Molecular Monitoring of Enteropathogens in Sewage During NATO Exercise Trident Juncture 2018: Potential Tool in Early Outbreak Warning?

INTRODUCTION: Outbreaks of gastrointestinal disease among military service personnel can have severe impact on operational effectivity and force readiness. Thus, early outbreak detection is critical to minimize spread. This pilot study aimed to explore field-based molecular screening of sewage as a supplemental tool in early outbreak warning before disease is diagnosed in personnel seeking medical care. MATERIALS AND METHODS: Sewage from permanent (n = 3) and temporary (n = 3) military camps, hosting national and international military personnel, were sampled during the NATO Exercise TRJE18 taking place in southern Norway during fall 2018. Samples were screened for 22 gastrointestinal pathogens using multiplex PCR. RESULTS: Markers of multiple enteropathogens were detected in samples from all locations with some variations in diversity. Yersinia enterocolitica, pathogenic Escherichia coli, adenovirus, and Giardia were detected in sewage from all six camps during the exercise. Agent diversity seemed to increase with population size, regardless of nationality. Only a minor outbreak (n = 6) of norovirus was reported in one of the permanent camps. From the same camp, genetic markers of norovirus were detected in sewage 2 days before outbreak notification. No other outbreaks of gastrointestinal disease were reported during the exercise, indicating that markers of several enteropathogens can be normally found in sewage from healthy soldier populations. Thus, discriminating between true outbreaks and nonrelevant "background levels" would be of critical importance for correct decision-making in operational contexts. CONCLUSIONS: Molecular screening of sewage allows rapid detection of multiple gastrointestinal pathogens in biological waste from military camps. However, background levels of pathogens challenges interpretation of qualitative analyses in outbreak situations. As such, quantitative measures, as well as high-resolution sequence-based methods, which allows strain identification and broader target spectrum, should be further explored in future studies.

Multiplex polymerase chain reaction detection of enteropathogens in sewage in Norway.

The primary objective of this small-scale study was to investigate the occurrence of enteropathogens in sewage (municipal wastewater) in Norway using the commercially available FilmArray® multiplex polymerase chain reaction (PCR) system with the gastrointestinal (GI) panel. Our findings indicate that DNA/RNA of several enteropathogens are present simultaneously in Norwegian wastewater systems. The spectre was broad even in smaller communities. With some exceptions, occurrence corresponded more or less to the reported cases of infectious human gastrointestinal disease in the same geographical regions. The effects of different sewage purification techniques were assessed on a limited number of samples indicating that neither chemical nor biological treatment was sufficiently effective to reduce gene material from the pathogens to undetectable levels. Further studies are required to assess the performance and suitability FilmArray® multiplex PCR when used on collective sewage samples in outbreak situations. Additionally, screening sewage samples using multiplex-PCR could be valuable in order to detect new and emerging pathogens and for preliminary analysis of samples before proceeding to more work demanding confirmatory techniques.

National outbreak of Yersinia enterocolitica infections in military and civilian populations associated with consumption of mixed salad, Norway, 2014.

In May 2014, a cluster of Yersinia enterocolitica (YE) O9 infections was reported from a military base in northern Norway. Concurrently, an increase in YE infections in civilians was observed in the Norwegian Surveillance System for Communicable Diseases. We investigated to ascertain the extent of the outbreak and identify the source in order to implement control measures. A case was defined as a person with laboratory-confirmed YE O9 infection with the outbreak multilocus variable-number tandem repeat analysis (MLVA)-profile (5-6-9-8-9-9). We conducted a case-control study in the military setting and calculated odds ratios (OR) using logistic regression. Traceback investigations were conducted to identify common suppliers and products in commercial kitchens frequented by cases. By 28 May, we identified 133 cases, of which 117 were linked to four military bases and 16 were civilians from geographically dispersed counties. Among foods consumed by cases, multivariable analysis pointed to mixed salad as a potential source of illness (OR 10.26; 95% confidence interval (CI): 0.85-123.57). The four military bases and cafeterias visited by 14/16 civilian cases received iceberg lettuce or radicchio rosso from the same supplier. Secondary transmission cannot be eliminated as a source of infection in the military camps. The most likely source of the outbreak was salad mix containing imported radicchio rosso, due to its long shelf life. This outbreak is a reminder that fresh produce should not be discounted as a vehicle in prolonged outbreaks and that improvements are still required in the production and processing of fresh salad products.

Occurrence of Vibrio parahaemolyticus, V. cholerae, and V. vulnificus in Norwegian Blue Mussels (Mytilus edulis).

Vibrio parahaemolyticus, V. cholerae, and V. vulnificus were isolated from 10.3%, 1.0%, and 0.1% of 885 blue mussel samples, respectively. Four of the samples contained trh(+) V. parahaemolyticus, while no tdh-positive isolates were detected. The V. cholerae isolates were non-O:1/non-O:139 serotypes and were ctxA negative.

Lack of agreement between biochemical and genetic identification of Aeromonas spp.

Biochemical and genetic identification by RFLP (restriction fragment length polymorphism) of the PCR-amplified 16S r-RNA sequence were compared for a selection of 171 clinical and environmental isolates of Aeromonas spp. The investigation revealed large differences between the two methods. The species phenotypic identification scheme and the genetic technique applied to the environmental strains gave divergent results for 96% of the strains tested. There was 46% discrepancy between the two methods for the clinical isolates. The distribution of species differed between clinical and environmental isolates. A. hydrophila, A. caviae, A. jandaei and A. veronii dominated the clinical material (81% of isolates by RFLP), whilst only 21% of the environmental isolates belonged to those four species. From the environmental group A. salmonicida, A. bestiarum, A. sobria, A. media, and A. encheleia contributed 72% of the strains tested. The poor parity between the biochemical and the genetic identification of the environmental isolates, and to a lesser extent for the clinical isolates, underlines the fact that our current biochemical methods cannot adequately differentiate Aeromonas spp. This work also shows that the biochemical schemes derived from clinical isolates are incomplete for the identification of environmental strains.

Studies of aerolysin promoters from different Aeromonas spp.

Aeromonas spp. have been related to food and waterborne diseases. The pore forming toxin aerolysin is regarded as the most important virulence factor in Aeromonas food poisoning. In this work the aerolysin promoters from several Aeromonas spp. strains have been sequenced, and divided into two sequence groups. Further analyses of the promoters were carried out in a reporter-plasmid, pSTINA-II. This plasmid was constructed as a hybrid of pUC4K, pACYC184 and pKK232-8. We could conclude that our constructed reporter-gene plasmid was functional and was used to compare different promoters in an aerolysin negative Aeromonas spp. This construct made it possible to study the expression of the reporter gene using different aerolysin promoters under several conditions. We were able to show that the two obtained sequence groups of aerolysin promoters gave different expression of the reporter gene, and that this expression was dependent of temperature and osmolarity. Reducing the size of one promoter sequence from 254 to 148 bp and 102 bp gave a gradual reduction of the reporter gene expression under all conditions. According to our assay there seems to be more than one functional promoter upstream of the aerolysin gene, although the RT-PCR indicated one transcription starting point, under all test conditions.