A flow cytometry method for bacterial quantification and biomass estimates in activated sludge.

Absolute bacterial quantification receives little serious attention in the literature compared to sequencing, conceivably because it is considered unimportant and facile, or because existing methods are tedious, laborious and/or biased in nature. This is particularly true in engineered systems, including activated sludge, where such information underpins their design and operation. To overcome these limitations we built upon existing work and optimised and comprehensively validated, through comparison with epifluorescence microscopy (EFM), a rapid and precise flow cytometric protocol to enumerate total bacterial numbers in activated sludge. Insights into potential biases were evaluated using appropriate statistical analyses on this comparison, which spanned four orders of magnitude, as well as comparing volatile suspended solid (VSS) concentrations. The results suggest flow cytometry (FCM) is a rapid, reproducible and economical technique for quantifying total bacterial numbers and biomass concentrations in activated sludge, despite within order of magnitude discrepancies with EFM counts, which had inherent and evidently greater errors and biases than FCM. The use of FCM for routine monitoring over both EFM and VSS should help further understanding of the microbial ecology in, and the operation of, engineered systems.

Increased waterborne blaNDM-1 resistance gene abundances associated with seasonal human pilgrimages to the upper ganges river.

Antibiotic resistance (AR) is often rooted in inappropriate antibiotic use, but poor water quality and inadequate sanitation exacerbate the problem, especially in emerging countries. An example is increasing multi-AR due to mobile carbapenemases, such as NDM-1 protein (coded by blaNDM-1 genes), which can produce extreme drug-resistant phenotypes. In 2010, NDM-1 positive isolates and blaNDM-1 genes were detected in surface waters across Delhi and have since been detected across the urban world. However, little is known about blaNDM-1 levels in more pristine locations, such as the headwaters of the Upper Ganges River. This area is of particular interest because it receives massive numbers of visitors during seasonal pilgrimages in May/June, including visitors from urban India. Here we quantified blaNDM-1 abundances, other AR genes (ARG), and coliform bacteria in sediments and water column samples from seven sites in the Rishikesh-Haridwar region of the Upper Ganges and five sites on the Yamuna River in Delhi to contrast blaNDM-1 levels and water quality conditions between season and region. Water quality in the Yamuna was very poor (e.g., anoxia at all sites), and blaNDM-1 abundances were high across sites in water (5.4 ± 0.4 log(blaNDM-1·mL(-1)); 95% confidence interval) and sediment (6.3 ± 0.7 log(blaNDM-1·mg(-1))) samples from both seasons. In contrast, water column blaNDM-1 abundances were very low across all sites in the Upper Ganges in February (2.1 ± 0.6 log(blaNDM-1·mL(-1))), and water quality was good (e.g., near saturation oxygen). However, per capita blaNDM-1 levels were 20 times greater in June in the Ganges water column relative to February, and blaNDM-1 levels significantly correlated with fecal coliform levels (r = 0.61; p = 0.007). Given that waste management infrastructure is limited in Rishikesh-Haridwar, data imply blaNDM-1 levels are higher in visitor's wastes than local residents, which results in seasonally higher blaNDM-1 levels in the river. Pilgrimage areas without adequate waste treatment are possible "hot spots" for AR transmission, and waste treatment must be improved to reduce broader AR dissemination via exposed returning visitors.