Development and Validation of a Liquid Medium (M7H9C) for Routine Culture of Mycobacterium avium subsp. paratuberculosis To Replace Modified Bactec 12B Medium

Whittington, Richard J.; Whittington, Ann-Michele; Waldron, Anna M.; Begg, Douglas J.; Silva, Kumi de; Purdie, Auriol C.; Plain, Karren M.

doi:10.1128/jcm.01373-13

Cited by 56 publications

(52 citation statements)

References 42 publications

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“…A notable validation of NlpC/P60 proteins as antibiotic targets has been done for M. marinum IipA (homolog of MAP_1203), where the gene knockout was constructed and complemented to show a lack of growth and infectivity in macrophages. 13 Since MAP is resistant to many antibiotics, 28,29 novel approaches must be explored in treating infections by this organism. Furthermore, since NlpC/P60 proteins are found in all Eubacteria, any compounds that are capable of inhibiting these enzymes have the potential to be developed into Figure 5.…”

Section: Discussionmentioning

confidence: 99%

NlpC/P60 domain‐containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

et al. 2016

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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.

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Section: Discussionmentioning

confidence: 99%

NlpC/P60 domain‐containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

et al. 2016

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“…M7H9C liquid culture medium for MAP containing Middlebrook 7H9 broth base (Becton Dickinson [BD]), PANTA antibiotic preparation (BD), mycobactin J (Allied Monitor), Middlebrook ADC enrichment (containing bovine albumin fraction V, dextrose, and catalase; BD), and egg yolk was prepared and cultures were performed as previously described (4). Modified Bactec 12B medium (Becton Dickinson) containing egg yolk, PANTA Plus, and mycobactin J was prepared as described previously (7).…”

Section: Methodsmentioning

confidence: 99%

“…For MAP, liquid culture media, such as Bactec 12B medium, have been found to be more sensitive than solid culture media, such as Herrold's egg yolk medium (HEYM) supplemented with mycobactin J (4)(5)(6). MAP in cultures with positive growth is confirmed by the demonstration of mycobactin dependency or directly by molecular techniques (7).…”

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Efficient, Validated Method for Detection of Mycobacterial Growth in Liquid Culture Media by Use of Bead Beating, Magnetic-Particle-Based Nucleic Acid Isolation, and Quantitative PCR

et al. 2015

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Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 10 4 -fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n ‫؍‬ 54) and sheep fecal and tissue (n ‫؍‬ 90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis. P aratuberculosis, or Johne's disease, affects many economically important livestock species, including cattle, sheep, goats, and deer (1). It is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is characterized by chronic enteritis, wasting, and death. Control programs attempting to limit the impact and spread of the disease in many countries are based on the detection of infected animals by using fecal culture, which is one of the most specific antemortem tests for the diagnosis of paratuberculosis infection (2). The gold standard indications for disease confirmation are a pathological lesion consistent with paratuberculosis and a positive culture for MAP from the affected tissues (3).For many mycobacterial species, culture requires long incubation times, from weeks to months, and highly specialized media. For MAP, liquid culture media, such as Bactec 12B medium, have been found to be more sensitive than solid culture media, such as Herrold's egg yolk medium (HEYM) supplemented with mycobactin J (4-6). MAP in cultures with positive growth is confirmed by the demonstration of mycobactin dependency or directly by molecular techniques (7). The advantages of liquid culture media systems include shorter culture incubation times (5, 7), and, importantly, some liquid culture media are capable of supporting the growth of all strains of MAP, including the fastidious sheep strain (8). Monitoring of bacterial growth in liquid cul...

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“…The purpose of adding egg yolk to culture media used for MAP is still unclear, but neutralization of the chemical decontaminant HPC seems to be a common reason (Whittington 2010). Whittington et al (2013) subsequently suggested that egg yolk provides major carbon and energy sources as well as the surfactant lecithin. The question arises, are sub-lethally injured MAP cells likely to benefit from, or be adversely affected by, such a rich source of nutrients when trying to repair heat or dessication damage?…”

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confidence: 99%

Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer

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Tarrant³

et al. 2017

Journal of Dairy Science

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Development and Validation of a Liquid Medium (M7H9C) for Routine Culture of Mycobacterium avium subsp. paratuberculosis To Replace Modified Bactec 12B Medium

Cited by 56 publications

References 42 publications

NlpC/P60 domain‐containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

NlpC/P60 domain‐containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

Efficient, Validated Method for Detection of Mycobacterial Growth in Liquid Culture Media by Use of Bead Beating, Magnetic-Particle-Based Nucleic Acid Isolation, and Quantitative PCR

Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer

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