Before new, rapid quantitative PCR (qPCR) methods for assessment of recreational water quality and microbial source tracking (MST) can be useful in a regulatory context, an understanding of the ability of the method to detect a DNA target (marker) when the contaminant source has been diluted in environmental waters is needed. This study determined the limits of detection and quantification of the human-associated Bacteroides sp. (HF183) and human polyomavirus (HPyV) qPCR methods for sewage diluted in buffer and in five ambient, Florida water types (estuarine, marine, tannic, lake, and river). HF183 was quantifiable in sewage diluted up to 10 ؊6 in 500-ml ambient-water samples, but HPyVs were not quantifiable in dilutions of >10 ؊4 . Specificity, which was assessed using fecal composites from dogs, birds, and cattle, was 100% for HPyVs and 81% for HF183. Quantitative microbial risk assessment (QMRA) estimated the possible norovirus levels in sewage and the human health risk at various sewage dilutions. When juxtaposed with the MST marker detection limits, the QMRA analysis revealed that HF183 was detectable when the modeled risk of gastrointestinal (GI) illness was at or below the benchmark of 10 illnesses per 1,000 exposures, but the HPyV method was generally not sensitive enough to detect potential health risks at the 0.01 threshold for frequency of illness. The tradeoff between sensitivity and specificity in the MST methods indicates that HF183 data should be interpreted judiciously, preferably in conjunction with a more host-specific marker, and that better methods of concentrating HPyVs from environmental waters are needed if this method is to be useful in a watershed management or monitoring context. Fecal indicator bacteria (FIB), including fecal coliforms, Escherichia coli, and enterococci, have been approved indicators of sewage contamination in recreational waters for decades (49, 51). Swimmers exposed to waters contaminated with sewage are at a greater risk of infection by human pathogens, and subsequent gastrointestinal or other illnesses, than those exposed to unimpacted waters (57). The use of FIB to detect sewage contamination, however, relies on the assumptions that elevated FIB concentrations are representative of pathogen presence and that the fate of these bacteria mimics that of pathogens. Unfortunately, previous studies have shown that concentrations of FIB do not necessarily correlate well with bacterial, protozoan, and viral pathogens (2,20).To address the limitations of FIB, microbial source tracking (MST) methods have been developed to identify human fecal contamination (e.g., sewage) in recreational waters (7, 32). These methods are culture independent and are therefore more rapid than FIB methodologies that require a culture step. The potential for same-day results provides the possibility of advisories that reflect current conditions, rather than day-old ones, which allows more-immediate action to protect against public health risks posed by recreational water use (12, 56). The selecti...

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n ‫؍‬ 35) or clinical (n ‫؍‬ 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical "clinical" or "environmental" polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the "environmental" profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical "clinical" profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the "clinical" polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.Vibrio vulnificus is associated with serious wound infections or frequently fatal (mortality rates are generally Ͼ50%) septicemia related to consumption of raw shellfish, particularly oysters (5, 37). The bacterium is indigenous to temperate estuaries, and prevalence in oysters and seawater approaches 100% during warmer months (25,45). Most strains isolated from environmental reservoirs appear to be as virulent as clinical strains in animal models (13,35,36,39). Also, multiple virulence factors have been proposed for V. vulnificus but are generally present in most strains and do not provide predictive value (16,36,39,50). Virulent strains are distinguished by opaque colony morphology (34, 49), which reflects expression of a protective capsular polysaccharide (CPS); however, both clinical and environmental strains are generally encapsulated (45). Thus, appropriate markers to screen the virulence potential of V. vulnificus in environmental reservoirs are not available.Recently, DNA sequence polymorphisms at individual loci discriminated isolates from clinical versus oyster origin in several independent studies. Polymorphic variants generally included two genotypes, such as types ...

BackgroundMetabolic syndrome (MetS) is a highly prevalent condition that identifies individuals at risk for type 2 diabetes mellitus and atherosclerotic cardiovascular disease. Prevention of these diseases relies on early detection and intervention in order to preserve pancreatic β-cells and arterial wall integrity. Yet, the clinical criteria for MetS are insensitive to the early-stage insulin resistance, inflammation, cholesterol and clotting factor abnormalities that characterize the progression toward type 2 diabetes and atherosclerosis. Here we report the discovery and initial characterization of an atypical new biomarker that detects these early conditions with just one measurement.MethodsWater T2, measured in a few minutes using benchtop nuclear magnetic resonance relaxometry, is exquisitely sensitive to metabolic shifts in the blood proteome. In an observational cross-sectional study of 72 non-diabetic human subjects, the association of plasma and serum water T2 values with over 130 blood biomarkers was analyzed using bivariate, multivariate and logistic regression.ResultsPlasma and serum water T2 exhibited strong bivariate correlations with markers of insulin, lipids, inflammation, coagulation and electrolyte balance. After correcting for confounders, low water T2 values were independently and additively associated with fasting hyperinsulinemia, dyslipidemia and subclinical inflammation. Plasma water T2 exhibited 100% sensitivity and 87% specificity for detecting early insulin resistance in normoglycemic subjects, as defined by the McAuley Index. Sixteen normoglycemic subjects with early metabolic abnormalities (22% of the study population) were identified by low water T2 values. Thirteen of the 16 did not meet the harmonized clinical criteria for metabolic syndrome and would have been missed by conventional screening for diabetes risk. Low water T2 values were associated with increases in the mean concentrations of 6 of the 16 most abundant acute phase proteins and lipoproteins in plasma.ConclusionsWater T2 detects a constellation of early abnormalities associated with metabolic syndrome, providing a global view of an individual’s metabolic health. It circumvents the pitfalls associated with fasting glucose and hemoglobin A1c and the limitations of the current clinical criteria for metabolic syndrome. Water T2 shows promise as an early, global and practical screening tool for the identification of individuals at risk for diabetes and atherosclerosis.Electronic supplementary materialThe online version of this article (10.1186/s12967-017-1359-5) contains supplementary material, which is available to authorized users.

Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 10 2 to 10 3 V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R 2 ‫؍‬ >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n ‫؍‬ 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n ‫؍‬ 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics.Vibrio vulnificus is a gram-negative bacterium that is autochthonous to warm estuarine waters and is frequently isolated from shellfish harvested in the Gulf of Mexico. V. vulnificus infections have been noted as the leading cause of food-related mortality in Florida (12). In the United States, nearly all foodborne infections result from the consumption of oysters collected from the Gulf of Mexico (5). Immunocompromised individuals and those with diseases causing increased iron levels in the body, such as liver disease or hemochromatosis, are at relatively high risk for development of primary septicemia, with a mortality rate of around 50% (6). There is also a risk of acute gastroenteritis due to the consumption of raw or undercooked seafood such as oysters (9). Infections can also occur due to trauma associated with handling contaminated seafood or the contact of open wounds with water containing V. vulnificus. Wound infections can become fatal or so severe that amputation is necessary to stop the spread of infection (17). Although the health of the host is a factor, it is not an absolute determinant of infection or clinical outcome (26).Regulations for water quality in shellfish-harvesting areas in Florida rely on testing for indicator organisms, i.e., fecal coliforms (http://www.floridaaquaculture.com/SEAS/SEAS_intro .htm). For the most part, indicator bacteria have not been shown to correlate with the presence of...

Aims:  To determine the occurrence of the human pathogen, Vibrio vulnificus, in south Texas coastal waters. Methods and Results:  Coastal waters were sampled monthly between August 2006 and July 2007. Water temperature, dissolved oxygen, pH, salinity, conductivity and turbidity were measured during each sampling event. Culture‐based techniques utilizing Vibrio vulnificus agar (VVA) and membrane‐Enterococcus indoxyl‐β‐d‐glucoside agar (mEI) were used to assess the occurrence and levels of V. vulnificus and the faecal contamination indicator group, enterococci, respectively. Vibrio vulnificus isolates were confirmed using colony‐blot hybridization with the species‐specific VVAP probe. Vibrio vulnificus was isolated at all sites throughout the year even when the water temperature dropped to 9·71°C. Significant correlations were found between concentrations of V. vulnificus and the abiotic factors, water temperature (P = 0·002) and dissolved oxygen (P = 0·028), as well as between concentrations of V. vulnificus and enterococci (P < 0·001). Conclusions:  This study demonstrated the year‐round presence of V. vulnificus in coastal waters of south Texas. Significance and Impact of the Study:  These findings indicate that the potential for human exposure to the pathogen, V. vulnificus, exists throughout the year. It also suggests that routinely monitored data might be used to predict the occurrence of the pathogen.