Cryptosporidium are ubiquitous and significant enteropathogens of all classes of vertebrates and a major cause of human morbidity and mortality worldwide. Of the 24 recognized species, the zoonotic Cryptosporidium parvum and the host-specific Cryptosporidium hominis cause the majority of cases of human cryptosporidiosis. Here, we report on structural and transcriptional variability between C. parvum and C. hominis at the MIC1 locus, which encodes a microneme localized thrombospondin-like domain containing protein previously demonstrated to be critical for host cell infection by C. parvum. We demonstrate, using reverse transcription quantitative PCR with the aid of genomic data from the EuPathDB site, that the transcribed product in C. hominis is both truncated and significantly down-regulated in the sporozoite. We hypothesize that CpMIC1 may be a genetic factor involved in facilitating the wider host range of C. parvum in comparison with the specific host range of C. hominis. Furthermore, we show that the presence of a microsatellite (ML-2) within the C. parvum MIC-1 locus enables the development of a PCR marker that can rapidly distinguish the zoonotic C. parvum from C. hominis and other significant human infectious Cryptosporidium species due to reproducible PCR slippage across the ML-2 microsatellite. Additionally, we demonstrate that this locus is tightly linked to the GP60 locus, a locus commonly used in the genetic characterization of C. parvum and C. hominis isolates. This marker should provide a robust and additional tool to aid in the rapid identification of C. parvum from other Cryptosporidium species.

SUMMARY Cryptosporidium parvum are protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring within Cryptosporidium sporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37°C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.

Aims: To determine the effect of solar radiation on Cryptosporidium parvum in tap and environmental waters.Methods and Results: Outdoor tank experiments and a cell culture infectivity assay were used to measure solar inactivation of C. parvum oocysts in different waters. Experiments conducted on days with different levels of solar insolation identified rapid inactivation of oocysts in tap water (up to 90% inactivation within the first hour). Increased dissolved organic carbon content in environmental waters decreased solar inactivation. The role of solar ultraviolet (UV) in inactivation was confirmed by long-pass filter experiments, where UV-B was identified as the most germicidal wavelength. Reductions in oocyst infectivity following solar radiation were not related to a loss of excystation capacity.Conclusions: Solar UV can rapidly inactivate C. parvum in environmental waters.Significance and Impact of the Study: This is the first study to assess natural sunlight inactivation of C. parvum oocysts in surface waters and drinking water using an infectivity measure and determines the wavelengths of light responsible for the inactivation. The findings presented here provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in aquatic environments and identify solar radiation as a critical process affecting the oocyst survival in the environment.

Aims: To profile the fractions of bacteria in heat- treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. Methods and Results: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene- directed polymerase chain reaction denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis ( 99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H-2 g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. Conclusion: Clostridium species in heat- treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. Significance and Impact of the Study: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.

Biological sand filters were assessed for their ability to remove geosmin, 2-methylisoborneol (MIB) and microcystin-LR. Microcystin-LR was the most readily degradable metabolite with a maximum lag period of only 5 days before it was undetected in the filter effluent. Geosmin and MIB were difficult to degrade, with a period in excess of 75 days before greater than 95% removal was achieved. A microcystin-degrading gene was detected in the biofilm from one of the filters, confirming that the biofilm possessed the ability to degrade microcystin. A Sphingomonas sp. was identified as a potential geosmin degrader based on denaturing gradient gel electrophoresis (DGGE) analysis. DGGE analysis revealed a more complex bacterial community during the degradation of MIB, suggesting that more than one bacterium may be responsible for its degradation.

Aims: To isolate and identify bacteria from a sand filter column capable of degrading the taste and odour compound, geosmin. In doing so, to investigate if these organisms degrade geosmin either individually or if an alternative mechanism is utilized.Methods: Geosmin-degrading bacteria from a biologically active sand filter column were enriched by their growth in a minimal medium supplemented with geosmin as the sole carbon source. By day 51, 21.7 mg l(-1) of geosmin had been degraded as determined by solid-phase microextraction gas chromatography/mass spectrometry, and was accompanied by a 2.12 log(10) increase in active bacterial numbers as measured using the BacLight(TM) bacterial viability kit and flow cytometric enumeration. During the onset of geosmin degradation, the predominance of three bacteria, most similar to previously cultured species of Sphingopyxis alaskensis, Novosphingobium stygiae and Pseudomonas veronii based on 16S rRNA gene sequences was detected by denaturing gradient gel electrophoresis. Subsequent isolation of these organisms revealed that degradation of geosmin, when present as either the sole carbon source (ranging from 40 ng l(-1) to 20 mg l(-1)) or when spiked into sterile reservoir water (37 and 131 ng l(-1)), occurred only when all three isolates were present. None of the isolates was shown to be capable of degrading geosmin either individually or in any combination of two.Conclusions: This study has reported, for the first time, the cooperative degradation of geosmin by a consortium comprising three gram-negative bacteria isolated from a biologically active sand filter column.Significance and Impact of the Study: These results are important for researchers currently employing molecular-based approaches to further understand the biodegradation of geosmin by bacteria, as such studies may be complicated by the discovery of geosmin degradation occurring by a consortium. This study also advances the knowledge surrounding the types of bacteria capable of degrading the taste and odour compound, as investigations to date regarding this are limited.

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 x 10(5) bacteria ml(-1). The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 x 10(3) active AOB ml(-1) were detected in the membrane-intact fraction, and 1.40 x 10(4) active AOB ml(-1) were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml(-1) detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.

Aims: To profile fractions of active bacteria and of bacteria culturable with routine heterotrophic plate count (HPC) methods through a typical water treatment process and subsequent distribution system. In doing so, investigate how water treatment affects both bacterial abundance and diversity, and reveal the identities of active bacteria not detected by traditional HPC culture.Methods and Results: Profiling active fractions was performed by flow cytometric cell sorting of either membrane- intact (BacLight (TM) kit) or enzymatically active ( carboxyfluorescein diacetate, CFDA) bacteria, followed by eubacterial 16S rDNA-directed PCR and denaturing gradient gel electrophoresis (DGGE). Water treatment significantly reduced active bacterial numbers detected by the BacLight (TM) kit and CFDA assay by 2.89 and 2.81 log respectively. Bacterial diversity was also reduced from > 20 DGGE bands in the active fractions of reservoir water to only two bands in the active fractions of finished water. These two bands represented Stenotrophomonas maltophila, initially culturable by HPC, and a Burkholderia-related species. Both species maintained measurable traits of physiological activity in distribution system bulk water but were undetected by HPC.Conclusions: Flow cytometric cell sorting with PCR-DGGE, to assess water treatment efficacy, identified active bacteria from a variety of major phylogenetic groups undetected by routine HPC. Following treatment S. maltophila and a Burkholderia-related species retained activity and entered distribution undetected by HPC.Significance and Impact of the Study: Methods used here demonstrate how water treatment operators can better monitor water treatment plant efficacy and assess distribution system instability by the detection and identification of active bacteria recalcitrant to routine HPC culture.

Maintaining optimal conditions in catchments or distribution systems relies heavily on water authorities having access to rapid and accurate water quality data, including an indication of bacteriological quality. In this study, the BacLight bacterial viability kit and carboxyfluorescein diacetate (CFDA) were coupled with flow cytometry (FCM) for rapid detection of physiologically active bacteria from raw and potable waters taken from various locations around South Australia. Results were compared to the direct viable count (DVC) and quantitative DVC (qDVC), in addition to the culture-based methods of the heterotrophic plate count (HPC) and a commercial SimPlate technique. Raw and potable water analysis revealed that DVC and culture-based techniques reported significantly fewer viable bacteria compared to the number of physiologically active bacteria detected using the rapid FCM assays, where this difference appeared to be nonlinear across different samples. Inconclusive results were obtained using qDVC as a viability assay. In particular, HPC results were 2-4 log orders of magnitude below that reported by the FCM assays for raw waters. Few bacteria in potable waters examined were culturable by HPC, even though FCM assays reported between 5.56 x 10(2) and 3.94 x 10(4) active bacteria ml(-1). These differences may be attributed to the presence of nonheterotrophic bacteria, sublethal injury or the adoption of an active but nonculturable (ABNC) state.

Staining bacteria with esterified fluorogenic substrates followed by flow cytometric analysis offers a means for rapid detection of metabolically active bacteria. Flow cytometry (FCM) was used to assess carboxyfluorescein diacetate (CFDA) and carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) as indicators of bacterial activity for cultured bacteria, including Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and bacteria from environmental waters. In theory, CFDA/SE should be a better indicator of metabolic bacterial activity compared to CFDA due to greater intracellular retention of the fluorescent product. Qualitative and quantitative analysis of exponential phase cultures, mixtures of active and inactive cells and bacteria from environmental waters revealed CFDA was successful in detecting active bacteria, whereas CFDA/SE was not. CFDA/SE labelled inactive cells with intensities equal to that of the active population and could not even discriminate between bacteria in exponential phase growth and a fixed cell preparation. We propose that the specific mode of action of the succinimidyl ester (SE) group in combination with the nonenzymatic aqueous hydrolysis of the CFDA moiety results in the nonspecific labelling of all cells, irrespective of their metabolic state. This study shows that CFDA/SE is a poor marker of bacterial activity.