Comparing minimum inhibitory concentrations of amikacin for pulmonary Mycobacterium avium complex disease: An analysis of culture media differences.

Mycobacterium avium complex (MAC) is considered a paramount microbe, especially in East Asia, including Japan. The commonly used commercial Minimum Inhibitory Concentrations (MIC) assay using Middlebrook 7H9 (7H9) medium deviates from the latest Clinical and Laboratory Standards Institute (CLSI) guidelines. Alternatively, measurement with cation-adjusted Mueller-Hinton broth (CAMHB) that conforms to CLSI standards is not yet widely available. Following the approval and commercialization of amikacin liposome inhalation suspension (ALIS) in 2021, a more precise evaluation of amikacin (AMK) susceptibility in MAC is necessary for treatment decisions. In the present study, 33 sputum samples were extracted from 27 patients, and MICs of AMK were compared between the frequently used 7H9 and the recommended CAMHB of the isolated MAC strains. The history of exposure to aminoglycosides for each sample was also added as clinical information. The findings indicated that there was only an 18% concordance rate in MIC between the two media, with 19 samples (58%) indicating lower MICs in 7H9 relative to CAMHB. The 17 samples had a history of exposure to aminoglycosides for periods ranging from 1.5 to 28 months. Specifically, 10 samples were exposed to amikacin by inhalation and intravenous injection, and the remaining seven samples had a history of ALIS inhalation. Samples with a prior utilization of aminoglycosides were significantly predisposed to developing resistance to ALIS compared to those without such a history (P = 0.046). Physicians are encouraged to scrutinize the findings of susceptibility testing utilizing CLSI-endorsed MIC assay using CAMHB medium to ascertain the optimal therapeutic approach.

Manual liquid culture on simple Middlebrook 7H9 or MGIT for the diagnosis of smear-negative pulmonary tuberculosis.

OBJECTIVES: To compare the performance of liquid culture on simple Middlebrook 7H9 to the one of manual mycobacterial growth indicator tube (MGIT) and solid culture on Ogawa for the diagnosis of smear-negative tuberculosis (SN-TB) in a high-burden, resource-constrained setting. METHODS: Sputum samples from patients with clinical suspicion of SN-PTB admitted to two-third-level hospitals in Lima between September 2005 and May 2008 were cultured in parallel on simple Middlebrook 7H9, manual MGIT and Ogawa. A case of SN-TB was defined as one with a positive culture in any medium. RESULTS: Among samples from 542 patients, 151 (28%) cases of SN-TB were identified. The sensitivity of Middlebrook 7H9 (0.76, 95% CI 0.69-0.83) was not substantially different from that of MGIT (0.85, 95% CI 0.79-0.91). Ogawa had the lowest sensitivity (0.63, 95% CI 0.55-0.71). The median turnaround time was similar for both liquid media (18 days), and it was shorter than that of Ogawa (30 days). CONCLUSIONS: Culture on simple Middlebrook 7H9 performs almost as well as MGIT, at a probably more affordable cost. Further studies on the cost-effectiveness of this overlooked technique should be performed.

A novel biphasic approach to reactivation of dormant Mycobacterium avium subspecies paratuberculosis.

A new culture technique that involves a potato slice and enriched Middlebrook 7H9 in a two-part glass tube has been developed to revive dormant or persistent Mycobacterium avium subspecies paratuberculosis and acclimate it to Middlebrook 7H9 liquid media. This method is more efficient than directly introducing the bacteria into the liquid medium.

Proteome and phosphoproteome analysis of the serine/threonine protein kinase E mutant of Mycobacterium tuberculosis.

AIMS: Serine/threonine protein kinases (STPKs) have prominent roles in the survival mechanisms of Mycobacterium tuberculosis (M. tuberculosis). Previous studies from our laboratory underscored the role of PknE, an STPK in virulence, adaptation and the suppression of host cell apoptosis. In this study, two-dimensional gel electrophoresis was used to study the proteome and phosphoproteome profiles of wild type M. tuberculosis and its isogenic pknE deletion mutant (ΔpknE) during growth in Middlebrook 7H9 and nitric oxide stress. MAIN METHODS: Wild-type M. tuberculosis and its isogenic pknE deletion mutant strain were grown in Middlebrook 7H9 as well as subjected to nitric oxide stress using sodium nitroprusside. Whole cell lysates were prepared and analyzed by 2D-gel electrophoresis. Phosphoproteomes were analyzed using phospho serine and phospho threonine antibodies after subjecting the 2D-gels to western blotting. Proteins of interest were identified using mass spectrometry. KEY FINDINGS: Our analysis provides insights into the targets that impose pro-apoptotic as well as altered cellular phenotypes on ΔpknE, revealing novel substrates and functions for PknE. SIGNIFICANCE: For the first time, our proteome and phosphoproteome data decipher the function of PknE in cell division, virulence, dormancy, suppression of sigma factor B and its regulated genes, suppression of two-component systems and in the metabolic activity of M. tuberculosis.

Identification and characterization of starvation induced msdgc-1 promoter involved in the c-di-GMP turnover.

C-di-GMP [Bis-(3'-5')-cyclic-dimeric-guanosine monophosphate], a second messenger is involved in intracellular communication in the bacterial species. As a result several multi-cellular behaviors in both Gram-positive and Gram-negative bacteria are directly linked to the intracellular level of c-di-GMP. The cellular concentration of c-di-GMP is maintained by two opposing activities, diguanylate cyclase (DGC) and phosphodiesterase (PDE-A). In Mycobacterium smegmatis, a single bifunctional protein MSDGC-1 is responsible for the cellular concentration of c-di-GMP. A better understanding of the regulation of c-di-GMP at the genetic level is necessary to control the function of above two activities. In this work, we have characterized the promoter element present in msdgc-1 along with the +1 transcription start site and identified the sigma factors that regulate the transcription of msdgc-1. Interestingly, msdgc-1 utilizes SigA during the initial phase of growth, whereas near the stationary phase SigB containing RNA polymerase takes over the expression of msdgc-1. We report here that the promoter activity of msdgc-1 increases during starvation or depletion of carbon source like glucose or glycerol. When msdgc-1 is deleted, the numbers of viable cells are ~10 times higher in the stationary phase in comparison to that of the wild type. We propose here that msdgc-1 is involved in the regulation of cell population density.