Use of metagenomic microbial source tracking to investigate the source of a foodborne outbreak of cryptosporidiosis.

Cryptosporidium is a protozoan parasite of global public health importance that causes gastroenteritis in a variety of vertebrate hosts, with many human outbreaks reported yearly, often from ingestion of contaminated water or food. Despite the major public health implications, little is typically known about sources of contamination of disease outbreaks caused by Cryptosporidium. Here, we study a national foodborne outbreak resulted from infection with Cryptosporidium parvum via romaine lettuce, with the main goal to trace the source of the parasite. To do so, we combined traditional outbreak investigation methods with molecular detection and characterization methods (i.e. PCR based typing, amplicon and shotgun sequencing) of romaine lettuce samples collected at the same farm from which the contaminated food was produced. Using 18S rRNA typing, we detected C. parvum in two out of three lettuce samples, which was supported by detections in the metagenome analysis. Microbial source tracking analysis of the lettuce samples suggested sewage water as a likely source of the contamination, albeit with some uncertainty. In addition, the high degree of overlap in bacterial species content with a public human gut microbial database corroborated the source tracking results. The combination of traditional and molecular based methods applied here is a promising tool for future source tracking investigations of food- and waterborne outbreaks of Cryptosporidium spp. and can help to control and mitigate contamination risks.

The impact of temperature on the inactivation of enteric viruses in food and water: a review.

Temperature is considered as the major factor determining virus inactivation in the environment. Food industries, therefore, widely apply temperature as virus inactivating parameter. This review encompasses an overview of viral inactivation and virus genome degradation data from published literature as well as a statistical analysis and the development of empirical formulae to predict virus inactivation. A total of 658 data (time to obtain a first log(10) reduction) were collected from 76 published studies with 563 data on virus infectivity and 95 data on genome degradation. Linear model fitting was applied to analyse the effects of temperature, virus species, detection method (cell culture or molecular methods), matrix (simple or complex) and temperature category (<50 and ≥50°C). As expected, virus inactivation was found to be faster at temperatures ≥50°C than at temperatures <50°C, but there was also a significant temperature-matrix effect. Virus inactivation appeared to occur faster in complex than in simple matrices. In general, bacteriophages PRD1 and PhiX174 appeared to be highly persistent whatever the matrix or the temperature, which makes them useful indicators for virus inactivation studies. The virus genome was shown to be more resistant than infectious virus. Simple empirical formulas were developed that can be used to predict virus inactivation and genome degradation for untested temperatures, time points or even virus strains.

Growth potential of faecal bacteria in simulated psychrophilic/mesophilic zones during composting of organic waste.

AIM: This study investigated the growth potential of Salmonella serotype Typhimurium and faecal indicator organisms in compost materials and the correlation between bacterial growth potential and the physico-chemical composition of the compost substrate and temperature. METHODS AND RESULTS: Survival of Salm. Typhimurium, Enterococcus spp. and total coliforms at 14, 24 and 37 degrees C was determined in material of different degrees of maturity collected from composting plants for household waste and manure. All three micro-organisms showed the potential for growth in the material from active composts (Solvita index 4) but inactivation generally occurred over time in mature compost material (Solvita index 7-8). CONCLUSIONS: Salm. Typhimurium had the potential for growth in psychrophilic/mesophilic (P/M) zones of immature compost material and its growth potential correlated negatively with the maturity of the compost and the temperature within the simulated P/M zone. SIGNIFICANCE AND IMPACT OF THE STUDY: The risk of pathogen regrowth in P/M zones during organic waste composting further emphasizes the importance of good management practices and of avoiding P/M zones in combination with low compost maturity.

Sanitation of faeces from source-separating dry toilets using urea.

AIM: To develop a reliable and simple method to produce safe fertilizers from human excreta using urea for sanitation of faeces. METHODS AND RESULTS: Urea was added to faecal matter (17% dry matter) at concentrations of 0.5-2% (w/w) and inactivation of Salmonella enterica subspecies 1 serovar Typhimurium (Salm. Typhimurium), Enterococcus spp. and the Salm. Typhimurium bacteriophage 28B was monitored at 14, 24 and 34 degrees C. Urea additions enhanced inactivation and inactivation rates were positively related to increasing NH(3) (aq) concentration and temperature. Salm. Typhimurium was the most sensitive of the organisms studied, while Enterococcus spp. showed more persistence, especially at lower temperatures. The bacteriophage was the most resistant organism studied. CONCLUSIONS: Salmonella reduction levels that meet requirements for safe reuse of faeces as fertilizer (i.e. 6 log(10) reduction) can be achieved for 1% urea within 2 months at 14 degrees C or within 1 week at 24 degrees C and 34 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The relationships between organism inactivation rates and temperature, ammonia and pH were identified. Urea treatment proved to be a robust and efficient option for safe recycling of plant nutrients.

In situ ammonia production as a sanitation agent during anaerobic digestion at mesophilic temperature.

AIM: To measure the sanitizing effect of mesophilic (37 degrees C) anaerobic digestion in high ammonia concentrations produced in situ. METHODS AND RESULTS: Indicator organisms and salmonella were transferred to small-scale anaerobic batch cultures and D-values were calculated. Batch cultures were started with material from two biogas processes operating at high (46 mmol l(-1)) and low (1.6 mmol l(-1)) ammonia concentration. D-values were shortened from c. 3 days to <1 day for the bacteria. MS2 had the same D-value (1.3 days) independent of ammonia concentration whereas PhiX174 and 28B were faster inactivated in the control (1.1 and 7.9 days) than in the high ammonia (8.9 and 39 days) batch cultures. CONCLUSION: Running biogas processes at high levels of ammonia shortens the time to meet EU regulation concerning reduction of salmonella and enterococci (5 log). Unless a minimum retention time of 2 days, post-treatment digestion is needed to achieve sufficient sanitation in continuous biogas processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Running mesophilic biogas processes at high ammonia level produces residue with a high fertilizer value. With some stipulations concerning management parameters, such processes provide a method of bacterial sanitation without preceding pasteurization of the incoming organic waste.

Salmonella reduction in manure by the addition of urea and ammonia.

Animal manure may contain pathogenic microorganisms and disinfection is suggested to avoid reinfection of animal herds and introduction of zoonotic disease into the food chain. Ammonia and urea were tested for disinfection of bovine manure and Salmonella was found to be rapidly eliminated by the addition of 0.5% aqueous ammonia or 2% w/w urea (s). Treatments (2% urea and 0.5% ammonia), temperature (4 degrees C or 14 degrees C) and combinations of these factors significantly affected the inactivation rate. Decimal values (T(90)) were reduced from 8.3 days in the control to 2.0 days and 0.4 days at 14 degrees C after the addition of urea and ammonia, respectively. At 4 degrees C, the decimal values were reduced from 34 to 4.8 and 1.1 days, respectively. Recommended treatments of bovine manure based on Monte Carlo simulations are 0.5% ammonia followed by storage for one week or 2% urea followed by storage for two weeks at 14 degrees C, one month at 4 degrees C. Storage without additives should include at least one summer in temperate regions. Enterococci were evaluated as indicators for Salmonella but significantly slower decay rate and different behaviour in the material made them unsuitable as indicators for Salmonella in manure disinfected by ammonia or urea. Free ammonia treatment of Salmonella-contaminated manure disinfects the material and raises its fertilizer value.

Removal of noro- and enteroviruses, Giardia cysts, Cryptosporidium oocysts, and fecal indicators at four secondary wastewater treatment plants in Sweden.

The aim of this study was to investigate variation in the occurrence and removal of enteroviruses, noroviruses, Giardia cysts, Cryptosporidium oocysts, and the most commonly used fecal indicators in four Swedish secondary wastewater treatment plants (WWTPs). Paired samples were taken from the inlet and outlet of each WWTP. (Oo)cysts and indicators were enumerated with standard methods and viruses with a reverse transcriptase polymerase chain reaction. Giardia cysts and enteroviruses were constantly detected (mean numbers were 10(3.31) cysts and 10(4.44) polymerase chain reaction (PCR) units L(-1), respectively). Oocysts were found in 5 out of 19 samples (mean number was 20 L(-1)). Noroviruses were found between November and February, with an average titer of 10(3.29( L(-1). Mean cyst removal was 2.6 log, while noroviruses and enteroviruses were removed by 0.9 and 1.3 log, respectively. There was no correlation between the removals of pathogens and indicators (p > 0.05). Coliphage removal resembled human viral removal better than did F-specific phage.

Removal of viruses, parasitic protozoa and microbial indicators in conventional and membrane processes in a wastewater pilot plant.

The aim of this study was to investigate variations in the occurrence and removal of enterovirus and norovirus genomes, Giardia cysts, Cryptosporidium oocysts and the most commonly used faecal indicators in a Swedish wastewater pilot plant. Paired samples were taken from the inlet and outlet of each treatment line: tertiary filtration, membrane bioreactor (MBR) and upflow anaerobic sludge blankets (UASB). (Oo)cysts and indicators were enumerated using standard methods and viruses using RT-PCR. Giardia cysts and enteroviruses were constantly detected, mean numbers 10(3.11) cysts and 10(4.0) PCR units L(-1), respectively. Oocysts were found in 5/19 samples, mean number 5 L(-1). Noroviruses were found in 6/7 influent samples, with an average titre of 10(3.28)L(-1), during winter, but only in 2/15 in the rest of the year (mean 200 L(-1)). MBR treatment removed indicators more efficiently than did the other two lines, with 5log removal of E. coli. Human virus genome removal did not differ between the MBR and tertiary treatment line. Microorganism removal in UASB was significantly lower for all the organisms studied. E. coli, enterococci and Cl. perfringens removal was correlated (p<0.05) with enterovirus genome removal, with R-values around 0.4. However, values for removal of indicators were more strongly correlated to each other. Removal of viruses based on enumeration using RT-PCR probably underestimates infectious virion removal.

Removal of micro-organisms in a small-scale hydroponics wastewater treatment system.

AIMS: To measure the microbial removal capacity of a small-scale hydroponics wastewater treatment plant. METHODS AND RESULTS: Paired samples were taken from untreated, partly-treated and treated wastewater and analysed for faecal microbial indicators, i.e. coliforms, Escherichia coli, enterococci, Clostridium perfringens spores and somatic coliphages, by culture based methods. Escherichia coli was never detected in effluent water after >5.8-log removal. Enterococci, coliforms, spores and coliphages were removed by 4.5, 4.1, 2.3 and 2.5 log respectively. Most of the removal (60-87%) took place in the latter part of the system because of settling, normal inactivation (retention time 12.7 d) and sand filtration. Time-dependent log-linear removal was shown for spores (k = -0.17 log d(-1), r(2) = 0.99). CONCLUSIONS: Hydroponics wastewater treatment removed micro-organisms satisfactorily. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigations on the microbial removal capacity of hydroponics have only been performed for bacterial indicators. In this study it has been shown that virus and (oo)cyst process indicators were removed and that hydroponics can be an alternative to conventional wastewater treatment.

Contextual influences on learning application in practice. An extended role for process evaluation.

Although process evaluation usually ends with the program, process itself does not end. This study analyzed process or contextual influences health professionals face when returning from continuing professional education (CPE) and attempting to apply learning. Follow-up survey data were analyzed for a sample (N = 549) of physicians, nurses, counselors, and rehabilitation specialists. Contextual influences studied include resources, encouragement, support, opportunity, and authority. Logistic regression analysis found support for changes to apply training ideas was a predictor of application for the sample as a whole and for all professional groups except one. Encouragement from others was a predictor of application for rehabilitation specialists, and opportunity to apply learning was a second predictor for nurses. Sufficient resources was not a predictor in any models tested. Results indicate that the practice context can affect educational outcomes and an extended vision of process evaluation is needed to incorporate such variables in the evaluation of CPE.

Relation of colloid concentration to volume expansion for resuscitation of rats subjected to intestinal ischemic shock.

This study was undertaken to determine the volumes of colloids (albumin and dextran 40) with concentrations varying from 0% (Ringer's solution) to 10% solutions required to obtain the same hemodilution. Shock was produced in rats by exteriorization of the small intestine with added complete occlusion of the superior mesenteric vessels with a rubber clamp. After 1 h the clamp was removed, the intestines returned, and the abdomen closed. Over a 6-h period, the fluids were infused continuously in volumes needed to maintain similar hematocrit. Each concentration was given over a wide range of volumes in order to obtain volume-hematocrit curves. Based on these curves, the effect of varying colloid concentration on blood volume expansion was calculated. Albumin and dextran 40 had similar volume expansion/g colloid substance regardless of concentration. When hematocrit of 50% was chosen, as an arbitrary endpoint for volume infusion, the colloid concentration in relation to volume required to be infused is expressed by the equation F50 (formula: see text) where C = colloid concentration and where F50 is the volume factor, or the number of times the volume required for a 10% solution, when a 10% solution is given the volume factor of 1. This relationship is now being used to find the optimal colloid concentration with regard to survival rate in animals subjected to the same intestinal shock.