Quantitative Microbial Risk Assessment of North Carolina Type 2 Reclaimed Water for Agricultural Reuse

As treated wastewater is increasingly used for agricultural purposes; questions remain about the microbiological quality of produce irrigated by these waters. This study conducted a quantitative microbial risk assessment (QMRA) using microbial data collected from North Carolina Type 2 reclaimed waters, which have been proposed as supplemental irrigation waters. Reclaimed waters were collected from four different water reclamation facilities located in central North Carolina and evaluated for five representative pathogens from the three groups of microorganisms (bacteria, virus, and protozoan parasites). Using these data, produce consumption scenarios were evaluated using a variety of irrigation techniques, including spray irrigation, drip irrigation, and subsurface drip irrigation, and the disability adjusted life years (DALYs) that result from illness by each pathogen as a result of produce consumption were compared to the acceptable level set by the World Health Organization. Based on the types of crop irrigation examined in this study using NC Type 2 reclaimed water, there were irrigation conditions and certain pathogens for which the annual risk of infection was not always reduced below the acceptable DALY risk level of <1 × 10−6 set by the WHO. The risks of viral infection by adenoviruses groups A–F were below the acceptable risk level; however, for Salmonella spp., Cryptosporidium, and Giardia, the annual risk of infection was sometimes greater than would be considered acceptable

Optimisation of methods for the collection and detection of bacterial pathogens from diarrhoeal human faecal samples using a novel stool collection kit

Bacterial pathogens such as Escherichia coli, Salmonella-, Shigella- and Vibrio species are known to be common causative agents for diarrhoeal disease in humans. This study aimed to develop a culture-independent PCR assay for the detection of bacterial pathogens present in human faecal samples collected using a less intrusive faecal collection technique, the Bio-wipe kit. A multiplex-PCR (m-PCR) was optimised targeting the E. coli mdh gene, the Salmonella IpaB gene, the Vibrio sodB gene, and the Ial and IpaH genes present in entero-invasive E. coli and Shigella spp. The influence of the DNA extraction method, and sensitivity and specificity of the m-PCR and the Bio-wipe storage conditions on the detection of the bacterial pathogens was investigated. A guanidium thiocyanate DNA extraction method used with laboratory-prepared spin columns could successfully extract DNA from 93% of the samples analysed. The m-PCR could successfully identify and differentiate between the various pathogens tested and was specific for the selected pathogens. Faecal matter was successfully recovered from used Bio-wipes and the bacterial DNA could be detected from these samples at concentrations of 10 cfu. Bacterial DNA could be recovered from the Bio-wipes 5 to 10 d after use when the Bio-wipes were stored at 30°C and 14 d after usage when stored at ambient temperature. Thus the Bio-wipe kit, along with the m-PCR, can be used for collection and detection of bacterial pathogens during outbreaks and in rural settings.Keywords: Bio-wipe kit, bacterial pathogens, faecal matter, PCR, DNA extractio

Microbial reductions and physical characterization of chitosan flocs when using chitosan acetate as a cloth filter aid in water treatment

The World Health Organization (WHO) estimates 2.1 billion people lack access to safely managed water. Cloth filtration is often employed in rural and developing communities of South Asia for point-of-use water treatment, but bacteria and viruses are too small for efficient removal by this filtration method. Chitosan is a biodegradable, cationic, organic polymer derived from the chemical treatment of chitin that acts as a coagulant and flocculant of contaminant of microbes and other particles in water, thereby facilitating filtration of microbes. This research 1) evaluated the use of chitosan acetate as a pre-treatment coagulation-flocculation process followed by cloth filtration for microbial reductions and 2) assessed floc particle size under three stirring conditions. E. coli KO11 bacteria and MS2 coliphage virus removals were quantified using culture-based methods. Chitosan acetate coagulation-flocculation pre-treatment of water, followed by cloth filtration, met or exceeded the protective (2-star) WHO performance levels for bacteria (2 log10 reduction) and viruses (3 log10 reduction), and filtrate turbidity was consistently reduced to &lt; 1 NTU, meeting United States Environmental Protection Agency (EPA) and WHO targets

Assessment of E. coli partitioning behavior via both culture-based and qPCR methods

Quantitative polymerase chain reaction (qPCR) offers a rapid, highly sensitive analytical alternative to the traditional culture-based techniques of microbial enumeration typically used in water quality monitoring. Before qPCR can be widely applied within surface water monitoring programs and stormwater assessment research, the relationships between microbial concentrations measured by qPCR and culture-based methods must be assessed across a range of water types. Previous studies investigating fecal indicator bacteria quantification using molecular and culture-based techniques have compared measures of total concentration, but have not examined particle-associated microorganisms, which may be more important from a transport perspective, particularly during the calibration of predictive water quality models for watershed management purposes. This study compared total, free-phase, and particle-associated Escherichia coli concentrations as determined by the Colilert defined substrate method and qPCR targeting the uidA gene in stream grab samples partitioned via a calibrated centrifugation technique. Free-phase concentrations detected through qPCR were significantly higher than those detected using Colilert although total concentrations were statistically equivalent, suggesting a source of analytical bias. Although a specimen processing complex was used to identify and correct for inhibition of the qPCR reaction, high particle concentrations may have resulted in underestimation of total cell counts, particularly at low concentrations. Regardless, qPCR-based techniques will likely have an important future role in stormwater assessment and management

Applicability of Bio-wipes for the collection of human faecal specimens for detection and characterisation of enteric viruses

OBJECTIVE : To determine whether gastroenteritis viruses and other enteric viruses could be detected in faecal specimens collected with Bio-wipes. METHODS : Faecal specimens, self-collected with Bio-wipes, from 190 individuals (94 diarrhoeal, 93 non-diarrhoeal, 3 unknown) were screened for eight human enteric viruses (enterovirus, hepatitis A virus, adenovirus, astrovirus, norovirus GI and GII, sapovirus and rotavirus) by real-time (reverse transcription)-polymerase chain reaction. Rotaviruses and noroviruses from positive specimens were genotyped. results At least one enteric virus could be detected in 82.6% (157/190) of faecal specimens. Mixed infections of up to four different viruses could be detected in both diarrhoeal and non-diarrhoeal specimens. Enteroviruses were detected most frequently (63.7%), followed by adenoviruses (48.4%) and noroviruses (32.2%). Genotyping was successful for 78.6% of rotaviruses and 44.8% of noroviruses. CONCLUSIONS : Bio-wipes provide a user friendly, easier method for stool collection that facilitates enteric virus detection and genetic characterisation.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-3156hb201

Use of viral pathogens and indicators to differentiate between human and non-human fecal contamination in a microbial source tracking comparison study

Assays for the detection and typing of adenoviruses, enteroviruses and F+ specific coliphages were performed on samples created as part of a national microbial source tracking methods comparison study. The samples were created blind to the researchers, and were inoculated with a variety of types of fecal contamination source (human, sewage, dog, seagull and cow) and mixtures of sources. Viral tracer and pathogen assays demonstrated a general ability to discriminate human from non-human fecal contamination. For example, samples inoculated with sewage were correctly identified as containing human fecal contamination because they contained human adenovirus or human enterovirus. In samples containing fecal material from individual humans, human pathogen analysis yielded negative results probably because the stool samples were taken from healthy individuals. False positive rates for the virus-based methods (0-8%) were among the lowest observed during the methods comparison study. It is suggested that virus-based source tracking methods are useful for identification of sewage contamination, and that these methods may also be useful as an indication of the public health risk associated with viral pathogens. Overall, virus-based source tracking methods are an important approach to include in the microbial source tracking 'toolbox'

Occurrence of male-specific and somatic coliphages and relationship with rainfall in privately-owned wells from peri‑urban and rural households

Privately-owned drinking water wells serving fewer than 25 people (private wells) are prevalent and understudied across most of the US. Private wells primarily serve rural households located outside of municipal drinking water and sewerage service coverage areas. These wells are not regulated by United States Environmental Protection Agency (EPA) under the Safe Drinking Water Act, are not regularly monitored by any public agency or utility, and generally do not undergo disinfection treatment. Coliphages are a group of viruses that infect coliform bacteria and are useful viral surrogates for fecal contamination in water systems in much the same way that fecal indicator bacteria (FIB), such as E. coli and to a lesser extent total coliforms, are used to quantify fecal contamination. Coliphages are approved by the EPA for regulatory monitoring in groundwater wells in the USA, but are not routinely used for this purpose. The present study characterizes the occurrence of male-specific and somatic coliphages, along with FIB, in private wells (n = 122) across two different counties in North Carolina. While occurrences of E. coli were rare and frequency of total coliform was generally low (~20%), male-specific and somatic coliphages were detectable in 66% and 54% of samples, respectively. Concentrations of male-specific coliphages were higher than somatics at each county and on a monthly basis. Rainfall appears to be partly influencing higher coliphage concentrations in December, January and February. This research underscores the need for increased surveillance in private wells and consideration of using coliphages in order to better characterize occurrence of fecal contamination at the time of sampling, especially during rainier months

Round robin investigation of methods for the recovery of poliovirus from drinking water.

Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS

Multilaboratory evaluation of methods for detecting enteric viruses in soils.

Two candidate methods for the recovery and detection of viruses in soil were subjected to round robin comparative testing by members of the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group. Selection of the methods, designated "Berg" and "Goyal," was based on results of an initial screening which indicated that both met basic criteria considered essential by the task group. Both methods utilized beef extract solutions to achieve desorption and recovery of viruses from representative soils: a fine sand soil, an organic muck soil, a sandy loam soil, and a clay loam soil. One of the two methods, Goyal, also used a secondary concentration of resulting soil eluants via low-pH organic flocculation to achieve a smaller final assay volume. Evaluation of the two methods was simultaneously performed in replicate by nine different laboratories. Each of the produced samples was divided into portions, and these were respectively subjected to quantitative viral plaque assay by both the individual, termed independent, laboratory which had done the soil processing and a single common reference laboratory, using a single cell line and passage level. The Berg method seemed to produce slightly higher virus recovery values; however, the differences in virus assay titers for samples produced by the two methods were not statistically significant (P less than or equal to 0.05) for any one of the four soils. Despite this lack of a method effect, th

The BioWipe: a non-invasive method to detect intestinal carriage of multi-drug resistant GRAM-negative bacteria

Colonization precedes infection and facilitates spread of several clinically important multidrug resistant organisms (MDRO). Reliable detection of carriage is important to improve our understanding of risk factors and spread of MDRO. Bacterial culture of stool samples obtained from peri-rectal swabs or whole stool is often used for this purpose. The previously described BioWipe method is a non-invasive stool collection method that resembles the use of toilet paper, and can be self-administered. The BioWipe consists of a 100×160 mm square of soft, absorbent synthetic fiber material attached to a plastic backing layer (Fisher Scientific, USA). It is used prior to using toilet paper after a bowel movement. The wipe with collected stool sample is placed onto the surface of an absorbent pad (3M™ Petroleum Sorbent Pads, Fisher Scientific, USA) containing modified Cary Blair transport media. The two parts are then folded together and placed inside a plastic bag. Prior to use, both components are treated with ultraviolet light irradiation in a biological safety cabinet for 30 minutes. After sample collection, the BioWipe is eluted with 20 mL mix of Phosphate Buffer Saline solution (PBS, pH=7.2) and 0.1% Tween 80 (vol/vol) directly in its original bag in a biosafety cabinet, until the stool sample is completely eluted. The resulting suspended stool sample is used for further processing