Characterisation and control of the biosolids storage environment: Implications for E. coli dynamics.

E. coli survival in biosolids storage may present a risk of non-compliance with guidelines designed to ensure a quality product safe for agricultural use. The storage environment may affect E. coli survival but presently, storage characteristics are not well profiled. Typically biosolids storage environments are not actively controlled or monitored to support increased product quality or improved microbial compliance. This two-phased study aimed to identify the environmental factors that control bacterial concentrations through a long term, controlled monitoring study (phase 1) and a field-scale demonstration trial modifying precursors to bacterial growth (phase 2). Digested and dewatered biosolids were stored in operational-scale stockpiles to elucidate factors controlling E. coli dynamics. E. coli concentrations, stockpile dry solids, temperature, redox and ambient weather data were monitored. Results from ANCOVA analysis showed statistically significant (p < 0.05) E. coli reductions across storage periods with greater die-off in summer months. Stockpile temperature had a statistically significant effect on E. coli survival. A 4.5 Log reduction was measured in summer (maximum temperature 31 °C). In the phase 2 modification trials, covered stockpiles were able to maintain a temperature >25 °C for a 28 day period and achieved a 3.7 Log E. coli reduction. In winter months E. coli suppression was limited with concentrations >6 Log10 CFU g-1 DS maintained. The ANCOVA analysis has identified the significant role that physical environmental factors, such as stockpile temperature, has on E. coli dynamics and the opportunities for control.

Quantification of relative proportions of intact cells in microbiological samples using the example of Cryptosporidium parvum oocysts.

UNLABELLED: The fast analysis of relative proportions of live and dead cells can be of great value whether for comparing inactivation efficiencies of different biocidal treatments or for monitoring organisms of interest in environmental samples. We introduce here a straightforward method to determine the percentage of intact cells based on treatment of samples with the viability dye propidium monoazide (PMA). PMA selectively enters membrane-damaged cells and suppresses their PCR detection through modification of their DNA. The study was performed using Cryptosporidium parvum oocysts as a model although the principle should be applicable to other organisms. Validation was performed with defined mixtures of live and heat-killed oocysts and by exposing oocysts to a heat stress gradient. The method correctly indicated increasingly lower proportions of intact cells with increasing temperatures. When comparing the loss of membrane integrity of UV-killed (40 mJ cm(-2) ) oocysts during storage in nonsterile tap water, results suggested that integrity declines slowly (over weeks) and at a rate comparable to non-UV-exposed oocysts. For all experiments, the amplification of longer DNA sequences was found beneficial. In the UV experiment, longer amplicons revealed not only higher sensitivity in excluding membrane-damaged oocysts, but also in excluding DNA with UV-induced damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Whether in the context of microbial ecology or in an industrial context, many questions in microbiology are linked to microbial viability. As cultivation of micro-organisms can be long or may not be possible, fast methods to assess the numbers of live cells are in great demand. We present here a straightforward strategy to determine the relative proportions of intact cells. The PCR-based rapid method is expected to be useful where relative information is sufficient (e.g. for comparing the effect of different antimicrobial treatments on known numbers of micro-organisms) or when the presence of PCR inhibitors does not allow absolute quantification.

Evaluation of engineered nanoparticle toxic effect on wastewater microorganisms: current status and challenges.

The use of engineered nanoparticles (ENPs) in a wide range of products is associated with an increased concern for environmental safety due to their potential toxicological and adverse effects. ENPs exert antimicrobial properties through different mechanisms such as the formation of reactive oxygen species, disruption of physiological and metabolic processes. Although there are little empirical evidences on environmental fate and transport of ENPs, biosolids in wastewater most likely would be a sink for ENPs. However, there are still many uncertainties in relation to ENPs impact on the biological processes during wastewater treatment. This review provides an overview of the available data on the plausible effects of ENPs on AS and AD processes, two key biologically relevant environments for understanding ENPs-microbial interactions. It indicates that the impact of ENPs is not fully understood and few evidences suggest that ENPs could augment microbial-mediated processes such as AS and AD. Further to this, wastewater components can enhance or attenuate ENPs effects. Meanwhile it is still difficult to determine effective doses and establish toxicological guidelines, which is in part due to variable wastewater composition and inadequacy of current analytical procedures. Challenges associated with toxicity evaluation and data interpretation highlight areas in need for further research studies.

Mycobacterium avium subsp. paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) is known to be a very slow-growing organism. The fact that cells typically need several weeks to form visible colonies severely compromises the suitability of plate counting for assessment of viable cell counts. This problem might be overcome by the application of fast molecular methods containing a viability component. We have evaluated a promising technology combining sample treatment with propidium monoazide (PMA) prior to DNA extraction for selective detection of cells with intact cell membranes with detection of sequence element F57 by quantitative PCR (F57 qPCR). Element F57 is unique for MAP and is not known to exist in any other bacterial species. Conditions of PMA treatment were optimised for MAP isolate 7082 using live and heat-killed cells and comparing different DNA extraction procedures. The subsequent successful application of the optimised protocol to four other MAP isolates of different origins suggested that the optimised protocol might be broadly applicable to different MAP strains. Furthermore, different equations were compared to use the data resulting from this technology to optimally predict the percentage of live MAP cells in mixtures containing both live and dead cells. The presented protocol holds promise to be used routinely for detecting MAP with intact cell membranes in research applications.

Retention of a model pathogen in a porous media biofilm.

The inadvertent or the deliberate introduction of pathogens into drinking water can lead to public health consequences. Distribution system sampling strategies are needed to provide information on the identity, source and fate of the introduced pathogens. Porous media biofilm reactors conditioned with undefined drinking water biofilms were tested for their ability to immobilize Escherichia coli 0157:H7. Biofilms were established by applying continuous flow of biologically activated carbon treated water with natural microflora and supplemented nutrient solution (0.5 mg l(-1) C) for 2 or 3 weeks. Control reactors were clean and were not colonized with biofilm. All reactors were injected with slug doses of approximately 1 x 10(9) cfu E. coli O157:H7. On the basis of the plate count enumeration of the introduced pathogen, reactors pre-colonized for 2 or 3 weeks retained significantly more cells (0.75 and 9.37% of the introduced spike dose, respectively) compared with uncolonized control reactors (0.22%). Compared with cultivation, microscopic direct counts and quantitative PCR suggested significantly higher and lower numbers of pathogens, respectively. Plate counts were thus considered as the method of choice for pathogen enumeration in this study. In addition to providing general insights into interactions between pathogens and drinking water biofilms, the study concluded that engineered biofilm systems may be considered as a device to capture pathogens from the bulk flow for monitoring purposes.

A mRNA-based thermosensor controls expression of rhizobial heat shock genes.

Expression of several heat shock operons, mainly coding for small heat shock proteins, is under the control of ROSE (repression of heat shock gene expression) in various rhizobial species. This negatively cis-acting element confers temperature control by preventing expression at physiological temperatures. We provide evidence that ROSE-mediated regulation occurs at the post-transcriptional level. A detailed mutational analysis of ROSE(1)-hspA translationally fused to lacZ revealed that its highly conserved 3'-half is required for repression at normal temperatures (30 degrees C). The mRNA in this region is predicted to form an extended secondary structure that looks very similar in all 15 known ROSE elements. Nucleotides involved in base pairing are strongly conserved, whereas nucleotides in loop regions are more divergent. Base substitutions leading to derepression of the lacZ fusion at 30 degrees C exclusively resided in potential stem structures. Optimised base pairing by elimination of a bulged residue and by introduction of complementary nucleotides in internal loops resulted in ROSE elements that were tightly repressed not only at normal but also at heat shock temperatures. We propose a model in which the temperature-regulated secondary structure of ROSE mRNA influences heat shock gene expression by controlling ribosome access to the ribosome-binding site.

ROSE elements occur in disparate rhizobia and are functionally interchangeable between species.

Expression of at least ten genes in Bradyrhizobium japonicum, seven of which code for small heat shock proteins (sHsps), is under the control of ROSE (repression of heat shock gene expression). This negatively cis-acting DNA element confers temperature control to a sigma(70)-type promoter. Here, we show that ROSE elements are not restricted to B. japonicum but are also present in Bradyrhizobium sp. (Parasponia), Rhizobium sp. strain NGR234 and Mesorhizobium loti. An overall alignment of all ROSE sequences reveals a highly conserved and probably functionally important region towards the 3'-end of the element. Moreover, we provide genetic evidence for the previously proposed presence of multiple sHsps in these organisms. Primer-extension data of five newly identified ROSE-associated operons show that transcription is repressed at low temperatures and induced after a temperature upshift. Translational ROSE-hsp'-'lacZ fusions of Bradyrhizobium sp. (Parasponia) and Rhizobium sp. strain NGR234 integrated into the chromosome of B. japonicum were heat-responsive. The functionality of these heterologous ROSE elements hints at a common regulatory principle conserved in various rhizobia.

Multiple small heat shock proteins in rhizobia.

Seven genes coding for small heat shock proteins (sHsps) in Bradyrhizobium japonicum have been identified. They are organized in five operons that are coordinately regulated by ROSE, a negatively cis-acting DNA element. The deduced sHsps can be divided into two separate classes: class A, consisting of proteins that show similarity to Escherichia coli IbpA and IbpB, and class B, whose members display significant similarity to other sHsps from prokaryotes and eukaryotes. Two-dimensional gel electrophoresis and Edman sequencing revealed the presence of at least 12 sHsps in B. japonicum, indicating a remarkable abundance of sHsps in this organism. Three additional members of class A and two potentially novel heat shock proteins were identified on the basis of their amino termini. The presence of multiple sHsps was also demonstrated for a variety of Rhizobium and Bradyrhizobium species by immunoblot analysis and two-dimensional gel electrophoresis. An extensive database survey revealed that, in contrast to the rhizobia, other bacteria contain maximally two sHsps whereas many plants have been reported to possess a sHsp superfamily.

A novel DNA element that controls bacterial heat shock gene expression.

The hspArpoH1 and hspBCdegP heat shock operons of Bradyrhizobium japonicum are preceded by a novel, conserved DNA element of approximately 100 bp, which is responsible for the temperature-regulated transcription of their sigma70-type promoters. We designated this motif ROSE for repression of heat shock gene expression and found additional ROSE elements upstream of two newly identified heat shock operons. A critical core region in the hspA-associated ROSE1 was defined by introducing insertions or deletions. While four mutants retained the ability to repress transcription of the hspArpoH1 operon, five deletion mutants produced elevated hspA mRNA levels under low-temperature growth conditions. Derepression was confirmed by increased RpoH1 levels in non-heat-shocked cells from one of these mutants and by strains that contained a translational hspA-lacZ fusion associated with mutated ROSE1 elements. The hspArpoH1 operon was efficiently transcribed in vitro, and a deletion of ROSE1 did not impair this activity. Gel retardation experiments demonstrated that a protein in non-heat-shocked cells specifically binds to the intact ROSE1 element but not to a mutated element lacking the core region. Taken together, these results indicate that a central region of ROSE serves as a binding site for a repressor protein under standard growth conditions in order to prevent the undesired transcription of heat shock genes.