Review Article Defining Established and Emerging Microbial Risks in the Aquatic Environment: Current Knowledge, Implications, and Outlooks

This timely review primarily addresses important but presently undefined microbial risks to public health and to the natural environment. It specifically focuses on current knowledge, future outlooks and offers some potential alleviation strategies that may reduce or eliminate the risk of problematic microbes in their viable but nonculturable (VBNC) state and Cryptosporidium oocysts in the aquatic environment. As emphasis is placed on water quality, particularly surrounding efficacy of decontamination at the wastewater treatment plant level, this review also touches upon other related emerging issues, namely, the fate and potential ecotoxicological impact of untreated antibiotics and other pharmaceutically active compounds in water. Deciphering best published data has elucidated gaps between science and policy that will help stakeholders work towards the European Union's Water Framework Directive (2000/60/EC), which provides an ambitious legislative framework for water quality improvements within its region and seeks to restore all water bodies to "good ecological status" by 2015. Future effective risk-based assessment and management, post definition of the plethora of dynamic inter-related factors governing the occurrence, persistence and/or control of these presently undefined hazards in water will also demand exploiting and harnessing tangential advances in allied disciplines such as mathematical and computer modeling that will permit efficient data generation and transparent reporting to be undertaken by well-balanced consortia of stakeholders. Viable But Nonculturable Forms of Waterborne Bacteria 1.1. Background. Since the introduction of the concept or sublethally injured or viable but nonculturable (VBNC) cells by Byrd and Colwell in the 1980's [1], there is increasing evidence for the existence of such a state in microbes, particularly in the aquatic environment that elicits a myriad of interrelated sub-lethal microbial stresses such as nutrient starvation and osmotic stress Definition. According to Use of Fluorescent Redox and Other Enzymatic Probes. Direct visualization of actively respiring bacteria is gaining in popularity amongst research groups investigating this VBNC state International Journal of Microbiology 3 were more chlorine resistant than culturable bacteria. That said, a demonstration of active respiration does not necessarily infer that these stressed bacteria are capable of future growth. As numerous researchers continue to report on the use of redox stains for highlighting differences in agarplate counts, it is important that we holistically explore and identify specific microbial cues at the cellular level which govern the transition to the VBNC state along with exploiting commensurate advances in media formulations that are tailored for optimal resuscitation of these sublethally stressed cells (cited in VBNC State and Cell Suicide Phenomenon. Advancing the earlier pioneering work of Dodd et al. [26], who previously postulated that selfdestruction or "cell suicide" may be attributed to sublethally stressed or damage microbes being incapable of coping with oxidative burst when rapidly growing on nutrient rich media, it is important that we also exploit advances in toxicology (such as methods exploring cellular apoptosis and necrosis, cell membrane lipid peroxidation assays, and nuclear chromatin/comet assays) that will provide valuable insights into the possible role of intracellular free radicals in combination with the direct physical action of the applied environmental stress on the generation and persistence of VBNC organisms in water. Indeed, Servais and coworkers Molecular-Based Techniques for Rapid Detection and [15] who recently found that autoinducer-(AI-) 2 quorumsensing inhibitors affected starvation response and reduce virulence in several Vibrio species, most likely by interfering the signal transduction pathway at the level of LuxPQ. Risk Assessment and Predictive Modeling for Management of VBNC State Organisms in Water. The aforementioned detection methods are not as yet commonly used for routine measurement as they either are not simple enough and/or the equipment is too expensive. Therefore, in terms of enabling effective risk-based assessment and environmental management to address the occurrence and persistence of VBNC pathogens in water, critical data must be acquired to convert VBNC-phase potential pathogens from unknown to known and defined hazards. In terms of satisfying these information gaps, accurate enumeration of total microbial load and real time identification to the various types present in water are pivotal to hazard identification and characterization, which will contribute significantly to assuring water safety. Regarding the latter, Smeets et al. Use of Advanced Detection Techniques to Investigate Virulence Potential in VBNC State Organisms. Greater information is also required on elucidating the existence of commonly shared cellular mechanisms (and associated gene expression regulators and gene markers) that govern cellular conversion to this VBNC state. Moreover, there is a dearth of knowledge regarding specific underlying molecular and associated cellular mechanisms governing transition and persistence of waterborne microorganisms in this VBNC state, in addition to obviously establishing what specific environmental conditions or triggers cause these changes in culturable state. Greater studies are also required to investigate the presence and role of this VBNC state in microbial pathogens that cause disease in the aquatic natural setting such as in fish.Taking for example, Flavobacterium psychrophilum, the causative agent of rainbow trout fly syndrome and cold water disease in salmonids, where subpopulations were still culturable after starvation for 300 days in sterilised fresh water. The virulence of starved F. psychrophilum was maintained for at least 7 days after the transfer of the bacterial cells to fresh water Addressing Scientific Shortfalls in VBNC to Inform Policy. To comprehensively address such issues, detailed analysis of the proteome that arise during this morphological transition will enable identification and characterization of key proteins that are responsible for this VBNC state. In addition, tandem use of microarray gene analysis along with real-time quantitative PCR will help unravel specific gene functions and will pin-point over-arching regulatory framework(s). The aforementioned studies should also be carried out in parallel with developing improved protocols for resuscitating VBNC organisms, particularly availing of advances made in fermentation technology such as exploiting chemostat-based bioreactor approaches that can simulate and monitor multiple environmental stresses (either applied simultaneously or sequentially) over extended time periods. Indeed, Kooi and coworkers The Waterborne Enteroparasite: Cryptosporidium 2.1. Background. Cryptosporidium species have emerged over the past decades as major waterborne pathogens causing gastroenteritis in humans Alternative or Complementary Decontamination Methods. Development of alternative methods of Cryptosporidium disinfection for water applications (such as ozone and/or UV) has been hindered by the uncertainty surrounding efficacy of using in vitro surrogate viability assays due to their overestimation of oocysts survivors posttreatments and the lack of critical data on the preferred use of in vitro cell culture and/or in vivo animal-based infectivity assays to determine interrelated factors governing repeatable disinfection of oocysts suspended in water Development of PUV has recently received attention as a potentially novel strategy for decontaminating water as it offers many benefits including rapid microbial reductions and efficiency of energy usage due to underpinning high peak-power dissipation during treatments PUV is produced using techniques that multiply power manifold by storing electricity in a capacitor over relatively long times (fractions of a second) and releasing it in a short time (millionths or thousandths of a second) using sophisticated pulse compression techniques [69] recently reported that Cryptosporidium spp. oocysts could neither repair UV light-induced damage nor regain infectivity under standard conditions used for storage and distribution of treated drinking water. This is despite the fact that both C. parvum and C. hominis were shown to harbour genes encoding UV repair proteins for mechanisms such as nucleotide excision repair and photolyase enzymes Use of ozone is gaining in popularity as an alternative or complementary approach for disinfection in drinking water facilities worldwide Pulsed plasma gas-discharge (PPGD) technology involves applying high voltage pulses to gas-injected test liquids resulting in the formation of a plasma that causes free radicals such as dissolved ozone and hydrogen peroxide, free electrons, ultraviolet light (UV), acoustic shock waves, and electric fields at levels between 10-50 kV/cm to be generated in the test liquid