The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis.
A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases. Keywords: Mycobacterium; Johne's disease; crystal structure; peptidoglycan; proteins; antigens; paratuberculosis Additional Supporting Information may be found in the online version of this article.Importance: Johne's disease in ruminant livestock is caused by the bacterium Mycobacterium avium subspecies paratuberculosis. This research describes the functional aspects of two proteins that show promise in a subunit vaccine for Johne's disease. Through crystal structure determination and amino acid modification, we demonstrate that although both proteins have a similar structure, one of them lacked hydrolytic activity on peptidoglycan. We show that a specific amino acid is likely responsible for this lack of hydrolytic activity.
Background The 3M™ Petrifilm™ Rapid E. coli/Coliform Count Plate is a selective and differential sample-ready-culture medium designed for the rapid enumeration of Escherichia coli (E. coli) and coliforms in the food and beverage industries. Objective The 3M Petrifilm Rapid E. coli/Coliform Count Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) Chapter 4 Enumeration of Escherichia coli and the Coliform Bacteria, the International Organization of Standards (ISO) 4832:2006 Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of coliforms—Colony-count technique, and ISO 16649-2:2017 Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli—Part 2 Colony-count technique at 44 degrees C using bromo-4-chloro-3- indolyl beta-D-glucuronide methods for the enumeration of E. coli and coliforms in dry dog kibble. Method The candidate method was evaluated using two diluents, Butterfield's phosphate buffered diluent and peptone salt solution, in a paired study design with each reference method in a multi-laboratory collaborative study following the current AOAC Validation Guidelines. Three target contamination levels and an uninoculated control level were evaluated. Results The candidate and reference methods were not statistically different at each contamination level. Reproducibility values obtained during the collaborative study were similar between the candidate and reference methods. Conclusion These results demonstrate that the candidate method is equivalent to the reference methods. Highlight 3M Petrifilm Rapid E. coli/Coliform Count Plate was recommended for Official First Action status for enumeration of E. coli and coliforms in a broad range of foods and environmental surfaces.
Rapid ATP testing and microbiological enumeration are two common methods to monitor the effectiveness of cleaning and sanitation in the food industry. In this study, ATP testing and microbiological enumeration were implemented at a tofu production facility with the goal of improving cleaning practices and overall plant hygiene. Results from ATP monitoring were used to target areas of the production environment needing additional cleaning; ATP results were verified by microbiological enumeration of aerobic microorganisms, lactic acid bacteria, and yeasts and molds. Products from the production line were enumerated for the same microorganisms to determine if there was an impact on product quality. After the implementation of ATP monitoring and targeted cleaning, there was a statistically lower proportion of swabs that failed to meet established sanitary requirements for ATP, aerobic microorganisms, and lactic acid bacteria (p < 0.05), but not for yeasts and molds. ATP swabs and microbiological enumeration agreed on site hygiene 75.1% (72.3-77.7%, 95% CI) of the time. Product data indicated that unpasteurized finished products contained a statistically lower microbial load of the three groups of organisms following implementation of the practices (p < 0.05). Importance Cleaning and sanitation are critical to maintaining safe and high-quality food production. Monitoring these activities is important to ensure proper execution of procedure and to assure compliance with regulatory guidelines. The results from monitoring activities can direct targeted cleaning of areas with higher risk of contamination from foodstuffs and microorganisms. The results of this study show that ATP monitoring and microbiological enumeration are useful tools to verify and improve the efficacy of cleaning and sanitation practices, which can have a positive impact on both plant hygiene and product quality. However, testing regimes and critical parameters will vary based on the product and facility.
Background The 3M™ Petrifilm™ Rapid Yeast and Mold (RYM) Count Plate is a sample-ready culture medium system which contains nutrients supplemented with antibiotics, a cold-water-soluble gelling agent, and an indicator system that facilitates yeast and mold enumeration. Objective The 3M Petrifilm RYM Plate was validated for the enumeration of yeast and mold in dried cannabis flower through the AOAC Emergency Response Validation process. Methods The performance of the 3M Petrifilm RYM Plate was compared to Dichloran Rose Bengal Chloramphenicol (DRBC) agar. Matrix data were normalized by log10 transformation and performance indicators included repeatability, difference of means, and inclusivity/exclusivity. Results These studies demonstrated the 3M Petrifilm RYM Plate method detects and enumerates yeasts and molds from dried cannabis flower at low, medium, and high contamination levels. The average log counts at 25 or 28 °C for 60 to 72 h were equivalent to the average log counts of the DRBC reference method at low, medium and high levels. In strain studies, all 71 yeasts and molds tested, produced typical colony morphology on 3M Petrifilm RYM Plates. Of the 32 non-target bacterial strains tested, none were detected on 3M Petrifilm RYM Plates. Conclusions The 3M Petrifilm RYM Plate is a reliable method for the enumeration of live yeast and mold in dried cannabis flower. Highlights The 3M Petrifilm RYM Plate allows for rapid detection of yeast and mold within 60 to 72 h of incubation. Up to 40 sample-ready plates can be stacked during incubation to save space.
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