Validation of the bag-mediated filtration system for environmental surveillance of poliovirus in Nairobi, Kenya.

AIMS: This study compared the bag-mediated filtration system (BMFS) and standard WHO two-phase separation methods for poliovirus (PV) environmental surveillance, examined factors impacting PV detection and monitored Sabin-like (SL) PV type 2 presence with withdrawal of oral polio vaccine type 2 (OPV2) in April 2016. METHODS AND RESULTS: Environmental samples were collected in Nairobi, Kenya (Sept 2015-Feb 2017), concentrated via BMFS and two-phase separation methods, then assayed using the WHO PV isolation algorithm and intratypic differentiation diagnostic screening kit. SL1, SL2 and SL3 were detected at higher rates in BMFS than two-phase samples (P < 0·05). In BMFS samples, SL PV detection did not significantly differ with volume filtered, filtration time or filter shipment time (P > 0·05), while SL3 was detected less frequently with higher shipment temperatures (P = 0·027). SL2 was detected more frequently before OPV2 withdrawal in BMFS and two-phase samples (P < 1 × 10-5 ). CONCLUSIONS: Poliovirus was detected at higher rates with the BMFS, a method that includes a secondary concentration step, than using the standard WHO two-phase method. SL2 disappearance from the environment was commensurate with OPV2 withdrawal. SIGNIFICANCE AND IMPACT OF THE STUDY: The BMFS offers comparable or improved PV detection under the conditions in this study, relative to the two-phase method.

Salmonella Knowledge, Attitudes and Practices: A Survey of Backyard Poultry Owners Residing in Seattle, Washington and the Surrounding Metropolitan Area.

Raising poultry flocks in urban backyard settings is becoming increasingly popular across the United States, but carries a risk of zoonotic infection. In the United States from 1990 to 2014, 53 outbreaks of human salmonellosis linked to live poultry have been documented resulting in 2611 known illnesses, 387 known hospitalizations and five known deaths (Centers for Disease Control and Prevention 2015a, http://www.cdc.gov/healthypets/resources/dont-play-chicken-with-your-health-poster-24x36_508.pdf). A cross-sectional descriptive study was developed to better understand knowledge, attitudes and practices of urban backyard poultry owners regarding Salmonella risk and prevention. The study included a survey of bird health, animal husbandry and hygiene practices, and knowledge, attitudes and practices relating to Salmonella risk. Participants were videotaped while caring for their birds, and the recordings were transcribed using notational analysis to determine whether reported practices differed from observed practices. The results indicated that while a large proportion of participants knew that exposure to Salmonella is an inherent risk associated with raising poultry and harvesting eggs, their reported and observed practices would not consistently reduce risk of transmission of Salmonella and other zoonotic diseases. Approximately one in four participants reported performing practices that increase risk of inoculation, such as snuggling and kissing birds or eating/drinking near them. None of the participants were observed kissing their birds on video; however, snuggling (holding birds to clothes) or touching their face during routine care was observed in approximately two-thirds of the video recordings. The video data provided a unique opportunity to compare reported practices with actions recorded during site visits. While the differences were not statistically significant, findings from our study suggest that flock owners may not accurately report the frequency with which risky practices are performed during routine animal care. Education and outreach targeting backyard flock owners should aim to improve husbandry and hygiene practices and reduce risk of zoonotic diseases associated with raising poultry in the backyard setting.

Sensitivity of quantitative microbial risk assessments to assumptions about exposure to multiple consumption events per day.

Quantitative microbial risk assessments (QMRAs) of contaminated drinking water usually assume the daily intake volume is consumed once a day. However, individuals could consume water at multiple time points over 1 day, so the objective was to determine if the number of consumption events per day impacted the risk of infection from Campylobacter jejuni during short-term contamination events. A probabilistic hydraulic and risk model was used to evaluate the impact of multiple consumption events as compared to one consumption event on the health risk from the intake of contaminated tap water. The fraction of the population that experiences greater than 10(-4) risk of infection per event at the median dose was 6.8% (5th-95th percentile: 6.5-7.2%) for one consumption event per day, 18.2% (5th-95th: 17.6-18.7%) for three consumption events per day, and 19.8% (5th-95th: 14.0-24.4%) when the number of consumption events varied around 3.49 events/day. While the daily intake volume remained consistent across scenarios, the results suggest that multiple consumption events per day increases the probability of infection during short-term, high level contamination events due to the increased coincidence of a consumption event during the contamination peak. Therefore, it will be important to accurately characterize this parameter in drinking water QMRAs.

Prevalence, concentrations, and antibiotic sensitivities of Salmonella serovars in poultry from retail establishments in Seattle, Washington.

Poultry have been identified as one of the major sources of salmonellosis, with estimates ranging from 10 to 22% of total cases. Despite several advances in the industry and new performance standards, the incidence of salmonellosis in the population has not declined over the last 15 years. Salmonella is pervasive in a wide variety of foods, and thus, estimating its burden resulting from specific food categories has been challenging and plagued with uncertainty due to critical data gaps. The objective of this study was to conduct a year-long market survey (1,322 samples) to help bridge the data gaps on the contamination rates and levels of Salmonella on raw poultry by product type (i.e., breast, thighs, drums, wings, and split breast) and production method (conventional versus organic). The isolates recovered were serotyped and tested for antibiotic sensitivities. A PCR method was utilized for initial screening of samples after an overnight enrichment in tryptic soy broth. Three-tube most-probable-number (MPN) assays and anti-Salmonella immunomagnetic separation methods were utilized to determine the levels of Salmonella and aid with the recovery of Salmonella species, respectively. Eleven percent of the samples were positive for Salmonella. Significant differences in percent positive rates by product type included up to a 4-fold difference in percent positive rates between establishments, ranging from 7 to 31%. Of the samples positive for Salmonella species, 94% had <30 MPN/100 g. Production methods identified as organic or as not using antibiotics had significantly higher rates of recovery of Salmonella. On the other hand, all of the Salmonella isolates that were resistant to two or more antibiotics originated from conventional processing establishments where antibiotics were utilized. In addition, a significant proportion of isolates from conventionally processed products were serotypes clinically relevant to humans.

Amplification of viral RNA from drinking water using TransPlex™ whole-transcriptome amplification.

AIMS: Viral pathogens in environmental media are generally highly diffuse, yet small quantities of pathogens may pose a health risk. This study evaluates the ability of TransPlex™ whole transcriptome amplification (WTA) to amplify small quantities of RNA viruses from complex environmental matrices containing background nucleic acids. METHODS AND RESULTS: DNA extracts from mock drinking water samples containing mixed microbial populations were spiked with small quantities of echovirus type 13 (EV) RNA. Samples were amplified using a Transplex™ WTA kit, and EV-specific quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify target pathogens before and after application of WTA. Samples amplified by WTA demonstrated a decreased limit of detection. The log-linear relationship between serial dilutions was maintained following amplification by WTA. CONCLUSIONS: WTA is able to increase the quantity of target organism RNA in mixed populations, while maintaining log linearity of amplification across different target concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: WTA may serve as an effective preamplification step to increase the levels of RNA prior to detection by other molecular methods such as PCR, microarrays and sequencing.

Characterization of Methicillin-resistant Staphylococcus aureus isolated from public surfaces on a university campus, student homes and local community.

AIM: Isolation and characterization of methicillin-resistant Staphylococcus aureus (MRSA) from frequently touched nonhospital environmental surfaces at a large university, student homes and community sites. METHODS AND RESULTS: Twenty-four isolates from 21 (4·1%, n = 509) surfaces were MRSA positive and included 14 (58%, n = 24) SCCmec type IV, two (8%, n = 24) type I, and eight (33%, n = 24) were not type I-IV (NT). Six different multilocus sequencing types were identified by PCR and sequencing. PCR assays identified one (4·2%, n = 24) Panton-Valentine leukocidin (PVL) positive, 22 (92%, n = 24) arginine catabolic mobile element (ACME) positive and 23 (96%, n = 24) multidrug-resistant (kanamycin, macrolide, tetracycline) MRSA isolates. Eleven (46%, n = 24) USA300 isolates were determined by pulsed-field gel electrophoresis. CONCLUSION: The MRSA-positive environmental surfaces were identified in student homes (11·8%, n = 85), the community (2·3%, n = 130) and the university (2·7%, n = 294). USA300 strains were isolated from the university, student homes and community samples. This is the first report of the animal clone ST97 on urban environmental surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights the distribution of USA300 on frequently touched surfaces. Whether contact with these MRSA contaminated environmental surfaces are associated with increased risk of transmission of MRSA to people needs further research.

Inactivation of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) on various environmental surfaces by mist application of a stabilized chlorine dioxide and quaternary ammonium compound-based disinfectant.

Contaminated environmental surfaces are important reservoirs in the transmission of many human pathogens. Although several options exist for disinfecting contaminated environmental surfaces, few are compatible with use on both hard smooth non-porous (hard) and soft porous surfaces (soft) while still offering significant disinfection of the contaminating organisms. This study evaluated the efficacy of mist application of a stabilized chlorine dioxide and quaternary ammonium compound-based disinfectant (Cryocide20) for inactivation of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) on various environmental surfaces. MRSA and VRE were applied to hard and soft surfaces (glass, steel, tile, carpet, and cotton fabric), allowed to dry, and exposed to a uniform mist application of the disinfectant solution. After 1 hr of contact time, the residual disinfectant was neutralized, and the bacteria were recovered and enumerated on brain heart infusion (BHI) agar. Reduction of both test bacteria was observed on most of the hard and soft surfaces tested. Log(10) reduction of the organisms tended to be higher on steel, tile, and carpet than glass or cotton. Overall, these results suggest that mist application of Cryocide20 disinfectant may be an effective option for reduction of low levels of infectious bacterial pathogens from contaminated environmental surfaces.

Evaluation of a novel electropositive filter for the concentration of viruses from diverse water matrices.

Human enteric viruses are important agents of waterborne illness. They are diffusely distributed in environmental waters, necessitating concentration of tens to hundreds of litres for effective detection. This study evaluates the novel ViroCap disposable capsule filter for concentration of coliphage MS2 and poliovirus (PV1) from deionised (DI) water and artificial seawater, as well as natural ground, surface, and seawater. Retention and recoveries for the ViroCap were compared with two well-characterised filters: the 1MDS for DI water, and the OptiCap XL for artificial seawater. The mean adsorption for MS2 by the ViroCap was 88%. Recovery of MS2 was significantly greater (p< or=0.01) than alternative filters tested: 65% from DI water and 63% from artificial seawater, compared to 30% for the 1MDS and 15% for the OptiCap for the respective matrices. Recovery of PV1 from DI water (37%) was similar to that of the 1MDS (51%). PV1 recoveries from artificial seawater were significantly greater (p< or =0.01) for the ViroCap (44%) than the OptiCap (11%). Recovery of MS2 from seeded environmental samples yielded 44% from groundwater, 53% from surface water, and 51% from seawater. ViroCap disposable filter is efficient for concentrating MS2 and PV1 from diverse matrices and is robust across a range of ionic concentrations.

Characterisation of the water quality from open and rope-pump shallow wells in rural Cambodia.

An 8 month investigation into the quality of water from open and rope-pump shallow wells in rural Cambodia was conducted. Wells were analysed for indicators of the health (arsenic, fluoride, manganese, nitrate, total coliforms, E. coli, male-specific coliphage) and aesthetic (iron, chloride, conductivity, total dissolved solids, hardnesss, turbidity, pH) quality of the water, and referenced to the Cambodian Drinking Water Standard when available. The shallow aquifer was chemically less of a health risk than the deep aquifer; however, microbial contamination was considerable for both shallow well types with mean E. coli loads of 10(3) CFU/100 mL and male-specific coliphage contamination of 10(2) PFU/eluate. Temporal variation in microbial contamination was significant (p<0.05), with overall loads decreasing during the dry season. The aesthetic quality of the water was poor for all samples, but worsened during the dry season. No significant difference was observed in the quality of water from open and rope-pump wells, despite their classification as unimproved and improved respectively by the WHO/UNICEF Joint Monitoring Programme. Contaminants present in both well types may readily be removed by simple water treatment, suggesting that household treatment may be more beneficial to rural Cambodian households than shallow aquifer source improvements.

Vancomycin-resistant Enterococcus spp. in marine environments from the West Coast of the USA.

AIMS: The aim of the study was to determine if vancomycin-resistant Enterococcus spp. [VRE] carrying vanA and/or vanB genes were present in public marine beaches and a fishing pier [2001-2003, 2008] from Washington and California [2008]. METHODS: PCR assays for the vanA and/or vanB genes with verification by DNA-DNA hybridization of the PCR products were used. Positive isolates were speciated using the BD BBL Crystal Identification and/or by sequencing the 16S ribosomal region. RESULTS: Eighteen (8%) of 227 isolates including Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus/gallinarum and a Staphylococcus epidermidis carrying vanA and/or vanB genes, from four of six Washington and one of two California sites, were identified. Selected VRE and the S. epidermidis were able to transfer their van genes to an E. faecalis recipient at frequencies ranging from 1.9 x 10(-6) to 6.7 x 10(-9). CONCLUSIONS: Vancomycin-resistant Enterococcus spp. was isolated from five of the seven sites suggesting that other North America public beaches could be the reservoirs for VRE and should be assessed. SIGNIFICANCE & IMPACT OF THE STUDY: This is the first report of isolation and characterization of VRE strains (and a vanB Staphylococcus sp.) from North American environmental sources suggesting that public beaches may be a reservoir for possible transmission of VRE to beach visitors.

A conjugative macrolide resistance gene, mef(A), in environmental Clostridium perfringens carrying multiple macrolide and/or tetracycline resistance genes.

AIMS: To determine if environmental Clostridium perfringens carry antibiotic resistance genes and if the genes are mobile. METHODS AND RESULTS: Clostridium perfringens from water, soil and sewage (2003-2006) were screened for the tetracycline and macrolide resistance genes previously described in animal and human C. perfringens [erm(B), erm(Q), tetA(P), tetB(P) and tet(M) genes] and the macrolide resistance mef(A) gene. Of the 160 isolates, 108 (67.5%) carried > or =1 of the six antibiotic resistance gene(s). The tetA(P), tetB(P) and tet(M) genes were in 53%, 22% and 8%, and the erm(B), erm(Q) and mef(A) genes in 26%, 1% and 18% of the isolates, respectively. The mef(A) gene and flanking regions were sequenced. The tet(M), erm(B), erm(Q) and mef(A) genes transfer independently from C. perfringens donors to the Enterococcus faecalis recipient. CONCLUSIONS: Six resistance genes were found in the environmental C. perfringens with the most common being the tetA(P) gene and the erm(Q) gene the least common. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time conjugal transfer of macrolide resistance genes and/or the tet(M) gene from C. perfringens has been demonstrated. The data presented supports the hypothesis that antibiotic-resistant environmental C. perfringens are capable of acting as reservoirs for these antibiotic resistance genes.

Hypothesis: a motor neuron toxin produced by a clostridial species residing in gut causes ALS.

We hypothesize that a yet-to-be-identified motor neuron toxin produced by a clostridial species causes sporadic amyotrophic lateral sclerosis (ALS) in susceptible individuals. This clostridial species would reside undetected in the gut and chronically produce a toxin that targets the motor system, like the tetanus and botulinum toxins. After gaining access to the lower motor neuron, the toxin would be transported back to the cell body, as occurs with the tetanus toxin, and destroy the lower motor neuron - the essential feature of ALS. Again like the tetanus toxin, some of the toxin would cross to neighboring cells and to the upper motor neuron and similarly destroy these motor neurons. Weakness would relentlessly progress until not enough motor neurons remained to sustain life. If this hypothesis were correct, treatment with appropriate antibiotics or antitoxins might slow or halt progression of disease, and immunization might prevent disease.

Comparative reduction of Norwalk virus, poliovirus type 1, F+ RNA coliphage MS2 and Escherichia coli in miniature soil columns.

Norwalk-like viruses (NLVs) are important agents of waterborne illness and have been linked to several groundwater-related outbreaks. The presence of human enteric viruses, in particular the presence of NLVs, is difficult to detect in the environment. Consequently, surrogate organisms are typically used as indicators of viruses from faecal contamination. Whether traditional bacterial indicators are reliable indicators for viral pathogens remains uncertain. Few studies have directly compared mobility and reduction of bacterial indicators (e.g. coliforms, Escherichia coli) and other surrogate indicators (coliphages) with pathogenic human viruses in soil systems. In this study the mobility and comparative reduction of the prototype NLV, Norwalk Virus (NV), was compared to poliovirus 1 (PV1), a bacterial indicator (E coli, EC) and a viral indicator (coliphage MS2) through miniature soil columns. Replicate, 10 cm deep, miniature columns were prepared using three soils representing a range of soil textures (sand, organic muck, and clay). Columns were initially conditioned, then incubated at 10-14 degrees C, dosed twice weekly for 8 weeks with one column pore volume of virus-seeded groundwater per dose, followed by 8 weeks of dosing with one column pore volume per dose of unseeded, simulated rainwater. Columns were allowed to drain after each dosing until an effluent volume equivalent to an applied dose was collected. Column effluents and doses were assayed for all viruses and EC. Rapid mobility with minimal reduction was observed for all organisms in the sand. Similar reductions were observed in organic muck for most organisms but NV showed a greater reduction. No organisms were shown to pass through the clay columns. Elution of viruses, in particular PV1, from the columns was gradual. After cessation of microbe dosing, E. coli was less detectable than viruses in column effluents and, therefore, unreliable as a virus indicator.

F-specific RNA coliphages: occurrence, types, and survival in natural waters.

A small, well-defined watershed was investigated over a 2-year period to determine the prevalence of F-specific RNA coliphage (F + RNA) serotypes as indicators of animal fecal contamination. Sampling sites collected runoff from areas of urban and agricultural land use patterns. F-specific coliphages were concentrated from 2-L freshwater samples by polyethylene glycol precipitation, isolated using the double agar layer (DAL) method, confirmed as F + RNA by RNAse suppression, and serotyped. A subset of serotyped F + RNA were confirmed by genotyping. To determine relative survival, 10 confirmed F + RNA field isolates and 5 prototypic F + RNA were spiked into surface water and incubated at 25 degrees C for 36 days. F-specific coliphage isolation was strongly associated with rainfall events and was infrequent from primarily animal impacted surface waters. Field isolates were predoffiinantly Type I F + RNA (81%) and raw sewage isolates were predominantly Type III F + RNA (57%). Genotyping from either the watershed or raw sewage samples never positively identified Type IV F + RNA. Results from laboratory studies showed that F + RNA differ in their survival in water and that Type IV strains were the least persistent. Type III F + RNA were found to be reliably related to the release of uncontrolled human fecal material in the watershed, but the results of this study suggest that further study is required before utilizing for fecal source identification in natural waters.