Sewage

The bacteriophages were isolated from sewage water sampled at the Käppala waste water treatment plant (location: WGS84: 59°21’22.2"N 18°13’45.3"E), recipient of waste water from Stockholm city including some hospitals, and kept at +4°C before processing. The sampling of water was approved by the owner, Käppalaförbundet, Box 3095, 181 03 Lidingö, Sweden, kappala@kappala.se (Phages are not considered to be protected species). The phages were amplified by mixing 50 ml of waste water with the same amount of double strength LB and 10 ml of a single ECOR strain bacteria cultured overnight. After incubation overnight at 30°C, 10 ml of the mixture was shaken with 1% v/v chloroform and left at room temperature for 30 minutes to kill the bacteria, centrifuged at 3000×g at +4°C for 15 minutes, and sterile filtered through a 0.45 μm membrane filter (Whatman, ref. no. 10462100). After checking the lysates for phages, the titre was measured in plaque assays. Sterile filtered phage lysates were diluted in SM buffer [30 ] or LB to five different dilutions (10^-5–10^-9). 100 μl of diluted phage and 200 μl of target bacteria were mixed with 2 ml SA, spread on pre-warmed LA plates, and incubated overnight at 30°C [31 (link)]. The harvested phages were selected according to their plaque morphology. Phages displaying large, clear and non-turbid plaques without a halo were classified as virulent. Phages were re-isolated by plaque purification from the LA plates when several phages on the same plate could be suspected. After additional plaque purifications, 25 virulent phages were saved and stored in 50% glycerol at -70°C as well as in LB at +4°C [32 (link)]. These phages were named according to guidelines in Kropinski et al. [33 (link)]. The six phages showing the broadest host range in the spot test (see below) were thus named as follows; vB_EcoP_SU10, vB_EcoP_SU16, vB_EcoP_SU27, vB_EcoP_SU32, vB_EcoP_SU57, and vB_EcoP_SU63, but only the last part of names is used in the following text, e.g. SU10, SU16 and so on.

We conducted studies at three marine beaches affected by treated sewage discharge from nearby Publicly Owned Treatment Works (POTW). In 2005, we studied Edgewater Beach in Biloxi, Mississippi, and in 2007 we studied Goddard Beach in Goddard Memorial State Park in West Warwick, Rhode Island and Fairhope Municipal Beach in Fairhope, Alabama (Figure 1). Each beach site was located within 7 miles or less of a treated sewage discharge outfall from facilities that served populations of at least 15,000. Based on historical records, each beach site showed variability in water quality, but were generally in compliance with local and federal water quality guidelines.
Data collection procedures for the health survey have been described previously [6 (link),7 (link)]. In brief, we conducted surveys on weekends and holidays between May and September. Upon arrival beachgoers were provided a pamphlet describing the study. After reviewing the pamphlet, interviewers approached the household group or individual and offered them the opportunity to enroll in the study. On most days, all beachgoers arriving between approximately 11 AM and 4 PM were offered enrollment. Respondents were ineligible if they had completed the study in the previous 30 days, or if there was no adult (18 years of age or older) household member present. Households provided verbal consent and completed an enrollment questionnaire consisting of demographic information, swimming exposures in the previous two weeks, and the presence of underlying health conditions, such as chronic diarrhea, asthma, or skin conditions. As they left the beach for the day, participants completed a questionnaire to ascertain the extent and duration of their contact with water and other activities during their visit to the beach such as contact with sand and food consumption. Ten to twelve days following the beach visit, we telephoned participants and asked about the occurrence of new gastrointestinal, skin, respiratory, eye, or ear symptoms.
The study procedures, questionnaires, protocols and consent process were reviewed and approved by the Institutional Review Board of the Centers for Disease Control and Prevention.