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Nucleic acid-based analytical methods, ranging from species-targeted PCRs to metagenomics, have greatly expanded our understanding of microbiological diversity in natural samples. However, these methods provide only limited information on the activities and physiological states of microorganisms in samples. Even the most fundamental physiological state, viability, cannot be assessed cross-sectionally by standard DNA-targeted methods such as PCR. New PCR-based strategies, collectively called molecular viability analyses, have been developed that differentiate nucleic acids associated with viable cells from those associated with inactivated cells. In order to maximize the utility of these methods and to correctly interpret results, it is necessary to consider the physiological diversity of life and death in the microbial world. This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology.Yet it hath happened that the veritable body without the spirit hath walked.-Ambrose Bierce, The Death of Halpin Frayser M icrobiologists, like characters in zombie fiction, quickly learn the critical importance of distinguishing the living from the dead. In addition to characterizing the numbers and species of microorganisms in samples, it is important to collect data on their physiological states. The most fundamental physiological state of microbial cells is their viability, defined here as the capacity to form progeny. For ecologists, pathobiologists, metagenomicists, food or water safety analysts, infectious-disease clinicians, and virtually every other stripe of microbiologist, the observation of a viable microorganism in a sample means something entirely different from the observation of a dead one. Despite its importance, this distinction remains extremely challenging by current microbiological methods.Microbiological culture meets this requirement, as it selectively detects viable organisms. However, because only a small percentage of species can be cultured, this strategy underestimates microbial diversity (1-6). In contrast, nucleic acid-based methods, ranging from species-specific PCR to metagenomic methods, have greatly advanced our ability to detect diverse microorganisms independently of microbiological culture (7,8). However, these methods provide only limited information on the physiology of microorganisms in samples. They can assess microbial viability retrospectively, by measuring quantitative changes over time, but they cannot discern viability cross-sectionally (in single measurements). Traditional PCR is notoriously poor at differentiating DNA associated with a viable bacterial cell from DNA associated with an inactivated one or from a free DNA fragment. All of these analytes register as "hits" in PCR, despite their very distinct meanings.In order to address this limitation, alternative PCR-based strategies have been developed. This article reviews two complementary strategies. One strategy, term...

Innovative research relating oceans and human health is advancing our understanding of diseasecausing organisms in coastal ecosystems. Novel techniques are elucidating the loading, transport and fate of pathogens in coastal ecosystems, and identifying sources of contamination. This research is facilitating improved risk assessments for seafood consumers and those who use the oceans for recreation. A number of challenges still remain and define future directions of research and public policy. Sample processing and molecular detection techniques need to be advanced to allow rapid and specific identification of microbes of public health concern from complex environmental samples. Water quality standards need to be updated to more accurately reflect health risks and to provide managers with improved tools for decision-making. Greater discrimination of virulent versus harmless microbes is needed to identify environmental reservoirs of pathogens and factors leading to human infections. Investigations must include examination of microbial community dynamics that may be important from a human health perspective. Further research is needed to evaluate the ecology of non-enteric water-transmitted diseases. Sentinels should also be established and monitored, providing early warning of dangers to ecosystem health. Taken together, this effort will provide more reliable information about public health risks associated with beaches and seafood consumption, and how human activities can affect their exposure to diseasecausing organisms from the oceans.

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This is the first report of MRSA and MRCoNS isolated from marine water and intertidal beach sand. The MLST types and antibiotic carriage of five MRSA isolates were similar to hospital MRSA isolates rather than US community-acquired MRSA isolates. Our results suggest that public marine beaches may be a reservoir for transmission of MRSA to beach visitors as well as an ecosystem for exchange of antibiotic resistance genes among staphylococci and related genera.

Ratiometric pre-rRNA analysis (RPA) detects the replenishment of rRNA precursors that occurs rapidly upon nutritional stimulation of bacterial cells. In contrast to DNA detection by PCR, RPA distinguishes viable from inactivated bacteria. It exhibits promise as a molecular viability test for pathogens in water and other environmental samples.As a tool for detecting bacteria in environmental samples, the PCR is limited in part by the false-positive detection of nonviable bacteria and free DNA. One solution to this problem involves treating bacteria with DNA intercalators that penetrate inactivated cells and inhibit PCR amplification (15). These methods require careful titration, and performance varies with sample and disinfection conditions (8,16). An alternative is to detect microbial RNA, which is less stable than DNA in the environment (10-12, 14, 17-19, 23). However, mRNA can be difficult to detect, while mature rRNA is stable within inactivated cells.Microbial rRNA precursors (pre-rRNA) comprise an alternative RNA target (2-4, 11, 17, 23). Pre-rRNAs have leader and tail sequences that are enzymatically removed during rRNA maturation. Pre-rRNA sequences are phylogenetically specific, which facilitates their detection in complex samples. In growing bacterial cells, pre-rRNAs constitute a significant fraction of the total rRNA and are much easier to detect than mRNA. Upon cessation of growth, pre-rRNA synthesis ceases but maturation continues, draining pre-rRNA pools. PrerRNA has been used as a steady-state indicator of bacterial physiology (2, 11); however, this strategy is compromised by the complex interplay of pre-rRNA synthesis and processing (2).The present study exploited the replenishment of pre-rRNA that occurs immediately upon the nutritional stimulation of nutrient-limited bacterial cells. Species-specific pre-rRNA was measured in samples after brief exposure to culture medium. Values that exceeded those seen in nonstimulated control samples indicated the presence of viable cells. This ratiometric pre-rRNA analysis (RPA) approach was tested on the rapidly growing opportunistic pathogen Aeromonas hydrophila (generation time [g] ϭ 1 h) and the slowly growing actinomycete Mycobacterium avium (g ϭ 20 h).For both organisms, real-time quantitative PCRs (RTqPCRs) targeted the 5Ј pre-rRNA leader region. Primers were designed to straddle the 5Ј mature rRNA terminus. Primers for cDNA synthesis and reverse PCR primers recognized semiconserved sequences within the mature rRNA. Forward primers recognized predicted species-specific sequences within the 5Ј leader. Therefore, amplification required intact specific prerRNA as templates (see Table S1 in the supplemental material).Primers targeted to the M. avium complex (MAC) consistently yielded the expected amplification products when applied to 19 genotypically diverse isolates of M. avium subsp. hominissuis and M. intracellulare (1, 9), the two most significant human pathogens within the MAC. Nucleic acids from M. terrae, M. gastri, M. smegmatis, M. nonchromogenic...

The application of quantitative microbial risk assessments (QMRAs) to understand and mitigate risks associated with norovirus is increasingly common as there is a high frequency of outbreaks worldwide. A key component of QMRA is the dose-response analysis, which is the mathematical characterization of the association between dose and outcome. For Norovirus, multiple dose-response models are available that assume either a disaggregated or an aggregated intake dose. This work reviewed the dose-response models currently used in QMRA, and compared predicted risks from waterborne exposures (recreational and drinking) using all available dose-response models. The results found that the majority of published QMRAs of norovirus use the F hypergeometric dose-response model with α = 0.04, β = 0.055. This dose-response model predicted relatively high risk estimates compared to other dose-response models for doses in the range of 1-1,000 genomic equivalent copies. The difference in predicted risk among dose-response models was largest for small doses, which has implications for drinking water QMRAs where the concentration of norovirus is low. Based on the review, a set of best practices was proposed to encourage the careful consideration and reporting of important assumptions in the selection and use of dose-response models in QMRA of norovirus. Finally, in the absence of one best norovirus dose-response model, multiple models should be used to provide a range of predicted outcomes for probability of infection.

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