Phytotherapeutic potential against MRSA: mechanisms, synergy, and therapeutic prospects.

BACKGROUND: Rising resistance to antimicrobials, particularly in the case of methicillin-resistant Staphylococcus aureus (MRSA), represents a formidable global health challenge. Consequently, it is imperative to develop new antimicrobial solutions. This study evaluated 68 Chinese medicinal plants renowned for their historical applications in treating infectious diseases. METHODS: The antimicrobial efficacy of medicinal plants were evaluated by determining their minimum inhibitory concentration (MIC) against MRSA. Safety profiles were assessed on human colorectal adenocarcinoma (Caco-2) and hepatocellular carcinoma (HepG2) cells. Mechanistic insights were obtained through fluorescence and transmission electron microscopy (FM and TEM). Synergistic effects with vancomycin were investigated using the Fractional Inhibitory Concentration Index (FICI). RESULTS: Rheum palmatum L., Arctium lappa L. and Paeonia suffructicosaas Andr. have emerged as potential candidates with potent anti-MRSA properties, with an impressive low MIC of 7.8 µg/mL, comparable to the 2 µg/mL MIC of vancomycin served as the antibiotic control. Crucially, these candidates demonstrated significant safety profiles when evaluated on Caco-2 and HepG2 cells. Even at 16 times the MIC, the cell viability ranged from 83.3% to 95.7%, highlighting their potential safety. FM and TEM revealed a diverse array of actions against MRSA, such as disrupting the cell wall and membrane, interference with nucleoids, and inducing morphological alterations resembling pseudo-multicellular structures in MRSA. Additionally, the synergy between vancomycin and these three plant extracts was evident against MRSA (FICI < 0.5). Notably, aqueous extract of R. palmatum at 1/4 MIC significantly reduced the vancomycin MIC from 2 µg/mL to 0.03 µg/mL, making a remarkable 67-fold decrease. CONCLUSIONS: This study unveil new insights into the mechanistic actions and pleiotropic antibacterial effectiveness of these medicinal plants against resistant bacteria, providing robust evidence for their potential use as standalone or in conjunction with antibiotics, to effectively combat antimicrobial resistance, particularly against MRSA.

Cytokine expression in subjects with Mycobacterium avium ssp. paratuberculosis positive blood cultures and a meta-analysis of cytokine expression in Crohn's disease.

Objectives: 1) Culture Mycobacterium avium ssp. paratuberculosis (MAP)from blood, 2) assess infection persistence, 3) determine Crohn's disease (CD) cytokine expression, 4) compare CD cytokine expression to tuberculosis, and 5) perform a meta-analysis of cytokine expression in CD. Methods: The Temple University/Abilene Christian University (TU/ACU) study had a prospective case control design with 201 subjects including 61 CD patients and 140 non-CD controls. The culture methods included MGIT, TiKa and Pozzato broths, and were deemed MAP positive, if IS900 PCR positive. A phage amplification assay was also performed to detect MAP. Cytokine analysis of the TU/ACU samples was performed using Simple Plex cytokine reagents on the Ella ELISA system. Statistical analyses were done after log transformation using the R software package. The meta-analysis combined three studies. Results: Most subjects had MAP positive blood cultures by one or more methods in 3 laboratories. In our cytokine study comparing CD to non-CD controls, IL-17, IFNγ and TNFα were significantly increased in CD, but IL-2, IL-5, IL-10 and GM-CSF were not increased. In the meta-analysis, IL-6, IL-8 and IL-12 were significantly increased in the CD patients. Conclusion: Most subjects in our sample had MAP infection and 8 of 9 subjects remained MAP positive one year later indicating persistent infection. While not identical, cytokine expression patterns in MAP culture positive CD patients in the TU/ACU study showed similarities (increased IL-17, IFNγ and TNFα) to patterns of patients with Tuberculosis in other studies, indicating the possibilities of similar mechanisms of pathogen infection and potential strategies for treatment.

Culture of Mycobacterium avium subsp. paratuberculosis: challenges, limitations and future prospects.

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants and is suspected to be involved in the development of Crohn's disease and several autoimmune disorders. As such, sensitive and specific MAP detection methods are required to confirm infection in animals and identify potential sources of animal and human exposure. Despite recent developments in immunological and nucleic acid-based detection methods, culture-based detection of MAP remains the 'gold standard' against which the sensitivity and specificity of other detection methods are measured. However, not all culture-based approaches are equivalent in terms of detection capability, which can lead to errors in the evaluation of other detection methods. This review will provide an overview of the chronological development of culture methods for MAP, and will consider the unique growth requirements of MAP, the merits of solid versus liquid culture media, the relative performance of the commonly used MAP culture media, and sample preparation/decontamination protocols for different sample types. The limitations of current MAP culture methods and prospects for improvements are discussed.

A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows' milk.

Bacteriophage-based methods for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) in veterinary specimens are a recent addition to the Johne's disease diagnostic toolbox. Here, we report the use of D29 mycobacteriophage-coated tosylactivated paramagnetic beads to capture and concentrate MAP cells from samples (termed phagomagnetic separation, PhMS) and then naturally lyse viable MAP cells (from the inside out) to provide DNA for IS900 qPCR purposes. Transmission electron microscopy confirmed that D29 phages had bound to beads in the correct orientation and that the phage-coated beads captured MAP cells from a suspension. During test optimization, conventional IS900 PCR results were used to subjectively assess the effect of different phage:bead coating ratios, differing amounts of coated beads during PhMS, optimal incubation time post-PhMS to obtain maximal MAP DNA, and the potential benefit of a brief heat shock (55 °C/1 min) prior to IS900 TaqMan qPCR. The limit of detection 50% (LOD50%) of the optimised PhMS-qPCR assay was 10.00 MAP cells/50 ml milk (95% CI 1.20-82.83). Finally, in order to demonstrate the new assay's ability to detect viable MAP in naturally contaminated milk, bulk tank milk samples from 100 dairy farms were tested. Forty-nine (49%) of these tested PhMS-qPCR-positive, with viable MAP numbers detected ranging from 3-126 MAP/50 ml. The novel PhMS-qPCR assay is a sensitive, specific and easy-to-apply phage-based assay for viable MAP, with potential application for milk surveillance or diagnosis of Johne's disease. KEY POINTS: • Phage-coated magnetic beads could capture, concentrate and lyse MAP cells from milk. • PhMS-qPCR assay proved to be a rapid, sensitive and specific test for viable MAP. • A potential application of PhMS-qPCR assay for milk surveillance was demonstrated.

Mechanisms of Antimicrobial Action of Cinnamon and Oregano Oils, Cinnamaldehyde, Carvacrol, 2,5-Dihydroxybenzaldehyde, and 2-Hydroxy-5-Methoxybenzaldehyde against Mycobacterium avium subsp. paratuberculosis (Map).

The antimicrobial modes of action of six naturally occurring compounds, cinnamon oil, cinnamaldehyde, oregano oil, carvacrol, 2,5-dihydroxybenzaldehyde, and 2-hydroxy-5-methoxybenzaldehyde, previously found to inhibit the growth of Mycobacterium avium subsp. paratuberculosis (Map) reported to infect food animals and humans and to be present in milk, cheese, and meat, were investigated. The incubation of Map cultures in the presence of all six compounds caused phosphate ions to leak into the extracellular environment in a time- and concentration-dependent manner. Cinnamon oil and cinnamaldehyde decreased the intracellular adenosine triphosphate (ATP) concentration of Map cells, whereas oregano oil and carvacrol caused an initial decrease of intracellular ATP concentration that was restored gradually after incubation at 37 °C for 2 h. Neither 2,5-dihydroxybenzaldehyde nor 2-hydroxy-5-methoxybenzaldehyde had a significant effect on intracellular ATP concentration. None of the compounds tested were found to cause leakage of ATP to the extracellular environment. Monolayer studies involving a Langmuir trough apparatus revealed that all anti-Map compounds, especially the essential oil compounds, altered the molecular packing characteristics of phospholipid molecules of model membranes, causing fluidization. The results of the physicochemical model microbial membrane studies suggest that the destruction of the pathogenic bacteria might be associated with the disruption of the bacterial cell membrane.

Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation.

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.

Production and evaluation of antibodies and phage display-derived peptide ligands for immunomagnetic separation of Mycobacterium bovis.

This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 10(5) CFU/ml) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis.

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.

Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa.

BACKGROUND: Interactions between Mycobacterium avium subsp. paratuberculosis (Map) and free-living protozoa in water are likely to occur in nature. The potential impact of ingestion of Map by two naturally occurring Acanthamoeba spp. on this pathogen's survival and chlorine resistance was investigated. RESULTS: Between 4.6 and 9.1% of spiked populations of three Map strains (NCTC 8578, B2 and ATCC 19698), which had been added at a multiplicity of infection of 10:1, were ingested by Acanthamoeba castellanii CCAP 1501/1B and A. polyphaga CCAP 1501/3B during co-culture for 3 h at 25 degrees C. Map cells were observed to be present within the vacuoles of the amoebae by acid-fast staining. During extended co-culture of Map NCTC 8578 at 25 degrees C for 24 d with both A. castellanii and A. polyphaga Map numbers did not change significantly during the first 7 days of incubation, however a 1-1.5 log10 increase in Map numbers was observed between days 7 and 24 within both Acanthamoeba spp. Ingested Map cells were shown to be more resistant to chlorine inactivation than free Map. Exposure to 2 mug/ml chlorine for 30 min resulted in a log10 reduction of 0.94 in ingested Map but a log10 reduction of 1.73 in free Map (p < 0.001). CONCLUSION: This study demonstrated that ingestion of Map by and survival and multiplication of Map within Acanthamoeba spp. is possible, and that Map cells ingested by amoebae are more resistant to inactivation by chlorine than free Map cells. These findings have implications with respect to the efficacy of chlorination applied to Map infected surface waters.