Kristen E. Gibson

A Norovirus Outbreak at a Long-Term-Care Facility: The Role of Environmental Surface Contamination

BACKGROUND The role of environmental surface contamination in the propagation of norovirus outbreaks is unclear. An outbreak of acute gastroenteritis was reported among residents of a 240-bed veterans long-term-care facility. OBJECTIVES To identify the likely mode of transmission, to characterize risk factors for illness, and to evaluate for environmental contamination in this norovirus outbreak. METHODS An outbreak investigation was conducted to identify risk factors for illness among residents and employees. Stool and vomitus samples were tested for norovirus by reverse transcription polymerase chain reaction (RT-PCR). Fourteen days after outbreak detection, ongoing cases among the residents prompted environmental surface testing for norovirus by RT-PCR. RESULTS One hundred twenty-seven (52%) of 246 residents and 84 (46%) of 181 surveyed employees had gastroenteritis. Case-residents did not differ from non-case-residents by comorbidities, diet, room type, or level of mobility. Index cases were among the nursing staff. Eight of 11 resident stool or vomitus samples tested positive for genogroup II norovirus. The all-cause mortality rate during the month of the outbreak peak was significantly higher than the expected rate. Environmental surface swabs from case-resident rooms, a dining room table, and an elevator button used only by employees were positive for norovirus. Environmental and clinical norovirus sequences were identical. CONCLUSION Extensive contamination of environmental surfaces may play a role in prolonged norovirus outbreaks and should be addressed in control interventions.

Antibiotic-Resistant Enterococci and Fecal Indicators in Surface Water and Groundwater Impacted by a Concentrated Swine Feeding Operation

Background The nontherapeutic use of antibiotics in swine feed can select for antibiotic resistance in swine enteric bacteria. Leaking swine waste storage pits and the land-application of swine manure can result in the dispersion of resistant bacteria to water sources. However, there are few data comparing levels of resistant bacteria in swine manure–impacted water sources versus unaffected sources. Objectives The goal of this study was to analyze surface water and groundwater situated up and down gradient from a swine facility for antibiotic-resistant enterococci and other fecal indicators. Methods Surface water and groundwater samples (n = 28) were collected up and down gradient from a swine facility from 2002 to 2004. Fecal indicators were isolated by membrane filtration, and enterococci (n = 200) were tested for susceptibility to erythromycin, tetracycline, clindamycin, virginiamycin, and vancomycin. Results Median concentrations of enterococci, fecal coliforms, and Escherichia coli were 4- to 33-fold higher in down-gradient versus up-gradient surface water and groundwater. We observed higher minimal inhibitory concentrations for four antibiotics in enterococci isolated from down-gradient versus up-gradient surface water and groundwater. Elevated percentages of erythromycin- (p = 0.02) and tetracycline-resistant (p = 0.06) enterococci were detected in down-gradient surface waters, and higher percentages of tetracycline- (p = 0.07) and clindamycin-resistant (p < 0.001) enterococci were detected in down-gradient groundwater. Conclusions We detected elevated levels of fecal indicators and antibiotic-resistant enterococci in water sources situated down gradient from a swine facility compared with up-gradient sources. These findings provide additional evidence that water contaminated with swine manure could contribute to the spread of antibiotic resistance.

Airborne Multidrug-Resistant Bacteria Isolated from a Concentrated Swine Feeding Operation

The use of nontherapeutic levels of antibiotics in swine production can select for antibiotic resistance in commensal and pathogenic bacteria in swine. As a result, retail pork products, as well as surface and groundwaters contaminated with swine waste, have been shown to be sources of human exposure to antibiotic-resistant bacteria. However, it is unclear whether the air within swine operations also serves as a source of exposure to antibiotic-resistant bacterial pathogens. To investigate this issue, we sampled the air within a concentrated swine feeding operation with an all-glass impinger. Samples were analyzed using a method for the isolation of Enterococcus. A total of 137 presumptive Enterococcus isolates were identified to species level using standard biochemical tests and analyzed for resistance to erythromycin, clindamycin, virginiamycin, tetracycline, and vancomycin using the agar dilution method. Thirty-four percent of the isolates were confirmed as Enterococcus, 32% were identified as coagulase-negative staphylococci, and 33% were identified as viridans group streptococci. Regardless of bacterial species, 98% of the isolates expressed high-level resistance to at least two antibiotics commonly used in swine production. None of the isolates were resistant to vancomycin, an antibiotic that has never been approved for use in livestock in the United States. In conclusion, high-level multidrug-resistant Enterococcus, coagulase-negative staphylococci, and viridans group streptococci were detected in the air of a concentrated swine feeding operation. These findings suggest that the inhalation of air from these facilities may serve as an exposure pathway for the transfer of multidrug-resistant bacterial pathogens from swine to humans.

Measuring and mitigating inhibition during quantitative real time PCR analysis of viral nucleic acid extracts from large-volume environmental water samples

Naturally-occurring inhibitory compounds are a major concern during qPCR and RT-qPCR analysis of environmental samples, particularly large volume water samples. Here, a standardized method for measuring and mitigating sample inhibition in environmental water concentrates is described. Specifically, the method 1) employs a commercially available standard RNA control; 2) defines inhibition by the change in the quantification cycle (C(q)) of the standard RNA control when added to the sample concentrate; and 3) calculates a dilution factor using a mathematical formula applied to the change in C(q) to indicate the specific volume of nuclease-free water necessary to dilute the effect of inhibitors. The standardized inhibition method was applied to 3,193 large-volume water (surface, groundwater, drinking water, agricultural runoff, sewage) concentrates of which 1,074 (34%) were inhibited. Inhibition level was not related to sample volume. Samples collected from the same locations over a one to two year period had widely variable inhibition levels. The proportion of samples that could have been reported as false negatives if inhibition had not been mitigated was between 0.3% and 71%, depending on water source. These findings emphasize the importance of measuring and mitigating inhibition when reporting qPCR results for viral pathogens in environmental waters to minimize the likelihood of reporting false negatives and under-quantifying virus concentration.

Tangential-Flow Ultrafiltration with Integrated Inhibition Detection for Recovery of Surrogates and Human Pathogens from Large-Volume Source Water and Finished Drinking Water

ABSTRACT Tangential-flow ultrafiltration was optimized for the recovery of Escherichia coli, Enterococcus faecalis, Clostridium perfringens spores, bacteriophages MS2 and PRD1, murine norovirus, and poliovirus seeded into 100-liter surface water (SW) and drinking water (DW) samples. SW and DW collected from two drinking water treatment plants were then evaluated for human enteric viruses.

Viral pathogens in water: occurrence, public health impact, and available control strategies

The public health impact of the transmission of viruses in water is significant worldwide. Waterborne viruses can be introduced into our recreational and finished drinking water sources through a variety of pathways ultimately resulting in the onset of illness in a portion of the exposed population. Although there have been advances in both drinking water treatment technologies and source water protection strategies, waterborne disease outbreaks (WBDOs) due to viral pathogens still occur each year worldwide. By highlighting the prevalence of viral pathogens in water as well as (1) the dominant viruses of concern, (2) WBDOs due to viruses, and (3) available water treatment technologies, the goal of this review is to provide insight into the public health impact of viruses in water.

Evaluation of human enteric viruses in surface water and drinking water resources in southern Ghana.

An estimated 884 million people worldwide do not have access to an improved drinking water source, and the microbial quality of these sources is often unknown. In this study, a combined tangential flow, hollow fiber ultrafiltration (UF), and real-time PCR method was applied to large volume (100 L) groundwater (N = 4), surface water (N = 9), and finished (i.e., receiving treatment) drinking water (N = 6) samples for the evaluation of human enteric viruses and bacterial indicators. Human enteric viruses including norovirus GI and GII, adenovirus, and polyomavirus were detected in five different samples including one groundwater, three surface water, and one drinking water sample. Total coliforms and Escherichia coli assessed for each sample before and after UF revealed a lack of correlation between bacterial indicators and the presence of human enteric viruses.

Detection of Bacterial Indicators and Human and Bovine Enteric Viruses in Surface Water and Groundwater Sources Potentially Impacted by Animal and Human Wastes in Lower Yakima Valley, Washington

ABSTRACT Tangential flow ultrafiltration (UF) was used to concentrate and recover bacterial indicators and enteric viruses from 100 liters of groundwater (GW; n = 10) and surface water (SW; n = 11) samples collected in Lower Yakima Valley, WA. Human and bovine enteric viruses were analyzed in SW and GW concentrates by real-time PCR by using integrated inhibition detection.

Removal and transfer of viruses on food contact surfaces by cleaning cloths.

ABSTRACT Contamination of food contact surfaces with pathogens is considered an important vehicle for the indirect transmission of food-borne diseases. Five different cleaning cloths were assessed for the ability to remove viruses from food contact surfaces (stainless steel surface and nonporous solid surface) and to transfer viruses back to these surfaces. Cleaning cloths evaluated include two different cellulose/cotton cloths, one microfiber cloth, one nonwoven cloth, and one cotton terry bar towel. Four viral surrogates (murine norovirus [MNV], feline calicivirus [FCV], bacteriophages PRD1 and MS2) were included. Removal of FCV from stainless steel was significantly greater (P ≤ 0.05) than that from nonporous solid surface, and overall removal of MNV from both surfaces was significantly less (P ≤ 0.05) than that of FCV and PRD1. Additionally, the terry towel removed significantly fewer total viruses (P ≤ 0.05) than the microfiber and one of the cotton/cellulose cloths. The cleaning cloth experiments were repeated with human norovirus. For transfer of viruses from cloth to surface, both cellulose/cotton cloths and microfiber transferred an average of 3.4 and 8.5 total PFU, respectively, to both surfaces, and the amounts transferred were significantly different (P ≤ 0.05) from those for the nonwoven cloth and terry towel (309 and 331 total PFU, respectively). There was no statistically significant difference (P > 0.05) in the amount of virus transfer between surfaces. These data indicate that while the cleaning cloths assessed here can remove viruses from surfaces, some cloths may also transfer a significant amount of viruses back to food contact surfaces.

Genes Indicative of Zoonotic and Swine Pathogens Are Persistent in Stream Water and Sediment following a Swine Manure Spill

ABSTRACT Manure spills into streams are relatively frequent, but no studies have characterized stream contamination with zoonotic and veterinary pathogens, or fecal chemicals, following a spill. We tested stream water and sediment over 25 days and downstream for 7.6 km for the following: fecal indicator bacteria (FIB), the fecal indicator chemicals cholesterol and coprostanol, 20 genes for zoonotic and swine-specific bacterial pathogens by presence/absence PCR for viable cells, one swine-specific Escherichia coli toxin gene (STII gene) by quantitative PCR (qPCR), and nine human and animal viruses by qPCR or reverse transcription-qPCR. Twelve days postspill, and 4.2 km downstream, water concentrations of FIB, cholesterol, and coprostanol were 1 to 2 orders of magnitude greater than those detected before, or above, the spill, and genes indicating viable zoonotic or swine-infectious Escherichia coli were detected in water or sediment. STII gene levels increased from undetectable before or above the spill to 105 copies/100 ml of water 12 days postspill. Thirteen of 14 water (8/9 sediment) samples had viable STII-carrying cells postspill. Eighteen days postspill, porcine adenovirus and teschovirus were detected 5.6 km downstream. FIB concentrations (per gram [wet weight]) in sediment were greater than in water, and sediment was a continuous reservoir of genes and chemicals postspill. Constituent concentrations were much lower, and detections less frequent, in a runoff event (200 days postspill) following manure application, although the swine-associated STII and stx 2e genes were detected. Manure spills are an underappreciated pathway for livestock-derived contaminants to enter streams, with persistent environmental outcomes and the potential for human and veterinary health consequences.