Novel microbiological and spatial statistical methods to improve strength of epidemiological evidence in a community-wide waterborne outbreak

Abstract

Failures in the drinking water distribution system cause gastrointestinal outbreaks with multiple pathogens. A water distribution pipe breakage caused a community-wide waterborne outbreak in Vuorela, Finland, July 2012. We investigated this outbreak with advanced epidemiological and microbiological methods. A total of 473/2931 inhabitants (16%) responded to a web-based questionnaire. Water and patient samples were subjected to analysis of multiple microbial targets, molecular typing and microbial community analysis. Spatial analysis on the water distribution network was done and we applied a spatial logistic regression model. The course of the illness was mild. Drinking untreated tap water from the defined outbreak area was significantly associated with illness (RR 5.6, 95% CI 1.9-16.4) increasing in a dose response manner. The closer a person lived to the water distribution breakage point, the higher the risk of becoming ill. Sapovirus, enterovirus, single Campylobacter jejuni and EHEC O157:H7 findings as well as virulence genes for EPEC, EAEC and EHEC pathogroups were detected by molecular or culture methods from the faecal samples of the patients. EPEC, EAEC and EHEC virulence genes and faecal indicator bacteria were also detected in water samples. Microbial community sequencing of contaminated tap water revealed abundance of Arcobacter species. The polyphasic approach improved the understanding of the source of the infections, and aided to define the extent and magnitude of this outbreak.

Figure 1. Schematic map of the water pipe of a defined outbreak area in Vuorela, July 2012.

The outbreak (boil water notice) area is indicated by dashed line (- - -), the water sampling points (1–9) are coded as (•) with a positive culture finding and (*) with a negative finding. Arrows indicate the inflow points of the water from the water plant (outside the figure). Points 1,2,3,6,7 and 9 are tap water sampling locations, point 4 is the water pipe line breakage point (surface water), point 5 represent drinking water from the upper water storage reservoir and point 8 is the municipal effluent sampling location.

Figure 2. Epidemic curve of a waterborne outbreak in Vuorela, July 2012 based on the reported onset date of illness of the cases, and E. coli bacteria counts and chlorine levels in the point 7 (See Fig. 1) of the water distribution network.

Figure 3. Distribution of the Bacteria domain as determined by taxonomic identification of partial 16S rRNA gene sequencing (at class level) in a waterborne outbreak in Vuorela, July 2012.

Charts and tables represent the cumulative distribution of total DNA and RNA extracted from samples of A) the upper storage reservoir before cleaning (point 5 in Figure 1), B) tap water during contamination (point 7 in Figure 1) and C) the upper storage reservoir after cleaning. Legend: Beta-proteobacteria (Bp); Gamma-proteobacteria (Gp); Alpha-proteobacteria (Ap); Acidobacteria (Ad); Delta-proteobacteria (Dp); Sphingobacteria (Sp); Planctomycetacia (Pl); Actinobacteria (At); Epsilon-proteobacteria (Ep); Nitrospira (Ni); Verrucomicrobia Subdivision3 (S3); Clostridia (Cl); Bacteroidia (Ba); Opitutae (Op); Spartobacteria (St); Verrucomicrobiae (Ve); Bacteroidetes (Bt); Others (classes each representing <1%); unclassified (Unc).

Figure 4. Phylogenetic relationships among OTUs (•) of the genus Arcobacter in a waterborne outbreak in Vuorela, July 2012.

The tree was inferred from a maximum likelihood analysis of aligned 16S rRNA gene sequence (≈255 bp) and nodes with a bootstrap value ≥50% of 1 000 replicates are identified. Sulfurospirillum deleyianum (NR_074378) and Campylobacter fetus (L04314) were used as outgroup. Number in bracket represents the total amount of DNA/RNA reads identified in samples from A) the upper storage reservoir before cleaning, B) tap water during contamination and C) the upper storage reservoir after cleaning. *A. butzleri, A. cryaerophilus and A. skirrowii have been associated with gastrointestinal diseases .