxyphenyl)naphthalene-2-carboxamide and N-[2-(but-2-yloxy)-phenyl]-3-hydroxynaphthalene-2-carboxamide had MIC = 12 µM against all methicillin-resistant S. aureus strains; thus their activity is 4-fold higher than that of ampicillin. The second mentioned compound as well as 3-hydroxy-N-[3-(prop-2-yloxy)phenyl]-naphthalene-2-carboxamide had MICs = 23 µM and 24 µM against M. tuberculosis respectively. N-[2-(But-2-yloxy)phenyl]-3-hydroxynaphthalene-2-carboxamide demonstrated higher activity against M. avium subsp. paratuberculosis than rifampicin. Screening of the cytotoxicity of the most effective antimycobacterial compounds was performed using THP-1 cells, and no significant lethal effect was observed for the most potent compounds. The compounds were additionally tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. N-(3-Ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide (IC50 = 4.5 µM) was the most active PET inhibitor. The structure-activity relationships are discussed.

Short communication: Application of an N-acetyl-L-cysteine-NaOH decontamination method for the recovery of viable Mycobacterium avium subspecies paratuberculosis from milk of naturally infected cows.

Mycobacterium avium ssp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne's disease and, therefore, is a route of transmission for infection in young stock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of MAP from individual bovine milk samples from known infected herds. Decontamination of milk samples (n = 17) with either 0.75% hexadecylpyridinium chloride for 5h or N-acetyl-L-cysteine-1.5% sodium hydroxide (NALC-1.5% NaOH) for 15 min before culture in BACTEC 12 B (Becton Dickinson, Franklin, NJ), para-JEM [Thermo Fisher Scientific (TREK Diagnostic Systems, Inc.), Cleveland, OH], and Herrold's egg yolk (HEY; Becton Dickinson) media was compared. Treatment with NALC-NaOH resulted in a lower percentage (6%) of contaminated samples than did treatment with hexadecylpyridinium chloride (47%), regardless of culture medium. The decontamination protocol (NALC-1.5% NaOH) was then applied to milk samples (n = 144) collected from cows at 7 US dairies. Recovery of viable MAP from the milk samples was low, regardless of culture medium, with recovery from 2 samples cultured in BACTEC 12 B medium, 1 sample cultured in para-JEM medium, and no viable MAP recovered on HEY medium. However, 32 cows were fecal culture positive and 13 milk samples were positive by direct PCR, suggesting that several cows were actively shedding MAP at the time of milk collection. Contamination rates were similar across media, with 39.6, 34.7, and 41.7% of samples contaminated after culture in BACTEC 12 B, para-JEM, and HEY media, respectively. Herd-to-herd variation had a major effect on sample contamination, with the percentage of contaminated samples ranging from 4 to 83%. It was concluded that decontamination of milk with NALC-1.5% NaOH before culture in BACTEC 12 B medium was the most efficacious method for the recovery of viable MAP from milk, although the ability to suppress the growth of contaminating microorganisms varied greatly between herds.

A comprehensive review on Phyto-MAP: A novel approach of drug discovery against Mycobacterium avium subspecies paratuberculosis using AYUSH heritage.

ETHNOPHARMACOLOGICAL RELEVANCE: Indian system of Traditional medicine, AYUSH (Ayurveda, Yoga, Unani, Siddha, and Homeopathy) has great potential with a History of Safe Use (HOSU) of thousands of medicinal plants included in pharmacopoeias. The multi-targeted approach of phytoconstituents present in different traditionally used medicinal plants makes them suitable candidates for research against various infective pathogens. MAP which is a dairy-borne pathogen is associated with the development of Johne's disease in ruminants and Crohn's disease like autoimmune disorders in human beings. There are no reliable treatment alternatives available against MAP, leaving surgical removal of intestines as the sole option. Hence, there exists an urgent need to search for leads against such infection. AIM OF THE STUDY: The present review has been conducted to find out the ethnopharmacological evidence about the potential of phytoconstituents against Mycobacterium avium subspecies paratuberculosis (MAP), along with the proposal of a potential phyto-MAP mechanism for the very first time taking anti-inflammatory, immunomodulatory, and anti-microbial traditional claims into consideration. MATERIALS AND METHODS: We have analyzed and reviewed different volumes of the two main traditional scriptures of India i.e. Ayurvedic Pharmacopoeia of India (API) and Unani Pharmacopoeia of India (UPI), respectively-for identification of potential anti-MAP plants based on their claims for related disorders. These plants were further investigated systematically for their scientific publications of the last 20 years (2002-2022) available through electronic databases including Google Scholar, Pubmed, and Scopus. The studies conducted in vitro, cell lines, and in vivo levels were taken into consideration along with the associated mechanisms of phytoconstituents. RESULTS: A total of 70 potential medicinal plants have been identified. Based on the ethnopharmacology, a potential phyto-paratuberculosis (Phyto-paraTB) mechanism has been proposed and out of 70, seven potential anti-MAP plants have been identified to have a great future as anti-MAP. CONCLUSION: A novel and scientifically viable plan has been proposed for addressing anti-MAP plants for stimulating research against MAP and related disorders using mass-trusted AYUSH medicine, which can be used as an alternative remedy in resistance cases otherwise can be advocated as an adjuvant with modern treatments for better management of the disease.

Genetic and chemical control of tuberculostearic acid production in Mycobacterium avium subspecies paratuberculosis.

Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and role in membrane dynamics. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a bfaA gene knockout encoding a flavin adenine dinucleotide (FAD)-binding oxidoreductase. Mycobacterium avium subspecies paratuberculosis (Map) growth and TBSA production were inhibited in 0.5-mg/mL pivalic acid-supplemented cultures, but higher concentrations were needed to have a similar effect in other mycobacteria, including Mycobacterium smegmatis. While Map C-type strains, isolated from cattle and other ruminants, will produce TBSA in the absence of pivalic acid, the S-type Map strains, typically isolated from sheep, do not produce TBSA in any condition. A SAM-dependent methyltransferase encoded by bfaB and FAD-binding oxidoreductase are both required in the two-step biosynthesis of TBSA. However, S-type strains contain a single-nucleotide polymorphism in the bfaA gene, rendering the oxidoreductase enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected only in S-type strains. Fatty acid methyl ester analysis of a C-type Map bfaA knockout revealed the loss of TBSA production, but the intermediate was present, similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between two primary genetic lineages of Map and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA should not be used as a diagnostic marker for Map.IMPORTANCEBranched-chain fatty acids are a predominant cell wall component among species belonging to the Mycobacterium genus. One of these is TBSA, which is a long-chain middle-branched fatty acid used as a diagnostic marker for Mycobacterium tuberculosis. This fatty acid is also an excellent biolubricant. Control of its production is important for industrial purposes as well as understanding the biology of mycobacteria. In this study, we discovered that a carboxylic acid compound termed pivalic acid inhibits TBSA production in mycobacteria. Furthermore, Map strains from two separate genetic lineages (C-type and S-type) showed differential production of TBSA. Cattle-type strains of Mycobacterium avium subspecies paratuberculosis produce TBSA, while the sheep-type strains do not. This important phenotypic difference is attributed to a single-nucleotide deletion in sheep-type strains of Map. This work sheds further light on the mechanism used by mycobacteria to produce tuberculostearic acid.

Optimization of hexadecylpyridinium chloride decontamination for culture of Mycobacterium avium subsp. paratuberculosis from milk.

A protocol was optimized for the isolation of Mycobacterium avium subsp. paratuberculosis (MAP) from milk and colostrum, with parameters including chemical decontamination, antibiotics, and different culture media. This study demonstrates that the efficiency of MAP recovery from milk is highly dependent upon the culturing protocol, and such protocols should be optimized to ensure that low concentrations of MAP in milk can be detected.

Survival of Mycobacterium avium subsp. paratuberculosis in synthetic human gastric juice and acidified porcine bile.

The bactericidal activities of synthetic gastric juice and acidified porcine bile on Mycobacterium avium subsp. paratuberculosis were assessed using propidium monoazide (PMA)-mediated quantitative reverse transcription-PCR, which allowed rapid relative quantitative analysis of viable M. avium subsp. paratuberculosis cells.

Synthesis and biological evaluation of 2-hydroxy-3-[(2-aryloxyethyl)amino]propyl 4-[(alkoxycarbonyl)amino]benzoates.

A series of twenty substituted 2-hydroxy-3-[(2-aryloxyethyl)amino]propyl 4-[(alkoxycarbonyl)amino]benzoates were prepared and characterized. As similar compounds have been described as potential antimycobacterials, primary in vitro screening of the synthesized carbamates was also performed against two mycobacterial species. 2-Hydroxy-3-[2-(2,6-dimethoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride, 2-hydroxy-3-[2-(4-methoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride, and 2-hydroxy-3-[2-(2-methoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride showed higher activity against M. avium subsp. paratuberculosis and M. intracellulare than the standards ciprofloxacin, isoniazid, or pyrazinamide. Cytotoxicity assay of effective compounds was performed using the human monocytic leukaemia THP-1 cell line. Compounds with predicted amphiphilic properties were also tested for their effects on the rate of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. All butyl derivatives significantly stimulated the rate of PET, indicating that the compounds can induce conformational changes in thylakoid membranes resulting in an increase of their permeability and so causing uncoupling of phosphorylation from electron transport.

Growth of M. avium subspecies paratuberculosis in culture is enhanced by nicotinic acid, nicotinamide, and α and ß nicotinamide adenine dinucleotide.

BACKGROUND: Without known mechanisms of action, Crohn's disease is exacerbated, and ulcerative colitis is improved, by the use of tobacco. Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. We hypothesized that tobacco components might alter the growth kinetics of MAP, explaining these divergent clinical observations. METHODS: The effect of nicotine, nicotinic acid, nicotinamide and α and ß nicotinamide adenine dinucleotide (α and ß NAD) were studied on eight strains of three mycobacterial species (MAP, M. avium and M. tb. complex). Data are obtained as "cumulative growth index," (cGI) and presented as "percent increase in cumulative GI" (% + ΔcGI). RESULTS: Nicotinic acid enhances the two human MAP isolates (Dominic; 225% + ΔcGI and UCF-4; 92% + ΔcGI) and M. avium (ATCC 25291; 175% + ΔcGI). Nicotinamide (at 6.4 µg/ml) enhances the human MAP isolates (Dominic; 156% + ΔcGI and UCF-4; 79% + ΔcGI) and M. avium (ATCC 25291; 144% + ΔcGI.) Both α and ß NAD enhance Dominic; (135 and 150 % + ΔcGI) and UCF-4; (81 and 79% + ΔcGI). At the doses tested, nicotine has no effect on any strain studied. CONCLUSIONS: We show enhancement of MAP growth by nicotinic acid, one of ≥4,000 tobacco-related molecules, its amide, nicotinamide and α and ß NAD. Pure nicotine has no enhancing effect at the doses studies.

In vitro antimicrobial activities of capuramycin analogues against non-tuberculous mycobacteria.

OBJECTIVES: To determine antibacterial activity of capuramycin analogues SQ997, SQ922, SQ641 and RKS2244 against several non-tuberculous mycobacteria (NTM). METHODS: In vitro antibiotic activities, i.e. MIC, MBC, rate of killing and synergistic interaction with other antibiotics, were evaluated. RESULTS: SQ641 was the most active compound against all the NTM species studied. The MIC of SQ641 was ≤0.06-4 mg/L for Mycobacterium avium complex (MAC; n = 20), 0.125-2 mg/L for M. avium paratuberculosis (MAP; n = 9), 0.125-2 mg/L for Mycobacterium kansasii (MKN;n = 2), 0.25-1 mg/L for Mycobacterium abscessus (MAB; n = 11), 4 mg/L for Mycobacterium smegmatis (MSMG; n = 1), and 1 and 8 mg/L for Mycobacterium ulcerans (MUL; n = 1), by microdilution and agar dilution methods, respectively. SQ641 was bactericidal against NTM, with an MBC/MIC ratio of 1 to 32, and killed all mycobacteria faster than positive control drugs for each strain. In chequerboard titrations, SQ641 was synergistic with ethambutol against both MAC and MSMG, and was synergistic with streptomycin and rifabutin against MAB. CONCLUSIONS: In vitro, SQ641 was the most potent of the capuramycin analogues against all NTM tested, both laboratory and clinical strains.

Growth rate retardation and inhibitory effect of para-JEM(R) BLUE on Mycobacterium avium subspecies paratuberculosis.

The effect of para-JEM(R) BLUE on Mycobacterium avium subspecies paratuberculosis (MAP) inoculated into broth-based culture media was evaluated by using 84 fecal samples with known MAP status. Results showed that growth of the organism in samples inoculated into the broth without the para-JEM BLUE was detectable 1-35 days (average of 6 days) earlier in 35 of the samples (42%) compared with the same samples inoculated in broth with para-JEM BLUE. Four additional samples (5%) that were MAP positive in the culture broth that lacked the para-JEM BLUE gave negative results when the reagent was included. Of the remaining 45 samples, growth of MAP was detected 1-4 days (average of 3 days) earlier in 4 of the samples (5%) inoculated in the broth with para-JEM BLUE compared with the same samples inoculated in the broth without the para-JEM BLUE, whereas 41 samples (49%) yielded equivalent results with respect to time-to-growth detection and negative growth, regardless of whether para-JEM BLUE was present in the culture broth. However, exclusion of para-JEM BLUE from the broth increased the number of samples that produced false-positive instrument signals compared with the number that produced false-positive signals when the reagent was added. Modification of the sample processing step had no measurable effect. Observations indicated that, although elimination of para-JEM BLUE from the broth increased false-positive instrument signals, its inclusion has an adverse effect on the growth of certain MAP, which suggests that its elimination from broth cultures may increase sensitivity.

An eco-friendly decontaminant to kill Mycobacterium avium subsp. paratuberculosis.

Mycobacteria are difficult to kill due to the complexity of their cell wall. Further, Mycobacterium avium subsp. paratuberculosis (MAP) has one of the more elaborate cell wall compositions of all the mycobacteria. As a working pathogen within a research laboratory setting or as an environmental contaminant shed in the manure from infected animals, MAP is highly resistant to typical disinfectants. In the past, the most successful disinfectants to kill mycobacteria were based upon phenolics, harsh compounds that can break down the lipids within the cell wall. New disinfectants have been developed that are less toxic to the environment, however, it is unknown how well they perform compared to more traditional disinfectants. In the present study, we present comparative data on the utility of a commercial eco-friendly disinfectant, Benefect®, compared to Amphyl®, a phenolic-based disinfectant, and Lysol®, a quaternary ammonium-based disinfectant, to kill MAP in pure culture, tissues, and manure. Results demonstrated that Benefect was highly effective with up to 100% kill of MAP within 30 min in all experiments, paralleling results obtained with Amphyl. Lysol performed the most poorly, requiring longer contact times to kill MAP. These results suggest that natural, nontoxic ingredients can be used to disinfect even hearty pathogens such as MAP effectively, both within the laboratory and on-farm.

Comparison of three methods for susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11 antimicrobial drugs.

OBJECTIVES: To evaluate the BACTEC(TM) MGIT(TM) 960/MGIT Para TB (MGIT) system for drug susceptibility testing of Mycobacterium avium subsp. paratuberculosis (MAP), a pathogen implicated in some forms of Crohn's disease. METHODS: MICs of 11 drugs for 10 MAP strains were determined using the MGIT system, the BACTEC(TM)460TB system (BACTEC) and conventional agar dilution methods. RESULTS: MICs determined by MGIT methods showed 80%-100% agreement (+/-1 log(2) dilution) with those determined by the BACTEC and agar dilution methods for ciprofloxacin, levofloxacin, azithromycin and clofazimine. The MGIT and BACTEC methods showed 70%, 80% and 90% agreement (+/-1 log(2) dilution) for MICs of ethambutol, rifabutin and rifampicin; agreement for all drugs increased to 100% at 2 log(2) dilution differences. For clarithromycin, the MGIT method had greater agreement with the agar dilution method (70% at the same dilution) than the BACTEC method (60% at +/-1 log(2) dilution); agreement increased to 100% at +/-2 log(2) dilutions in both cases. The MGIT and agar dilution methods agreed 60% and 100% for amikacin MICs at +/-1 log(2) dilution and +/-2 log(2) dilutions, respectively. By all methods MICs were higher than achievable serum concentrations for isoniazid and dapsone. There was 100% agreement between all three methods for azithromycin, clarithromycin and ciprofloxacin, and 80% agreement for rifampicin using published MIC thresholds available for M. avium complex strains. CONCLUSIONS: This study shows that the MGIT system can be used for rapid and reliable drug susceptibility testing of MAP.