Analysis of Ag-DP25/PET plasmonic nano-composites as a visible-light photocatalyst for wastewater treatment: Experimental/theoretical studies, and the DFT-MB degradation mechanism.

The development of polymeric-composites Agx%DP25-PET (x = 0,1,2,3) may significantly boost the potential application of Agx%DP25 (x = 0,1,2,3) photocatalytic powders. Producing large-scale nano-composites with hybrid-surfaces, that are also flexible materials and easy to employ in a variety of environments. A set of photocatalytic nan-composites embedded with the polymeric binder poly (acrylonitrile-co-butadiene)-dicarboxy terminated (C7H9N) were performed and evaluated for wastewater treatment applications. The results reveal that the flexible polymeric composites (Agx%DP25-PET, x = 0,1,2,3) have photocatalytic activity in aqua media to degrade methylene blue (MB) under visible-light. The addition of C7H9N to immobilize photocatalytic powders on the PET surface reduces photo-generated electron-hole recombination. The materials were characterized by HR-TEM, SEM/EDX, XRD, FT-IR, UV-Vis DRS and PL. The Agx%DP25-PET (x = 0,1,2,3) photocatalytic reactions exhibited productive discoloration/degradation rates, in both aerobic (AE) and anaerobic (AN) environments. The superior photodegradation of Ag2%DP25-PET was attributed to a combination of two effects: LSPR (localized surface plasmon resonance) and Ag-TiO2/environment affinities. The findings of molecular dynamics (MD) simulation and Fukui Function (FF) based on density functional theory (DFT) provide significant insight into the photocatalytic requirements for MB discoloration/degradation. The experimental/theoretical analysis aimed to offer an in-depth understanding of medium/surface interactions on decorated TiO2 materials, as well as how these interactions affect overall degradation behavior.

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.

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.

Rational Choice of Antibiotics and Media for Mycobacterium avium Complex Drug Susceptibility Testing.

The Clinical and Laboratory Standards Institute recommends the use of Mueller Hinton (MH) medium to perform drug susceptibility testing (DST) of Mycobacterium avium complex (MAC) using the microdilution method. For MAC, there has been no study on the impact of media on the determination of minimum inhibitory concentrations (MICs) of antibiotics other than clarithromycin. This study aimed at determining the impact of two media used for DST of MAC and at augmenting the number of pertinent MICs for MAC species encountered in clinical practice. MICs of antibiotics used for the treatment of MAC infections were determined for 158 clinical MAC isolates (80 M. avium, 40 M. intracellulare, 35 M. chimaera, two M. yongonense and one M. timonense) in MH and 7H9 broths using the SLOMYCO SensititreTM system (TREK Diagnostic Systems, East Grinstead, United Kingdom). The modal MICs determined in both media were the same for linezolid, moxifloxacin, rifabutin and amikacin but not for clarithromycin, rifampin and ethambutol. The kappa test for MICs converted to susceptibility categories showed an excellent agreement for clarithromycin, a moderate agreement for linezolid and a weak agreement for moxifloxacin and amikacin. For amikacin, 7H9 allowed a better distinction (fewer intermediate strains) of wild-type populations than MH. Existing breakpoints for linezolid and moxifloxacin are spread through the distribution of MICs for wild-type populations. The only breakpoints that can be used rationally are those for amikacin and clarithromycin. For amikacin, 7H9 performs better than MH, whereas both media perform equally for clarithromycin. Given that testing in 7H9, as opposed to MH, allows easier MIC measurements and yields greater reproducibility, we propose the use of 7H9 medium for DST of MAC.

Field study on bovine paratuberculosis using real-time PCR and liquid culture for testing environmental and individual fecal samples implemented in dairy cow management.

Bovine paratuberculosis (Johne's disease) is a bacterial, chronic, and wasting intestinal disease caused by Mycobacterium avium ssp. paratuberculosis (MAP). Johne's disease causes severe losses in dairy farm productivity and is also suspected to be a potential trigger for Crohn's disease in humans. The fecal-oral infection of MAP to neonates is recognized as an important within-herd transmission route. Our objective was to recommend diagnostic methods for herds with suspected paratuberculosis requiring fast results, as well as for herds with breeding programs or others that aim at being nonsuspected of paratuberculosis infection. We determined a period of 8 wk from sampling to diagnostic findings suitable for testing of cows during the dry period. We therefore tested environmental and individual fecal samples with one rapid and one highly sensitive diagnostic method. Environmental samples (boot swabs) were taken as a first step in 3 herds and tested using a DNA extraction protocol for feces and subsequent real-time PCR (referred to as fecal PCR). Additionally, cultivation in liquid medium for 6 wk was performed and verified with real-time PCR (referred to as liquid culture). Automation of DNA extraction based on magnetic beads and the PCR setup was performed with pipetting robots. As a result, we successfully detected MAP in boot swabs of all herds by both methods. In a second step, 245 individual fecal samples from the 3 herds were examined using also fecal PCR and liquid culture. The results obtained by fecal PCR were compared with detection of MAP using cultivation in liquid medium for 6 wk. Testing individual cows, we identified MAP-specific DNA in 53 fecal samples using the liquid culture. Using fecal PCR, we revealed 43 positive samples of which 39 also tested positive in the liquid culture, revealing MAP-positive cows in all 3 herds. The fecal PCR procedure allows rapid detection of MAP-specific DNA with 74% of the sensitivity of liquid culture. For the purpose of testing with maximal sensitivity, cultivation in liquid medium is recommended. Cultivation of MAP in liquid medium M7H9C means a significant time gain in comparison to cultivation on solid media, which requires twice as much time. Thus, this testing fits within the 6- to 8-wk dry period of gravid cows and provides test results before calving, a prerequisite to prevent fecal-oral transmission to newborn calves.

Enhanced sensitivity and fast turnaround time in laboratory diagnosis for bovine paratuberculosis in faecal samples.

Bovine paratuberculosis (Johne's disease in cattle) is caused by the pathogen Mycobacterium avium subsp. paratuberculosis (MAP) and is a widespread chronic bacterial infectious disease in cattle. Due to the peculiarities of the pathogen, detection of MAP in faeces remains difficult. DNA extraction and real-time PCR for detection of MAP in bovine faeces (direct PCR) have been refined and feasible procedures for rapid, sensitive and automatable detection of the pathogen agent have been developed. Accordingly, in a first step we tested 20 faecal samples using two MAP complete kits (DNA extraction kits based on magnetic beads combined with real-time PCR assays) and six other DNA extraction kits for faeces. MAP-specific DNA was detected by real-time PCR assays. Cultivation of MAP on the solid medium HEYM and in the liquid medium M7H9C served as reference standards. The two complete kits detected significantly more MAP-DNA positive samples than the other procedures applied (p < 0.04). Ct values of 37 and 38 served as cut-off for the respective real-time PCR assays calculated on the basis of standard curves and droplet digital PCR (ddPCR). In a second step, the two MAP complete kits were employed for a comprehensive study including 107 positive and 50 negative faecal samples which had been previously tested on HEYM cultivation. The MAP complete kits yielded sensitivity values of 86% and 89% and specificity values of 100% compared to cultivation of MAP in the liquid medium M7H9C. In detail, cultivation of MAP in M7H9C detected the pathogen in 97% and 100% of the samples tested after an incubation period of six and twelve weeks, respectively. However, the cultivation of MAP on HEYM succeeded in only 74% after twelve weeks of incubation. In all these solid culture positive samples, MAP was also detected using the two complete kits. Additionally, the impact of repeated freezing and thawing of samples on re-cultivation of MAP was tested using 20 faecal samples and resulted in a reduction to 75% and 25% of bacterial growth when using liquid medium M7H9C and solid medium HEYM, respectively. The results of this study show that complete kits with refined automatable protocols for DNA extraction in combination with real-time PCR assays for detection of MAP can compete with sensitive cultivation of the pathogen in liquid medium.

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.

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.