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Biofilms Resist Bleach
"Harmful microbes suspended in a biofilm were still alive and well after 60 minutes exposure to bleach."
Reference: Scientific American
By Denise I. Kennedy, Carlos E. Enriquez, Charles P. Gerba
This study identifies those sites in public restrooms where enteric bacteria are likely to occur. Forty-seven public restrooms were randomly selected and analyzed in this study. Samples collected with moistened-sterile cotton swabs were transferred to tubes containing Colilert media and incubated at 37° C for 24 hours, to determine the presence of total coliforms and Escherichia coli bacteria. Approximately 31 sites were sampled in 20 male restrooms and 28 sites in 27 female restrooms. Sinks, floors, toilet seats, and sanitary napkin disposals were the most contaminated sites based on occurrence. Coliform bacteria could be identified in more than 60% of sinks, and E.coli in 20%. Greater than 50% of restroom floors and sanitary napkin dispensers contained total coliforms. These areas were also the sites of greatest E. coli isolation. At least on one occasion Coliform or E. coli was isolated on all surfaces tested except the urinal top and flush handle.
Surfaces in high traffic restrooms (airports, bus terminals; educational institutions) were more likely to be contaminated with both coliforms (23.8%) and E. coli (5.6%), followed by fast food restaurants and hospitals. Female restrooms were more contaminated at all sites overall than male restrooms. Coliform bacteria were isolated twice as often in areas adjacent to the toilet in female restrooms vs. males. Restrooms with one stall were the most contaminated followed by those with four stalls/urinals. Restrooms with two or three stalls/urinals were less contaminated. Restrooms with a poor visual appearance were the most contaminated. No difference in the degree of contamination was seen between those judged excellent, good, or fair in appearance. Isolation of coliforms from all of the mops tested at fast food restaurants suggest them as one possible source of the coliforms or that coliforms grow in the mops after they are used.
The purpose of this study was to determine the occurrence of microbial "hotspots" in public restrooms. The presence of coliform bacteria and Escherichia coli, both indicators of potential fecal contamination, were used to identify these locations. Three types of public restrooms were analyzed in this study; high traffic (airports, bus terminals, educational institutions), hospital, and food service. Preselected surfaces in both male and female restrooms were sampled by wiping with cotton swabs at different locations.
There are a myriad of surfaces that could potentially become contaminated with enteric microorganisms during normal use. These surfaces include door handles, sink taps and faucets, toilet paper and paper towel dispensers. Such locations are all touched continually by patrons and may become contaminated by enteric organisms. Frequent touching of these areas can result in the transfer of potentially pathogenic microorganisms from the hand to the nose and mouth resulting in self-inoculation. In addition, the pathogens may be transferred to other vehicles involved in transmission such as foods.
During the course of this study, 20 male restrooms and 27 female restrooms were sampled. Data was analyzed based by descriptive categories in which all of the restrooms were located. The relative degree of contamination was determined in male and female restrooms as well as comparing differences in contamination among restrooms located in high traffic, hospital, and food service settings.
Materials and Methods
A cotton swab (individually wrapped and sterilized) was moistened with the hydrated (with 10 ml of sterilized distilled water) Colilert (IDEXX, Westbrook, Maine) solution. Each swab was removed from the wrapper and moistened by dipping the swab in the Colilert solution as aseptically as possible without touching any surface other than inside of the Colilert tube. An approximate area of 20 cm2 was swabbed for each selected site. Each site was swabbed without overlapping a previously swabbed area. Although it was difficult to know which restrooms had just been cleaned, restrooms that had a heavy lingering disinfectant odor and restrooms that looked like they had just been cleaned were avoided. Whenever possible, cooperation from the manager was obtained for information regarding cleaning routine. Only restrooms with fairly heavy traffic were sampled.
The areas sampled in each public restroom are listed in Table 1. [See original article for tables.]
Each Colilert (IDEXX, Westbrook, Maine) tube contains a dehydrated "formulation of salts, nitrogen, and carbon sources that are specific to total coliforms." (IDEXX, Westbrook, Maine). Non-coliform growth is suppressed and furthermore, cannot metabolize the target nutrients that indicate presence of coliforms. There are two nutrients that indicate the presence the target bacteria when metabolized; O-Nitrophenyl- B-d-Galactopyranoside (ONPG) and 4-Methyl umbelliferyl-B-d-Glucuronide (MUG). Viable total coliforms will cleave ONPG with the enzyme O-Nitrophenyl-B-d-Galacltopyranosidase. The remaining portion of ONPG, nitrophenol, is formed by that hydrolysis which produces a yellow color. Escherichia coli is confirmed by the presence of a component of MUG that fluoresces under UV light. MUG is cleaved through a hydrolysis reaction by Ecoli.
Twenty-seven female and 20 male restrooms were sampled completely. There were 28 sites sampled in each female restroom and 31 sites in the male restrooms. An additional survey of seven restrooms was conducted to gather data to determine the frequency of contamination of paper disposal bins and paper towel dispensers.
Extent of Contamination
The first objective of the study was to determine the locations most frequently contaminated by bacteria of fecal origin in public restrooms. The percentage of isolation for each selected site is shown in Table 2. Coliform bacteria were isolated on almost 21% of the 878 sites sampled in public restrooms. E. coli was isolated from 3.1% of the sites sampled. Coliforms were most often isolated on the floor in front of the toilet. Coliforms were isolated more than 50% of the time at three sites, i.e., floor in front of the toilet, drain of the sink basin, and the sanitary napkin disposal. The floor, the sink area and the toilet area were the most contaminated.
The only sites where coliforms were not isolated were the urinal flush handle and the top of the urinal. The sanitary napkin disposal was the site where E. coli was most commonly isolated (almost 29% of the time) followed by the area around the drain of the sink (21%). E. coli was also isolated 20% of the time inside the urinals. Other areas of isolation were associated with the floor and toilet area. Where the sampling sites were placed into groups the floor sites were the most contaminated followed by the sink sites and toilet sites (Table 3 ).
The restrooms were then grouped into 3 basic categories: high traffic (i.e., airports, bus terminal, educational), hospital, and food service restrooms (fast-food restaurants). For each type of restroom, each site was analyzed for frequency of Escherichia coli and total coliform occurrence. The results are shown in Table 4 (high traffic), 5 (hospitals), and 6 (food service), and summarized in Table 7. Overall, high traffic restrooms had the highest frequency of total coliform isolation and the highest E. coli contamination. In high traffic locations, E. coli was isolated 5.6% of the time and total coliforms were isolated 23.8% of the time. Fast food restrooms, however, had lower E. coli (1.5%), coliform (21.9%), contaminated sites. The lowest frequency of total coliform isolation was in hospital restrooms. Total coliforms could be isolated from only 17.2% of the sampled sites and E. coli was isolated from 2.0% of the sampled sites.
Male vs Female Restrooms
A comparison between the relative cleanliness of male and female restrooms (Table 8) showed that male restrooms were 1.5 times "cleaner" overall (1.5 times less total coliform contamination) and that female restrooms were contaminated with E. coli to a much greater extent. In female restrooms the sink (around the drain) was three times more likely to be contaminated with E. coli than the sink drains of the male restrooms. The trends are even more obvious when the toilet, sink, and floor areas were grouped for male and female restroom (Table 9). In frequency of total coliform occurrence, women out-do men nearly 2:1 in all areas. More specifically, in female restrooms, the floor area was 1.6 times more likely to be contaminated, the sink area was 1.9 times, and the toilet area was 1.8 times. The frequency of E. coli isolation was also greater in female restrooms. The E. coli was 4.5 times more often isolated in the sink area of female restrooms. There is a similar trend for the toilet area. Toilet area sites were contaminated with E. coli 1.9 times more often in female restrooms. The floors in male restrooms, however, are more often contaminated (1.7 times more often) with E. coli than the floors in the female restrooms.
Mops and Shoes
Both total coliforms and E coli appeared more often on the floors than on the toilet in public restrooms. In fact, there was a significantly greater frequency of occurrence of coliforms and E. coli on the floors as opposed to the sink and toilet sites (Table 6). However, no E. coli was detected on any shoes sampled. This indicates that shoes are not a likely source of E. coli contamination on floors. Most shoes, however, did have coliforms on the bottom of their soles and could serve as fomites for spreading contamination across restroom floors.
However, coliforms were detected in all of the mops obtained from fast food restaurants (Table 10), but no E. coli were detected. It is possible that mops may be picking up coliforms from around the toilet area and [coliforms were then] growing in the mop. It was observed that the same mops used in the restroom were also used in the food service and preparation areas at the establishments that were studied.
Rate of Usage
Restrooms were also grouped into the following categories depending on the combined number of stalls and urinals; 1 stall or urinal, 2 stalls and/or urinals, 3 stalls and/or urinals, and greater than 4 stalls and/or urinals. Restrooms in these groups were compared based on their total coliform and Escherichia coil contamination (Table 11). There was no trend evident except that restrooms with only one stall were by far the most contaminated with total coliforms (31.5% of sites contaminated) and were the most contaminated by E. coli by a small margin (5.2%). These results were expected because in bathrooms with one stall, the one stall will be used more frequently if there are many patrons. For the rest of the groups mentioned above, the relationship between contamination and number of stalls was proportional. Restrooms with 4 or more stalls are likely more frequently used (they are designed for areas that deal with more patrons) and had more sites contaminated with total coliforms and E. coil than the restrooms with 2 or 3 stalls and urinals. Locations in restrooms with 3 stalls and/or urinals were the next frequently contaminated. E.coli was found at 1.9% of the sites and total coliforms at 18.6%.
General Appearance Categories
Comparisons were done based on the general appearance of the restrooms sampled versus number of sites [where] the study bacteria were isolated. This comparison was done to assess whether appearance was a good indicator of contamination. The restrooms were placed into one of four categories. The restrooms were rated as excellent if all surfaces were free of debris and standing water, if there were no odors of mildew, feces, or urine, if the toilet seats were free of spots of human waste, if the floors did not have garbage or food particles on them, and if the sinks were not filled with hair and other clutter. Good restrooms were clean in most areas but could have debris on the floors, moisture around the sinks, and dust on other surfaces. Acceptable restrooms generally had a little human waste on the underside of the toilet seats, wet sinks that were not always clean, some bad odors (such as mildew), and debris (such as toilet paper) on the floor. Poor restrooms had deplorable conditions with broken or malfunctioning toilets, stall doors that were broken or had a large amount of graffiti, hair and dirt in the sinks, gum and scum stuck on the areas hand contact occurred on garbage cans, urine or feces odor, urine crusted in urinals and on the toilet seats, and toilet paper and garbage on the floors, stalls, and sink surfaces.
Following these guidelines based only on appearance, the bathrooms were grouped and the frequency of contaminated sites were determined for each group (Table 12-14). The results indicated that there was a clear distinction between restrooms rated poor in appearance and the ones judged excellent to good. Sampling sites in restrooms rated as poor were contaminated with E. coli 10.9% and with total coliforms 40.3% of the time. However, there were significantly fewer E. coli in restrooms rated excellent. Restrooms rated good and acceptable had 3-4 times more E. coli on surfaces than those rated excellent. Restrooms rated acceptable also had significantly more coliforms.
Restrooms that appear "excellent" must be cleaned often because one requirement for this category is that there be no dust on surfaces. Porcelain surfaces in these bathrooms were always clean to point where they shined. It was obvious that these restrooms were cleaned once a day if not more often. Restrooms rated poorly were often heavily used and not cleaned frequently. The high degree of total coliform and E. coli isolation is not surprising.
Paper Towel Dispensers and Towel Disposal Bins
Finally, a brief study was conducted to assess the contamination of paper towel dispensers and paper towel disposal bin doors in public bathrooms. Because there was no significant isolation of fecal bacteria on exit door latches and stall door latches (where the numbers of fecal bacteria might be expected to be more often detected), those locations were sampled again in a selected number of restrooms. Door latches, paper towel dispensers, and surprisingly paper disposal bins [were] found not to be frequently contaminated (data not shown). There is really not enough data, however, to conclude that fecal bacteria is not commonly isolated from paper disposal bins. We found very few paper disposal bins that require patrons to touch them to dispose of the paper towel. Most restrooms have the type of paper disposal bin that only requires that the paper be tossed into an open mouth. It was therefore difficult to get a large set of samples.
1. Overall, the most contaminated areas in public restrooms were the toilet, floor, sink and sanitary napkin disposal.
2. E. coli was most commonly isolated at the sanitary napkin disposal, drain in the sink basin, and inside the urinals.
3. Female restrooms were significantly more contaminated than male restrooms.
4. High traffic institutional restrooms were the most contaminated, followed by fast food restrooms and hospital public restrooms.
5. Coliform bacteria were isolated more than 50% of the time at three sites, i.e., floor in front of the toilet, drain of the sink basin, and the sanitary napkin disposal.
6. Coliforms or E. coli were isolated at least once on all sites sampled except the urinal flush handle and the top of the urinal.
7. Mops used to clean restroom floors contained coliform bacteria, but not E. coli.
8. Restrooms containing one stall and four stalls/urinals were the most contaminated.
9. Appearance was related to bacterial contamination for those judged poor and those judged excellent, good and acceptable.
10. Coliform bacteria could be detected on the paper towel trash bin door and paper towel lever.
Denise I. Kennedy
Carlos E. Enriquez
Charles P. Gerba
Department of Soil, Water, and Environmental Science
Department of Microbiology and Immunology
University of Arizona, Tucson, Arizona 8572
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