Evaluation of culture media for the recovery of Mycobacterium avium subsp. paratuberculosis from Cheddar cheese

Abstract

Aims:The study evaluated the efficacy of four Mycobacterium avium subsp. paratuberculosis (MAP) culture media in suppressing commonly used starter cultures and typical nonstarter microflora present during the manufacture and ripening of Cheddar cheese, with a view to identify a suitable medium for the enumeration of MAP during laboratory-scale Cheddar production. Methods and Results: Four Cheddar starter cultures and Cheddar cheese manufactured with these starters were inoculated onto Herrold's egg yolk medium (HEYM); HEYM supplemented with vancomycin, amphotericin B and nalidixic acid (HEYM/VAN); Middlebrook 7H10 agar containing polymyxin, amphotericin B, nalidixic acid, trimethoprim and azlocillin (PANTA) antibiotic supplement; and BACTEC 12B radiometric medium with and without a preliminary decontamination step (0AE75% w/v hexadecylpyridinium chloride (HPC), 5 h). The inclusion of a decontamination step inhibited all Cheddar cheese starter and nonstarter micro-organisms. The medium 7H10/PANTA and to a lesser extent HEYM/VAN were effective inhibitors of cheese microflora when no decontamination step was employed. Conclusions: Middlebrook 7H10 medium, supplemented with PANTA antibiotics, suppressed all microorganisms associated with ripening Cheddar cheese manufactured with pasteurized milk. Significance and Impact of the Study: A MAP culture medium has been identified, which may be used to enumerate this bacterium during the laboratory manufacture and ripening of Cheddar cheese and hence facilitate further research into the persistence of this pathogen in the product.

Comparison of Culture Methods for Isolation of Nontuberculous Mycobacteria from Surface Waters

“…(39). Reports have indicated that the sensitivity of Middlebrook 7H11 medium is improved by addition of mycobactine J (13,40), and the specificity can be improved by addition of the PANTA antibiotic cocktail. The PANTA additive was initially used for detection of mycobacteria in clinical samples using BACTEC (Becton Dickinson) liquid medium detection systems (36).…”

Decline in Mycobacterium bovis and Brucella abortus populations during the maturation of experimentally contaminated parmesan-type cheese

“…Note that the processing of milk into cheese reduced the population of the inoculated bacteria, which is probably associated with the loss of microorganisms in the serum fraction, in addition to a relative increase in remnant population, due to the concentration of solids. This phenomenon was also observed in the experimental production of cheddar and Caerphilly cheese inoculated with M. bovis (FORGRAVE et al, 2014), as well as in cheddar cheese inoculated with M. paratuberculosis (DONAGHY et al, 2004). The affinity of mycobacteria for fat (MCFADDEN et al, 1992) might result in a smaller reduction in M. bovis numbers during the processing of milk to cheese compared to B. abortus, but the difference between these bacteria was minor.…”

“…The variability in the survival of the pathogens during the maturation of various cheeses found by different studies can be explained by differences in the manufacturing technology, maturation temperature, physicochemical characteristics of the milk matrix, companion microbiota, and other aspects related to the methodology used in the study. milk, reinforcing the theory that the strains cultivated in the laboratory for long periods are less resistant (DONAGHY et al, 2004). Obs.…”

“…In another study, viable M. bovis was detected after 3-4 months of maturation of artificially contaminated blue cheeses (4 log CFU mL -1 ), despite a rapid reduction in the population in the first 14 days after the manufacture (LAFONT; LAFONT, 1981 (SUNG;COLLINS, 2000). In cheddar-type cheese, the D 10°C values were 90, 96, and 107 days, respectively, for a reference strain (NCTC) and two isolates from milk, reinforcing the theory that the strains cultivated in the laboratory for long periods are less resistant (DONAGHY et al, 2004).…”

“…For counting M. bovis colonies, serial tenfold dilutions were made in 0.1% peptone water, and 100 µL of each dilution in duplicate was seeded in Stonebrink-Leslie medium (CENTRO PANAMERICANO DE ZOONOSIS, 1985) containing antibiotics and incubated at 36°C for 45 days. To each 120 mL of Stonebrink-Leslie medium, 3.865 mg of 6,000 IU polymyxin B, 0.6 mg of amphotericin B, 2.4 mg of nalidixic acid, and 0.6 mg of trimethoprim were added, as adapted from Donaghy et al (2003).…”