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.

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.

Nilotinib: A Tyrosine Kinase Inhibitor Mediates Resistance to Intracellular Mycobacterium Via Regulating Autophagy.

Nilotinib, a tyrosine kinase inhibitor, has been studied extensively in various tumor models; however, no information exists about the pharmacological action of nilotinib in bacterial infections. Mycobacterium bovis (M. bovis) and Mycobacterium avium subspecies paratuberculosis (MAP) are the etiological agents of bovine tuberculosis and Johne's disease, respectively. Although M. bovis and MAP cause distinct tissue tropism, both of them infect, reside, and replicate in mononuclear phagocytic cells of the infected host. Autophagy is an innate immune defense mechanism for the control of intracellular bacteria, regulated by diverse signaling pathways. Here we demonstrated that nilotinib significantly inhibited the intracellular survival and growth of M. bovis and MAP in macrophages by modulating host immune responses. We showed that nilotinib induced autophagic degradation of intracellular mycobacterium occurred via the inhibition of PI3k/Akt/mTOR axis mediated by abelson (c-ABL) tyrosine kinase. In addition, we observed that nilotinib promoted ubiquitin accumulation around M. bovis through activation of E3 ubiquitin ligase parkin. From in-vivo experiments, we found that nilotinib effectively controlled M. bovis growth and survival through enhanced parkin activity in infected mice. Altogether, our data showed that nilotinib regulates protective innate immune responses against intracellular mycobacterium, both in-vitro and in-vivo, and can be exploited as a novel therapeutic remedy for the control of M. bovis and MAP infections.

Antimicrobial treatment with the fixed-dose antibiotic combination RHB-104 for Mycobacterium avium subspecies paratuberculosis in Crohn's disease: pharmacological and clinical implications.

INTRODUCTION: Crohn's disease (CD) is an inflammatory bowel disease of unknown etiology. However, increasing evidence suggests Mycobacterium avium subspecies paratuberculosis (MAP) as a putative causative agent: 1) MAP is the etiological agent of Johne's disease, a granulomatous enteritis affecting ruminants, which shares clinical and pathological features with CD; 2) MAP has been detected in tissues and blood samples from CD patients; 3) case reports have documented a favorable therapeutic response to anti-MAP antibiotics. Area covered: This review provides an appraisal of current information on MAP characteristics, diagnostic methodologies and emerging drug treatments. The authors focus on RHB-104, a novel oral formulation containing a fixed-dose combination of clarithromycin, clofazimine and rifabutin, endowed with synergistic inhibitory activity on MAP strains isolated from CD patients. Expert opinion: Based on encouraging in vitro data, RHB-104 has entered recently the clinical phase of its development, and is being investigated in a randomized, placebo-controlled phase III trial aimed at evaluating its efficacy and safety in CD. Provided that the overall clinical development will support the suitability of RHB-104 for inducing disease remission in CD patients with documented MAP infection, this novel antibiotic combination will likely take a relevant position in the therapeutic armamentarium for CD management.

A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival.

The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc(2) 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.

Serum BAFF levels, Methypredsinolone therapy, Epstein-Barr Virus and Mycobacterium avium subsp. paratuberculosis infection in Multiple Sclerosis patients.

Elevated B lymphocyte activating factor BAFF levels have been reported in multiple sclerosis (MS) patients; moreover, disease-modifying treatments (DMT) have shown to influence blood BAFF levels in MS patients, although the significance of these changes is still controversial. In addition, BAFF levels were reported increased during infectious diseases. In our study, we wanted to investigate on the serum BAFF concentrations correlated to the antibody response against Mycobacterium avium subspecies paratuberculosis (MAP), Epstein-Barr virus (EBV) and their human homologous epitopes in MS and in patients affected with other neurological diseases (OND), divided in Inflammatory Neurological Diseases (IND), Non Inflammatory Neurological Diseases (NIND) and Undetermined Neurological Diseases (UND), in comparison to healthy controls (HCs). Our results confirmed a statistically significant high BAFF levels in MS and IND patients in comparison to HCs but not NIND and UND patients. Interestingly, BAFF levels were inversely proportional to antibodies level against EBV and MAP peptides and the BAFF levels significantly decreased in MS patients after methylprednisolone therapy. These results implicate that lower circulating BAFF concentrations were present in MS patients with humoral response against MAP and EBV. In conclusion MS patients with no IgGs against EBV and MAP may support the hypothesis that elevated blood BAFF levels could be associated with a more stable disease.

The fibroblast growth factor-2 arrests Mycobacterium avium sp. paratuberculosis growth and immunomodulates host response in macrophages.

Mycobacterium tuberculosisis (M. tb) epidemic is one of the most severe health problem worldwide, while mechanisms underlying its pathogenesis and host immune responses remain unclear. Mycobacterium avium (M. avium), a mycobacterial species related to M. tb, shares similarities with M. tb in many ways. In this study, using M. avium infection of macrophages as a model, we systematically studied the effect of fibroblast growth factor-2 (FGF-2) on M. avium infection of macrophages. Our results showed that M. avium infection could increase FGF-2 expression on both mRNA and protein levels. M. avium infection elevated TNF-α and IFN-γ production while the addition of FGF-2 could further increase TNF-α but not IFN-γ level. M. avium infection could increase the expression of oxygen/nitrogen metabolism proteins iNOS and SOD-1, and FGF-2 had additive effect on the expression of these two proteins. M. avium infection had inhibitive effect on actin expression while FGF-2 could partly counteract such inhibition. Moreover, FGF-2 could inhibit M. avium proliferation in macrophages. Our results together indicate that macrophage-secreted FGF-2 upon M. avium infection could suppress M. avium proliferation through various ways including cytokine production, enhancement of phagocytosis as well as oxygen/nitrogen metabolism.

Resolution of Crohn's disease and complex regional pain syndrome following treatment of paratuberculosis.

A cohort of family members with various chronic diseases including Crohn's disease, asthma, complex regional pain syndrome, hypothyroidism, type 1 diabetes mellitus, and lymphangiomatosis and/or evidence of infection by Mycobacterium avium subsp. paratuberculosis (MAP) are described in this series of case reports. MAP was cultured from the blood of three members affected by the first five diseases and there was accompanying elevated anti-MAP IgG in two members. The patient affected by the sixth disease has a markedly elevated anti-MAP titer. The two patients affected by the first four diseases have been treated with a combination of anti-MAP antibiotics and ultraviolet blood irradiation therapy with resolution of the disease symptomatology and inability to culture MAP in post treatment blood samples. These case reports of patients with MAP infections provide supportive evidence of a pathogenic role of MAP in humans.

Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

Mycobacterium avium subspecies paratuberculosis in the etiology of Crohn's disease, cause or epiphenomenon?

The origin of inflammatory bowel disease is unknown. Attempts have been made to isolate a microorganism that could explain the onset of inflammation, but no pathological agent has ever been identified. Johne's disease is a granulomatous chronic enteritis of cattle and sheep caused by Mycobacterium avium subspecies paratuberculosis (MAP) and shows some analogies with Crohn's disease (CD). Several studies have tried to clarify if MAP has a role in the etiology of CD. The present article provides an overview of the evidence in favor and against the "MAP-hypothesis", analyzing the methods commonly adopted to detect MAP and the role of antimycobacterial therapy in patients with inflammatory bowel disease. Studies were identified through the electronic database, MEDLINE, and were selected based on their relevance to the objective of the review. The presence of MAP was investigated using multiple diagnostic methods for MAP detection and in different tissue samples from patients affected by CD or ulcerative colitis and in healthy controls. On the basis of their studies, several authors support a close relationship between MAP and CD. Although increasing evidence of MAP detection in CD patients is unquestionable, a clear etiological link still needs to be proven.

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.

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.

A Mycobacterium avium subsp. paratuberculosis LuxR regulates cell envelope and virulence.

Mycobacterium avium subsp. paratuberculosis adapts to the environment via the regulation of genes affecting its envelope's composition. Bacteria grown in milk (in vivo conditions) presented differences in the cell wall-associated lipids and in the expression of genes involved in lipid metabolism (FadE8, FadE6 and MAP1420) and host cell invasion (MAP1203, LprL). A different lipid profile was also observed in the envelope of intracellular bacteria after 1 h of infection. Intracellular bacteria showed up-regulation of a LuxR regulator which controls the envelope's composition by up-regulation of FadE8, MAP1420, MAP1203 and LprL and by down-regulation of pks12, mmpL2 and MAP2594. A LuxR-overexpressing strain with a lipid-deficient envelope phenotype, infected epithelial cells more efficiently than the wild-type bacteria; however, it was not more resistant than the wild-type strain to the action of bactericidal proteins. Here we show that LuxR regulates virulence determinants and is involved in mycobacteria adaptation to the host.

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.

Antibiotics and probiotics in inflammatory bowel disease: why, when, and how.

PURPOSE OF REVIEW: To summarize recent evidence on the role of intestinal bacteria in inflammatory bowel diseases, and of antibiotics and probiotics in their treatment. The implications connected with the use of antibiotics are also examined. RECENT FINDINGS: The hypothesis that Mycobacterium paratuberculosis could be a causative agent of Crohn's disease has not been confirmed by a large trial on symptomatic patients treated by a combination of antibiotics active against this bacterium. An increased number of adherent-invasive Escherichia coli have been found in the intestinal tissue of patients with Crohn's disease, but their role in the pathogenesis of this condition remains to be defined. The combination of metronidazole and azathioprine, associating the effects of a reduced bacterial load with immunosuppression, appears to be a therapeutic option to decrease the recurrence of postoperative Crohn's disease in high-risk patients. However, concerns are raised by the possibility that antibiotics may induce disease relapse due to Clostridium difficile infection. SUMMARY: Recent literature provides increasing support for the use of antibiotics in Crohn's disease, although the side effects limit their long-term use. The efficacy of antibiotics in ulcerative colitis is not confirmed by the available literature, except in severe colitis. More trials are needed to support the use of probiotics as therapy in inflammatory bowel disease.

On the effect of thalidomide on Mycobacterium avium subspecies paratuberculosis in culture.

BACKGROUND: Without known mechanisms of action, thalidomide is used to treat a variety of non-malignant 'idiopathic' diseases. There is increasing concern that Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. Recently, methotrexate, azathioprine, 6-mercaptopurine (6-MP), 5-aminosalicylic acid (5-ASA), cyclosporine A, rapamycin, and tacrolimus have been shown to inhibit MAP growth in culture, indicating that, unknowingly, MAP infections may have been treated for decades. We herein test the hypothesis that thalidomide may inhibit MAP growth. METHODS: Using the radiometric 14CO2 (Bactec) system we quantified growth kinetics of thalidomide (+/-), (+), and (-) and two components for thalidomide, phthalimide and 1-hydroxypiperidine-2,6-dione (HPD). We studied four MAP strains (three human isolates, 'Ben', 'Dominic', and UCF-4, and a bovine MAP isolate 19698) and three mycobacterial controls (Mycobacterium avium and bacillus Calmette-Guérin (BCG)). Growth was quantified as growth index (GI) and inhibition as percent decrease in cumulative GI (%-DeltacGI). RESULTS: Phthalimide had no dose-dependent inhibition on any strain. Neither thalidomide nor HPD inhibited M. avium or BCG. MAP inhibition varied; at 64 microg/ml, amongst human isolates, Dominic was most susceptible: thalidomide (+)=58%-DeltacGI and HPD=46%-DeltacGI. UCF-4 was next: thalidomide (-)=37%-DeltacGI and HPD=40%-DeltacGI. Ben was least susceptible: HPD=24%-DeltacGI. CONCLUSIONS: We have shown, in culture, the heretofore-undescribed inhibition of MAP growth by thalidomide and its enantiomers. Phthalimide was found to have no anti-MAP activity, whereas HPD was found to inhibit MAP growth. These data are compatible with the hypothesis that thalidomide, like other 'anti-inflammatories' and 'immunomodulators' may act, in part, as an anti-MAP antibiotic.

Antibacterial activities of naturally occurring compounds against Mycobacterium avium subsp. paratuberculosis.

The antibacterial activities of 18 naturally occurring compounds (including essential oils and some of their isolated constituents, apple and green tea polyphenols, and other plant extracts) against three strains of Mycobacterium avium subsp. paratuberculosis (a bovine isolate [NCTC 8578], a raw-milk isolate [806R], and a human isolate [ATCC 43015]) were evaluated using a macrobroth susceptibility testing method. M. avium subsp. paratuberculosis was grown in 4 ml Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase, 0.05% Tween 80 (or 0.2% glycerol), and 2 microg/ml mycobactin J supplemented with five concentrations of each test compound. The changes in the optical densities of the cultures at 600 nm as a measure of CFU were recorded at intervals over an incubation period of 42 days at 37 degrees C. Six of the compounds were found to inhibit the growth of M. avium subsp. paratuberculosis. The most effective compound was trans-cinnamaldehyde, with a MIC of 25.9 microg/ml, followed by cinnamon oil (26.2 microg/ml), oregano oil (68.2 microg/ml), carvacrol (72.2 microg/ml), 2,5-dihydroxybenzaldehyde (74 microg/ml), and 2-hydroxy-5-methoxybenzaldehyde (90.4 microg/ml). With the exception of carvacrol, a phenolic compound, three of the four most active compounds are aldehydes, suggesting that the structure of the phenolic group or the aldehyde group may be important to the antibacterial activity. No difference in compound activity was observed between the three M. avium subsp. paratuberculosis strains studied. Possible mechanisms of the antimicrobial effects are discussed.