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This paper highlights the current challenges with quantitative estimation of ammonia‐oxidizing bacteria (AOB) employing quantitative real‐time PCR (qPCR) in diverse natural‐ and engineered‐ environments. In this work we concentrates on: (i) identifying the coverage and specificity of the available primers and TaqMan probes for amoA gene based on current databases; (ii) the development of new primers and TaqMan probes for differential quantification of N. europaea and N. eutropha., Summary The choice of primer and TaqMan probes to quantify ammonia‐oxidizing bacteria (AOB) in environmental samples is of crucial importance. The re‐evaluation of primer pairs based on current genomic sequences used for quantification of the amoA gene revealed (1) significant misrepresentations of the AOB population in environmental samples, (2) and a lack of perfect match primer pairs for Nitrosomonas europaea and Nitrosomonas eutropha. We designed two new amoA cluster 7‐specific primer pairs and TaqMan probes to quantify N. europaea (nerF/nerR/nerTaq) and N. eutropha (netF/netR/netTaq). Specificity and quantification biases of the newly designed primer sets were compared with the most popular primer pair (amoA1f/amoA2r) using DNA from various AOB cultures as individual templates as well as DNA mixtures and environmental samples. Based on the qPCR results, we found that the newly designed primer pairs and the most popular one performed similarly for individual templates but differed for the DNA mixtures and environmental samples. Using the popular primer pair introduced a high underestimation of AOB in environmental samples, especially for N. eutropha. Thus, there is a strong need for more specific primers and probes to understand the occurrence and competition between N. europaea and N. eutropha in different environments.

Viruses 14(9), 1876 (2022). doi:10.3390/v14091876 special issue: "Special Issue "Molecular Epidemiology of SARS-CoV-2" / Special Issue Editors: Dr. Marta Giovanetti, Guest Editor; Dr. Luiz Carlos Junior Alcantara, Guest Editor", Published by MDPI, Basel

Treated wastewater irrigation (TWW) releases antibiotics and antibiotic resistance genes (ARGs) into the environment and might thus promote the dissemination of antibiotic resistance in groundwater (GW). We hypothesized that TWW irrigation increases ARG abundance in GW through two potential mechanisms: the contamination of GW with resistant bacteria and the accumulation of antibiotics in GW. To test this, the GW below a real-scale TWW-irrigated field was sampled for six months. Sampling took place before, during and after high-intensity TWW irrigation. Samples were analysed with 16S rRNA amplicon sequencing, qPCR of six ARGs and the class 1 integron-integrase gene intI1, while liquid chromatography tandem mass spectrometry was performed to detect antibiotic and pharmaceutical residues. Absolute abundance of 16S rRNA in GW decreased rather than increased during long-term irrigation. Also, the relative abundance of TWW-related bacteria did not increase in GW during long-term irrigation. In contrast, long-term TWW irrigation increased the relative abundance of sul1 and intI1 in the GW microbiome. Furthermore, GW contained elevated concentrations of sulfonamide antibiotics, especially sulfamethoxazole, to which sul1 confers resistance. Total sulfonamide concentrations in GW correlated with sul1 relative abundance. Consequently, TWW irrigation promoted sul1 and intI1 dissemination in the GW microbiome, most likely due to the accumulation of drug residues.

Wastewater-based epidemiology (WBE) is a great approach that enables us to comprehensively monitor the community to determine the scale and dynamics of infections in a city, particularly in metropolitan cities with a high population density. Therefore, we monitored the time course of the SARS-CoV-2 RNA concentration in raw sewage in the Frankfurt metropolitan area, the European financial center. To determine the SARS-CoV-2 RNA concentration in sewage, we continuously collected 24 h composite samples twice a week from two wastewater treatment plant (WWTP) influents (Niederrad and Sindlingen) serving the Frankfurt metropolitan area and performed RT-qPCR analysis targeting three genes (N gene, S gene, and ORF1ab gene). In August, a resurgence in the SARS-CoV-2 RNA load was observed, reaching 3 × 1013 copies/day, which represented similar levels compared to April with approx. 2 × 1014 copies/day. This corresponds to a continuous increase again in COVID-19 cases in Frankfurt since August, with an average of 28.6 incidences, compared to 28.7 incidences in April. Different temporal dynamics were observed between different sampling points, indicating local dynamics in COVID-19 cases within the Frankfurt metropolitan area. The SARS-CoV-2 RNA load to the WWTP Niederrad ranged from approx. 4 × 1011 to 1 × 1015 copies/day, the load to the WWTP Sindlingen from approx. 1 × 1011 to 2 × 1014 copies/day, which resulted in a preceding increase in these loading in July ahead of the weekly averaged incidences. The study shows that WBE has the potential as an early warning system for SARS-CoV-2 infections and a monitoring system to identify global hotspots of COVID-19.

Implementation of partial nitritation anammox (PNA) in the mainstream (municipal wastewater treatment) is still under investigation. Microbial community structure and reactor type can influence the performance of PNA reactor; yet, little is known about the role of the community composition of the inoculum and the reactor configuration under mainstream conditions. Therefore, this study investigated the community structure of inocula of different origin and their consecutive community dynamics in four different lab‐scale PNA reactors with 16S rRNA gene amplicon sequencing. These reactors were operated for almost 1 year and subjected to realistic seasonal temperature fluctuations as in moderate climate regions, that is, from 20°C in summer to 10°C in winter. The sequencing analysis revealed that the bacterial community in the reactors comprised: (1) a nitrifying community (consisting of anaerobic ammonium‐oxidizing bacteria (AnAOB), ammonia‐oxidizing bacteria (AOB), and nitrite‐oxidizing bacteria (NOB)); (2) different heterotrophic denitrifying bacteria and other putative heterotrophic bacteria (HB). The nitrifying community was the same in all four reactors at the genus level, although the biomasses were of different origin. Community dynamics revealed a stable community in the moving bed biofilm reactors (MBBR) in contrast to the sequencing batch reactors (SBR) at the genus level. Moreover, the reactor design seemed to influence the community dynamics, and reactor operation significantly influenced the overall community composition. The MBBR seems to be the reactor type of choice for mainstream wastewater treatment.