Validation of the MYChrOme™ Culture Plate for Detection and Differentiation of Rapid-Growing Nontuberculous Mycobacteria in Potable and Non-Potable Water: AOAC Performance Tested MethodSM 062101.

BACKGROUND: The MYChrOme™ Culture Plate is a chromogenic media for the detection and differentiation of rapid-growing nontuberculous mycobacteria (NTM) in water, aided by MYCOn™ decontamination to reduce background microbiota. OBJECTIVE: Evaluate the MYChrOme Culture Plates for the detection of rapid-growing NTM in potable and non-potable water as part of the AOAC Performance Tested Method(s)SM program. METHODS: Inclusivity and exclusivity of MYChrOme were evaluated with 50 target and 30 non-target organisms. Method robustness and lot stability of MYChrOme were analyzed. The candidate method was compared to a modified US Food and Drug Administration (FDA) Method: U.S. FDA-Isolation and Identification of Nontuberculous Mycobacteria in Tattoo Inks using an equivalency test. The matrix study consisted of artificially contaminated potable water and naturally contaminated non-potable water. Independent laboratory testing was conducted to verify method performance in non-potable water. RESULTS: The inclusivity of MYChrOme was 94% within one week, and 98% within two weeks. The exclusivity was 96% for untreated samples and 100% for treated samples. The candidate method remained statistically equivalent for robustness and a three-month shelf-life was confirmed. For both matrixes, the candidate and reference methods were not equivalent, with more colonies enumerated on the candidate method except for one contamination level of the potable matrix. CONCLUSION: The MYChrOme culture method can successfully detect and differentiate rapid-growing NTM in the matrixes tested, with sensitivity equivalent or higher than the reference method. HIGHLIGHTS: The MYChrOme culture plate offers differentiation of rapid-growing NTM colonies, improved detection in non-potable samples with MYCOn decontamination, and results within 7 days.

Inaccuracies of the ISO 11731 Method for Environmental Validation of Legionella in Building Water Systems: Opportunities to Improve Sensitivity and Detect Viable but Non-Culturable Legionella.

Current environmental diagnostics for the detection of Legionella fail to detect viable but non-culturable Legionella, have sensitivity limitations and are time-consuming (10-14 days to results). The objective of this study was to compare Legionella detection results between the standard ISO 11731 and an innovative Legionella detection method that utilizes a hybrid methodology of traditional microbiology and molecular detection. In this study, four hundred and seventy-six (476) potable building water samples were analyzed with ISO 11731 and the novel method in parallel. Of the 476 total samples that were tested, a discrepancy of 21% was observed when comparing the ISO 11731 method to the novel method. Separating the samples based on hazard control methods yielded a 15.4% discrepancy for chlorinated systems (n = 284) and a 29% discrepancy for monochloraminated systems (n = 192). The data presented here conclusively show inaccuracies in environmental validation for building water systems based on results returned by the standard ISO 11731 method. This is especially evident in systems primarily disinfected with monochloramines. Overall, these data highlight the need for new and innovative methods to overcome the inaccuracies of the traditional ISO 11731 spread plates to prevent disease and injury caused by Legionella.

Next Day Legionella PCR: a highly reliable negative screen for Legionella in the built environment.

The opportunistic, waterborne pathogen Legionella caused 9,933 cases of Legionnaires' disease in 2018 in the United States (CDC.gov). The incidence of Legionnaires' disease can be reduced by maintaining clean building water systems through water management programs (WMPs). WMPs often include validation testing to confirm the control of bacteria, but the traditional culture method for enumerating Legionella requires 10-14 days to obtain results. A rapid DNA extraction developed by Phigenics and a real-time PCR negative screen for the genus Legionella provided results the day after sampling. This study evaluated the Next Day Legionella PCR (Phigenics, LLC) compared with the traditional culture method (ISO 11731) on 11,125 building water samples for approximately 1 year. Two DNA extraction methods (Methods 1 and 2) were compared. The negative predictive value (NPV) of the Next Day Legionella PCR in comparison to traditional culture for Method 1 was 99.95%, 99.92%, 99.85%, and 99.17% at >10, >2, >1, and >0.1 CFU/ml limits of detection, respectively. The improved DNA extraction (Method 2) increased the NPV to 100% and 99.88% at >1 and >0.1 CFU/ml, respectively. These results demonstrate the reliability of the genus-level Legionella PCR negative screen to predict culture-negative water samples.

Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling.

Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes.