Transport and Retention of Fecal Indicator Bacteria in Unsaturated Porous Media: Effect of Transient Water Flow.

For production of clean drinking water, the processes governing bacterial remobilization in the unsaturated zone at transient water flow are critical. Although managed aquifer recharge is an effective way to dispose of pathogens, there are concerns about recontamination after heavy precipitation. To better understand how bacteria that were initially retained in porous media can be released to groundwater due to transient water content, transport experiments and modeling for Escherichia coli and Enterococcus moraviensis were conducted at the soil column scale. After inoculating dune sand columns with a bacteria suspension for 4 h, three rainfall events were performed at 24-h intervals. The effluent from sand columns was collected to analyze bacteria breakthrough curves (BTCs). After the rainfall experiments, the bacteria distribution in the sand column was determined. The collected BTCs and profile retentions were modeled with HYDRUS-1D, using different model concepts, including one-site kinetic attachment/detachment (M1), Langmuirian (M2), Langmuirian and blocking (M3), and two-site attachment/detachment (M4). After inoculation, almost 99% of the bacteria remained in the soil. The M1 and M2 bacteria models had a high agreement between observed and modeled concentrations, and attachment and detachment were two significant mechanisms for regulating bacteria movement in a porous medium with fluctuations in water flow. At the end of the experiment, the majority of bacteria were still found within the depth range of 5 cm to 15 cm. Our experiments show that E. coli is more mobile in sandy soils than E. moraviensis. The results of this study also suggest that the unsaturated zone is an important barrier between microbial contamination at the soil surface and groundwater. Follow-up studies are needed to completely understand the variables that regulate bacteria remobilization in the unsaturated zone of dune sands. IMPORTANCE At managed artificial recharge sites in the Netherlands, recontamination of infiltrated water with fecal indicator bacteria has been observed. The results of this study suggest that the unsaturated zone is an important barrier between microbial contamination at the soil surface and groundwater. Bacteria that accumulate in the unsaturated zone, on the other hand, can multiply to such an extent that they can be released into the saturated zone when saturation increases due to major rain events or a rise in groundwater level.

Monitoring the exposure and emissions of antibiotic resistance: Co-occurrence of antibiotics and resistance genes in wastewater treatment plants.

The COVID-19 pandemic has brought new momentum to 'wastewater-based epidemiology' (WBE). This approach can be applied to monitor the levels of antibiotic-resistant genes (ARGs), which in terms are used to make inferences about the burden of antimicrobial resistance (AMR) in human settlements. However, there is still little information about temporal variability in ARG levels measured in wastewater streams and how these influence the inferences made about the occurrence of AMR in communities. The goal of this study was hence to gain insights into the variability in ARG levels measured in the influent and effluent of two wastewater treatment plants in The Netherlands and link these to levels of antibiotic residues measured in the same samples. Eleven antibiotics were detected, together with all selected ARGs, except for VanB. Among the measured antibiotics, significant positive correlations (p > 0.70) with the corresponding resistance genes and some non-corresponding ARGs were found. Mass loads varied up to a factor of 35 between days and in concomitance with rainfall. Adequate sampling schemes need to be designed to ensure that conclusions are drawn from valid and representative data. Additionally, we advocate for the use of mass loads to interpret levels of AMR measured in wastewater.

International tempo-spatial study of antibiotic resistance genes across the Rhine river using newly developed multiplex qPCR assays.

The aim of this study was to capture and explain changes in antibiotic resistance gene (ARG) presence and concentration internationally across the Rhine river. Intl1 concentrations and national antibiotic usage were investigated as proxies to predict anthropogenic ARG pollution. Newly-developed multiplex qPCR assays were employed to investigate ARG profiles across 8 locations (L1-L8) in three countries (Switzerland, Germany, the Netherlands) and to detect potential regional causes for variation. Two of these locations were further monitored, over the duration of one month. A total of 13 ARGs, Intl1 and 16S rRNA were quantified. ARG presence and concentrations initially increased from L1(Diepoldsau) to L3(Darmstadt). A continuous increase could not be observed at subsequent locations, with the large river volume likely being a major contributing factor for stability. ARG presence and concentrations fluctuated widely across different locations. L2(Basel) and L3 were the two most polluted locations, coinciding with these locations being well-developed pharmaceutical production locations. We draw attention to the characteristic, clearly distinct ARG profiles, with gene presence being consistent and gene concentrations varying significantly less over time than across different locations. Five genes were Rhine-typical (ermB, ermF, Intl1, sul1 and tetM). Intl1 and sul1 were the genes with highest and second-highest concentration, respectively. Aph(III)a and blaOXA were permanently introduced downstream of L1, indicating no source of these genes prior to L1. We highlight that correlations between Intl1 and ARG concentrations (R2 = 0.72) were driven by correlations to sul1 and disappeared when excluding sul1 from the analysis (R2 = 0.05). Intl1 therefore seems to be a good proxy for sul1 concentrations but not necessarily for overall (anthropogenic) ARG pollution. Aminoglycoside usage per country correlated with concentrations of aph(III)a and several unrelated antibiotic resistance genes (blaOXA,ermB, ermF and tetM). This correlation can be explained by co-resistance caused by mobile genetic elements (MGEs), such as Tn1545.

Antibiotic resistance gene distribution in agricultural fields and crops. A soil-to-food analysis.

Despite the social concern about the generalization of antibiotic resistance hotspots worldwide, very little is known about the contribution of different potential sources to the global risk. Here we present a quantitative analysis of the distribution of Antibiotic Resistance Genes (ARGs) in soil, rhizospheric soil, roots, leaves and beans in tomato, lettuce and broad beans crops (165 samples in total), grown in nine commercial plots distributed in four geographical zones in the vicinity of Barcelona (North East Spain). We also analyzed five soil samples from a nearby forest, with no record of agricultural activities. DNA samples were analyzed for their content in the ARGs sul1, tetM, qnrS1, blaCTX-M-32, blaOXA-58, mecA, and blaTEM, plus the integron intI1, using qPCR methods. In addition, soil microbiomes from the different plots were analyzed by amplicon-targeted 16S rRNA gene sequencing. Our data show a decreasing gradient of ARG loads from soil to fruits and beans, the latter showing only from 0.1 to 0.01% of the abundance values in soil. The type of crop was the main determinant for both ARG distribution and microbiome composition among the different plots, with minor contributions of geographic location and irrigation water source. We propose that soil amendment and/or fertilization, more than irrigation water, are the main drivers of ARG loads on the edible parts of the crop, and that they should therefore be specifically controlled.

Characterization of wastewater effluents in the Danube River Basin with chemical screening, in vitro bioassays and antibiotic resistant genes analysis.

Averaged 7-day composite effluent wastewater samples from twelve wastewater treatment plants (WWTPs) in nine countries (Romania, Serbia, Hungary, Slovenia, Croatia, Slovakia, Czechia, Austria, Germany) in the Danube River Basin were collected. WWTPs' selection was based on countries' dominant technology and a number of served population with the aim to get a representative holistic view of the pollution status. Samples were analyzed for 2248 chemicals of emerging concern (CECs) by wide-scope target screening employing LC-ESI-QTOF-MS. 280 compounds were detected at least in one sample and quantified. Spatial differences in the concentrations and distribution of the compounds classes were discussed. Additionally, samples were analyzed for the possible agonistic/antagonistic potencies using a panel of in vitro transactivation reporter gene CALUX® bioassays including ERα (estrogenics), anti-AR (anti-androgens), GR (glucocorticoids), anti-PR (anti-progestins), PPARα and PPARγ (peroxisome proliferators) and PAH assays. The potency of the wastewater samples to cause oxidative stress and induce xenobiotic metabolism was determined using the Nrf2 and PXR CALUX® bioassays, respectively. The signals from each of the bioassays were compared with the recently developed effect-based trigger values (EBTs) and thus allowed for allocating the wastewater effluents into four categories based on their measured toxicity, proposing a putative action plan for wastewater operators. Moreover, samples were analyzed for antibiotics and 13 antibiotic-resistant genes (ARGs) and one mobile genetic element (intl1) with the aim to assess the potential for antibiotic resistance. All data collected from these various types of analysis were stored in an on-line database and can be viewed via interactive map at https://norman-data.eu/EWW_DANUBE.

QMRA of adenovirus in drinking water at a drinking water treatment plant using UV and chlorine dioxide disinfection.

According to the Dutch Drinking Water Act of 2011, Dutch drinking water suppliers must conduct a Quantitative Microbial Risk Assessment (QMRA) for infection by the following index pathogens: enterovirus, Campylobacter, Cryptosporidium and Giardia at least once every four years in order to assess the microbial safety of drinking water. The health-based target for safe drinking water is set at less than one infection per 10 000 persons per year. At Evides Water Company, concern has arisen whether their drinking water treatment, mainly based on UV inactivation and chlorine dioxide, reduces levels of adenovirus (AdV) sufficiently. The main objective was, therefore, to conduct a QMRA for AdV. Estimates of the AdV concentrations in source water were based on enumeration of total AdV by integrated cell culture PCR (iccPCR), most probable number PCR (mpnPCR) and quantitative PCR (qPCR), and on enumeration of AdV40/41 by mpnPCR and qPCR. AdV40/41 represents a large fraction of total AdV and only a small fraction of AdV is infectious (1/1700). By comparison of literature data and plant scale data, somatic coliphages appeared a good, conservative indicator for AdV disinfection by UV irradiation. Similarly, bacteriophage MS2 appeared to be a good, conservative indicator for disinfection by chlorine dioxide. Literature data on the efficiency of chlorine dioxide disinfection were fitted with the extended HOM model. Chlorine dioxide disinfection at low initial concentrations (0.05-0.1 mg/l) was found to be the major treatment step, providing sufficient treatment on its own for compliance with the health-based target. UV disinfection of AdV at 40 mJ/cm2 or 73 mJ/cm2 was insufficient without chlorine dioxide disinfection.

The impact of on-site hospital wastewater treatment on the downstream communal wastewater system in terms of antibiotics and antibiotic resistance genes.

This study quantified antibiotic and antibiotic resistance gene (ARG) concentrations in hospital and communal wastewaters as well as the influents and effluents of the receiving urban wastewater treatment plants (UWWTP) in two Dutch cities. In only one city, hospital wastewater was treated on-site using advanced technologies, including membrane bioreactor treatment (MBR), ozonation, granulated activated carbon (GAC) and UV-treatment. On-site hospital wastewater (HWW) treatment reduced gene presence of hospital-related antibiotic resistance genes and antibiotic concentrations in the receiving urban wastewater treatment plant. These findings support the need for on-site treatment of high-risk point sources of antibiotic resistance genes. 13 antibiotic resistance genes, Integrase Class 1 and 16S rRNA concentrations were quantified using multiplex quantitative real-time PCR (qPCR) assays and the presence and/or concentration of 711 antibiotics were analyzed. Hospital wastewater contained approximately 25% more antibiotics and gene concentrations between 0.4 log to 1.8-fold higher than communal wastewater (CWW). blaKPC and vanA could be identified as hospital-related genes and were reduced to under the limit of detection (LOD) during on-site treatment. Advanced on-site treatment removed between 0.5 and 3.6-fold more genes than conventional biological urban wastewater treatment (activated sludge). Advanced on-site treatment was able to eliminate 12 out of 19 detected antibiotics, while urban waste water treatment eliminated up to 1 (out of 21 detected). Different advanced treatment technologies were able to target different pollutants to varying extents, making sequential alignment more effective. MBR treatment was most efficient in antibiotic resistance gene reduction and ozonation in antibiotic reduction. blaKPC could only be detected in the influent of the urban wastewater treatment plant receiving untreated hospital wastewater. Similarly, vanA was only consistently detected in this treatment plant. These results indicate a positive effect of on-site treatment of hospital wastewater on the communal sewage system.

Distribution of antibiotic resistance genes in soils and crops. A field study in legume plants (Vicia faba L.) grown under different watering regimes.

Social concern has raised during the last years due to the development of antibiotic resistance hotspots in different environmental compartments, including the edible parts of crops. To assess the influence of the water quality used for watering, we collected samples from soil, roots, leaves and beans from the legume plant Vicia faba (broad beans) in three agricultural peri-urban plots (Barcelona, NE Spain), irrigated with either groundwater, river water, or reclaimed water. Antibiotic resistance genes (ARGs) sul1, tetM, qnrS1, blaCTX-M-32,blaOXA-58, mecA, and blaTEM were quantified by real-time PCR, along with 16S rDNA and intl1 sequences, as proxies for bacterial abundance and integron prevalence, respectively. Microbiome composition of all samples were analyzed by high-throughput DNA sequencing. Results show a gradient of bacterial species diversity and of ARG prevalence from highly diverse soil samples to microbially-poor beans and leaves, in which Rhizobiales essentially displaced all other groups, and that presented very small loads of ARGs and integron sequences. The data suggest that the microbiome and the associated resistome were likely influenced by agricultural practices and water quality, and that future irrigation water legal standards should consider the specific Physiology of the different crop plants.

Transport of bacteriophage MS2 and PRD1 in saturated dune sand under suboxic conditions.

Soil passage of (pretreated) surface water to remove pathogenic microorganisms is a highly efficient process under oxic conditions, reducing microorganism concentrations about 8 log10 within tens of meters. However, under anoxic conditions, it has been shown that removal of microorganisms can be limited very much. Setback distances for adequate protection of natural groundwater may, therefore, be too short if anoxic conditions apply. Because removal of microorganisms under suboxic conditions is unknown, this research investigated removal of bacteriophage MS2 and PRD1 by soil passage under suboxic conditions at field scale. At the field location (dune area), one injection well and six monitoring wells were installed at different depths along three suboxic flow lines, where oxygen concentrations ranged from 0.4 to 1.7 mg/l and nitrate concentrations ranged from 13 to 16 mg/L. PRD1 and MS2 were injected directly at the corresponding depths and their removal in each flow line was determined. The highest bacteriophage removal was observed in the top layer, with about 9 log removal of MS2, and 7 log removal of PRD1 after 16 meters of aquifer transport. Less removal was observed at 12 m below surface, probably due to a higher groundwater velocity in this coarser grained layer. MS2 was removed more effectively than PRD1 under all conditions. Due to short travel times, inactivation of the phages was limited and the reported log removal was mainly associated with attachment of phages to the aquifer matrix. This study shows that attachment of MS2 and PRD1 is similar for oxic and suboxic sandy aquifers, and, therefore, setback distances used for sandy aquifers under oxic and suboxic conditions provide a similar level of safety. Sticking efficiency and the attachment rate coefficient, as measures for virus attachment, were evaluated as a function of the physico-chemical conditions.

Models of the behaviour of (thermally stressed) microbial spores in foods: tools to study mechanisms of damage and repair.

The 'Omics' revolution has brought a wealth of new mechanistic insights in many fields of biology. It offers options to base predictions of microbial behaviour on mechanistic insight. As the cellular mechanisms involved often turn out to be highly intertwined it is crucial that model development aims at identifying the level of complexity that is relevant to work at. For the prediction of microbiologically stable foods insight in the behaviour of bacterial spore formers is crucial. Their chances of germination and likelihood of outgrowth are major food stability indicators, as well as the transition from outgrowth to first cell division and vegetative growth. Current available technology to assess these parameters in a time-resolved manner at the single spore level will be discussed. Tools to study molecular processes operative in heat induced damage will be highlighted.

On the origin of heterogeneity in (preservation) resistance of Bacillus spores: input for a 'systems' analysis approach of bacterial spore outgrowth.

Bacterial spores are the ultimate (stress) 'survival capsules'. They allow strains from the Bacillus and Clostridium species to survive harsh environmental conditions. In addition to the decision to enter sporulation the decision to do the reverse (germinate) is also a decisive event after which there is no return. Generally it is observed that the behaviour of spores towards the environment is not homogeneous. In fact in many cases it is even quite heterogeneous, certainly upon subjecting the spores to a thermal stress treatment. Genome information coupled to high resolution single-cell analysis techniques allow us currently to analyse signalling events of individual cells. In the area of food preservation the next challenge is to couple the newly acquired mechanistic data to the physiologically observed heterogeneity in spore behaviour. The current paper will introduce the background of physiological heterogeneity while discussing the molecular processes that likely contribute to the observed heterogeneity in outgrowth. The discussion is set in the framework of contemporary and future needs for single-cell data integration in order to enhance the mechanistic basis of food preservation and spoilage models targeting bacterial spores.

Role of germinant receptors in Caco-2 cell-initiated germination of Bacillus cereus ATCC 14579 endospores.

Spores obtained from Bacillus cereus ATCC 14579 and mutant strains lacking each of seven germinant receptor operons were exposed to differentiated Caco-2 cells and monitored for germination. Spores of the gerI and gerL mutants showed a reduced germination response, pointing to a role for these receptors in Caco-2-induced germination.

Influence of sporulation medium composition on transcription of ger operons and the germination response of spores of Bacillus cereus ATCC 14579.

Bacillus cereus ATCC 14579 endospores were produced in Y1 medium, a nutrient-rich, chemically defined sporulation medium, and in modified G medium, containing low amounts of nutrients. The average transcription level of the seven ger operons per cell was 3.5 times higher in Y1 medium, and the spores grown in this medium showed an enhanced germination response.

Characterization of germination receptors of Bacillus cereus ATCC 14579.

Specific amino acids, purine ribonucleosides, or a combination of the two is required for efficient germination of endospores of Bacillus cereus ATCC 14579. A survey including 20 different amino acids showed that l-alanine, l-cysteine, l-threonine, and l-glutamine are capable of initiating the germination of endospores of B. cereus ATCC 14579. In addition, the purine ribonucleosides inosine and adenosine can trigger germination of the spores. Advanced annotation of the B. cereus ATCC 14579 genome revealed the presence of seven putative germination (ger) operons, termed gerG, gerI, gerK, gerL, gerQ, gerR, and gerS. To determine the role of the encoded putative receptors in nutrient-induced germination, disruption mutants were constructed by the insertion of pMUTIN4 into each of the seven operons. Four of the seven mutants were affected in the germination response to amino acids or purine ribonucleosides, whereas no phenotype could be attributed to the mutants with disrupted gerK, gerL, and gerS loci. The strain with a disrupted gerR operon was severely hampered in the ability to germinate: germination occurred in response to l-glutamine but not in the presence of any of the other amino acids tested. The gerG mutant showed significantly reduced l-glutamine-induced germination, which points to a role of this receptor in the l-glutamine germination signaling pathway. gerR, gerI, and gerQ mutants showed reduced germination rates in the presence of inosine, suggesting a role for these operons in ribonucleoside signaling. Efficient germination by the combination of l-glutamine and inosine was shown to involve the gerG and gerI operons, since the germination of mutants lacking either one of these receptors was significantly reduced. Germination triggered by the combination of l-phenylalanine and inosine was lost in the gerI mutant, indicating that both molecules are effective at the GerI receptor.

Deletion of sigB in Bacillus cereus affects spore properties.

In Bacillus cereus and other gram-positive bacteria the alternative sigma factor sigma(B) is an important regulator of the stress response. Deletion of the sigB gene generally leads to a stress-sensitive phenotype of vegetative cells. In this study, we describe the effect of the deletion of the sigB gene in B. cereus on spore properties. In particular, spores of the sigB deletion mutant showed a defect in germination upon exposure to the germinants alanine and inosine.

Influence of glutamate on growth, sporulation, and spore properties of Bacillus cereus ATCC 14579 in defined medium.

A chemically defined medium in combination with an airlift fermentor system was used to study the growth and sporulation of Bacillus cereus ATCC 14579. The medium contained six amino acids and lactate as the main carbon sources. The amino acids were depleted during exponential growth, while lactate was metabolized mainly during stationary phase. Two concentrations of glutamate were used: high (20 mM; YLHG) and low (2.5 mM; YLLG). Under both conditions, sporulation was complete and synchronous. Sporulation started and was completed while significant amounts of carbon and nitrogen sources were still present in the medium, indicating that starvation was not the trigger for sporulation. Analysis of amino acids and NH4+ in the culture supernatant showed that most of the nitrogen assimilated by the bacteria was taken up during sporulation. The consumption of glutamate depended on the initial concentration; in YLLG, all of the glutamate was used early during exponential growth, while in YLHG, almost all of the glutamate was used during sporulation. In YLLG, but not in YLHG, NH4+ was taken up by the cells during sporulation. The total amount of nitrogen used by the bacteria in YLLG was less than that used by the bacteria in YLHG, although a significant amount of NH4+ was present in the medium throughout sporulation. Despite these differences, growth and temporal expression of key sigma factors involved in sporulation were parallel, indicating that the genetic time frames of sporulation were similar under both conditions. Nevertheless, in YLHG, dipicolinic acid production started later and the spores were released from the mother cells much later than in YLLG. Notably, spores had a higher heat resistance when obtained after growth in YLHG than when obtained after growth in YLLG, and the spores germinated more rapidly and completely in response to inosine, l-alanine, and a combination of these two germinants.

gerR, a novel ger operon involved in L-alanine- and inosine-initiated germination of Bacillus cereus ATCC 14579.

Bacillus cereus endospores germinate in response to particular nutrients. Spores are able to sense these nutrients in the environment by receptors encoded by the gerA family of operons. Analysis of the Bacillus cereus ATCC 14579 genome revealed seven gerA family homologues. Using a transposon Tn917-based insertional mutagenesis approach followed by an enrichment procedure to select for l-alanine-induced germination mutants, we isolated a mutant with a defect in the l-alanine germination pathway. The transposon disrupted the last gene of a tricistronic gerA family operon, designated gerR, with the order gerRA, gerRC, gerRB. A second mutant was created by insertion of pMUTIN4 in gerRC. Both mutants showed the same phenotype for nutrient-induced germination. Spores of the gerR mutant strains were blocked in their l-alanine-initiated germination pathway and showed a delayed inosine-induced germination response. Apparently, germination mediated by l-alanine and inosine cannot be compensated for completely by the other germinant receptors, and this points towards an essential role of the gerR-encoded receptor in the receptor complex. In food products, spores of the mutant strains showed a reduced germination response compared to spores of the parental strain. High-pressure-initiated germination was not affected by the gerR mutations, as experiments with 100 and 550 MPa showed no difference with spores of the parental strain.

Growth and sporulation of Bacillus cereus ATCC 14579 under defined conditions: temporal expression of genes for key sigma factors.

An airlift fermentor system allowing precise regulation of pH and aeration combined with a chemically defined medium was used to study growth and sporulation of Bacillus cereus ATCC 14579. Sporulation was complete and synchronous. Expression of sigA, sigB, sigF, and sigG was monitored with real-time reverse transcription-PCR, and the pattern qualitatively resembled that of Bacillus subtilis. This method allows reproducible production of stable spores, while the synchronous growth and defined conditions are excellently suitable for further gene expression studies of cellular differentiation of B. cereus.