The potential spread of infection caused by aerosol contamination of surfaces after flushing a domestic toilet

AIMS: To determine the level of aerosol formation and fallout within a toilet cubicle after flushing a toilet contaminated with indicator organisms at levels required to mimic pathogen shedding during infectious diarrhoea. METHODS AND RESULTS: A semisolid agar carrier containing either Serratia marcesens or MS2 bacteriophage was used to contaminate the sidewalls and bowl water of a domestic toilet to mimic the effects of soiling after an episode of acute diarrhoea. Viable counts were used to compare the numbers of Serratia adhering to the porcelain surfaces and those present in the bowl water before and after flushing the toilet. Air sampling and settle plates were used to determine the presence of bacteria or virus-laden aerosols within the toilet cubicle. After seeding there was a high level of contamination on the porcelain surfaces both under the rim and on the sides of the bowl. After a single flush there was a reduction of 2.0-3.0 log cycles cm(-2) for surface attached organisms. The number of micro-organisms in the bowl water was reduced by 2.0-3.0 log cycles ml(-1) after the first flush and following a second flush, a further reduction of c. 2.0 log cycles ml(-1) was achieved. Micro-organisms in the air were at the highest level immediately after the first flush (mean values, 1370 CFU m(-3) for Serratia and 2420 PFU m(-3) for MS2 page). Sequential flushing resulted in further distribution of micro-organisms into the air although the numbers declined after each flush. Serratia adhering to the sidewalls, as well as free-floating organisms in the toilet water, were responsible for the formation of bacterial aerosols. CONCLUSIONS: Although a single flush reduced the level of micro-organisms in the toilet bowl water when contaminated at concentrations reflecting pathogen shedding, large numbers of micro-organisms persisted on the toilet bowl surface and in the bowl water which were disseminated into the air by further flushes. SIGNIFICANCE AND IMPACT OF THE STUDY: Many individuals may be unaware of the risk of air-borne dissemination of microbes when flushing the toilet and the consequent surface contamination that may spread infection within the household, via direct surface-to-hand-to mouth contact. Some enteric viruses could persist in the air after toilet flushing and infection may be acquired after inhalation and swallowing.     

Toilet hygiene—review and research needs

The goal of good toilet hygiene is minimizing the potential for pathogen transmission. Control of odours is also socially important and believed to be a societal measure of cleanliness. Understanding the need for good cleaning and disinfecting is even more important today considering the potential spread of emerging pathogens such as SARS-CoV-2 virus. While the flush toilet was a major advancement in achieving these objectives, exposure to pathogens can occur from failure to clean and disinfect areas within a restroom, as well as poor hand hygiene. The build-up of biofilm within a toilet bowl/urinal including sink can result in the persistence of pathogens and odours. During flushing, pathogens can be ejected from the toilet bowl/urinal/sink and be transmitted by inhalation and contaminated fomites. Use of automatic toilet bowl cleaners can reduce the number of microorganisms ejected during a flush. Salmonella bacteria can colonize the underside of the rim of toilets and persist up to 50 days. Pathogenic enteric bacteria appear in greater numbers in the biofilm found in toilets than in the water. Source tracking of bacteria in homes has demonstrated that during cleaning enteric bacteria are transferred from the toilet to the bathroom sinks and that these same bacteria colonize cleaning tools used in the restroom. Quantitative microbial risk assessment has shown that significant risks exist from both aerosols and fomites in restrooms. Cleaning with soaps and detergents without the use of disinfectants in public restrooms may spread bacteria and viruses throughout the restroom. Odours in restrooms are largely controlled by ventilation and flushing volume in toilet/urinals. However, this results in increased energy and water usage. Contamination of both the air and surfaces in restrooms is well documented. Better quantification of the risks of infection are needed as this will help determine what interventions will minimize these risks.     

Isolation and characterization of an early colonizing Rhizobium sp. R8 from a household toilet bowl

The bacterial community structure was compared between the third days’, one week’, and three weeks’ biofilm samples from the surface of a household toilet bowl. It was found that the PCR-DGGE band pattern of 16S rRNA gene was dramatically changed after the third day and was not further changed until three weeks. This result suggests that there are early and late colonizing bacterial groups. One of the early colonizers isolated from the third days’ sample was Rhizobium sp. R8, a closest relative to Rhizobium giardinii, which exhibited the highest biofilm formation activity in an artificial urine condition. R8 produced extracellular polysaccharides containing galactose, glucose, and mannose at the molar ratio of 8:1:1, which were probably responsible for the biofilm formation. Its excelled biofilm formation and urease activities together with the lack of nodulation and nitrogen fixing genes in R8 suggest that this strain has been specifically adapted to urine condition in a toilet bowl.     

Microbiological Hazards of Household Toilets: Droplet Production and the Fate of Residual Organisms

Large numbers of bacteria and viruses when seeded into household toilets were shown to remain in the bowl after flushing, and even continual flushing could not remove a persistent fraction. This was found to be due to the adsorption of the organisms to the porcelain surfaces of the bowl, with gradual elution occurring after each flush. Droplets produced by flushing toilets were found to harbor both bacteria and viruses which had been seeded. The detection of bacteria and viruses falling out onto surfaces in bathrooms after flushing indicated that they remain airborne long enough to settle on surface throughout the bathroom. Thus, there is a possibility that a person may acquire an infection from an aerosol produced by a toilet.     

转座子随机插入鉴定肠炎沙门氏菌生物膜形成相关基因

[目的]生物膜在沙门氏菌的致病性和引起沙门氏菌食物中毒等方面起着重要作用,本研究为了鉴定影响沙门氏菌生物膜形成的基因.[方法]利用结晶紫染色定量法对74株鸡源的肠炎、鸡白痢和鸡伤寒沙门氏菌进行生物膜测定,选择生物膜生长较好的肠炎沙门氏菌C050041,采用转座子随机插入法构建突变株库.[结果]84%的鸡源沙门氏菌菌株可在塑料表面形成生物膜;通过转座子插入获得1924个突变株,筛选的生物膜降低突变株经生长曲线测定、测序和序列比对及Southern blot分析鉴定出15个插入基因,它们分别为metE、ompR、rpoS、,和G、rfaJ、rfaK、rfaP、rfbH、rhlE、spiA、steB、tpx、ybdN和2个未知功能的基因.[结论]我们鉴定出了多个影响生物膜形成的新基因,这些基因的发现为进一步研究沙门氏菌生物膜形成的调控机制,研制减毒沙门氏菌疫苗奠定了基础.     

Synergism in biofilm formation between Salmonella enteritidis and a nitrogen-fixing strain of Klebsiella pneumoniae

A laboratory reactor, which simulates biofilm formation in water pipes, was used to study interactions in biofilm formation between a nitrogen-fixing strain of Klebsiella pneumoniae and Salmonella enteritidis. The level of attachment of Salm. enteritidis was higher in the binary biofilm than in the single species biofilm. In the initial colonization phase the binary biofilm contained a much higher proportion of metabolically active cells than in single species biofilms formed by either Salm. enteritidis or Kl. pneumoniae. When a pulse of Salm. enteritidis was passed over an already established biofilm of Kl. pneumoniae it rapidly became integrated into the biofilm, from where it was subsequently released into the water column, along with Kl. pneumoniae. Klebsiella pneumoniae fixed nitrogen in the presence of Salm. enteritidis in both types of biofilm.     

Biofilm formation by the Enterobacteriaceae: a comparison between Salmonella enteritidis, Escherichia coli and a nitrogen-fixing strain of Klebsiella pneumoniae

A simple laboratory reactor, which simulates biofilm formation in pipes, was used to compare biofilm formation by three members of the Enterobacteriaceae, namely, an environmental, nitrogen-fixing strain of Klebsiella pneumoniae , a pathogen, Salmonella enteritidis , and a faecal indicator, Escherichia coli. All three attached to CVCP pipe surfaces in the reactor and formed substantial biofilm populations of over a million bacteria cm^-2 within 24 h. These populations increased by approximately 10-fold over the next 48 h. Estimates of the numbers of metabolically active cells and the ratios of viable to direct counts showed that Kl. pneumoniae formed the densest and most metabolically active biofilms, followed by Salm. enteritidis and E. coli , respectively. Nitrogen fixation and polysaccharide production (EPS) by Kl. pneumoniae occurred only in mature biofilms and were of no selective advantage in the initiation of biofilms. Despite producing more EPS the rate of attachment of Salm. enteritidis was lower than for Kl. pneumoniae .     

A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms

AIMS: The purpose of this study was to develop a laboratory biofilm growth reactor system that simulated the toilet bowl environment and which could be used for biocide efficacy testing. METHODS AND RESULTS: A microbial biofilm reactor system incorporating intermittent flow and nutrient provision was designed. The reactor system was open to the air and was inoculated with organisms collected from toilet bowl biofilms. Once per hour, reactors were supplied with a nutrient solution for a period of 5 min, then flushed and refilled with tap water or tap water amended with chlorine. Quantitative measures of the rate and extent of biofilm accumulation were defined. Biofilm accumulated in untreated reactors to cell densities of 108 cfu cm-2 after approximately 1 week. Biofilm accumulation was also observed in reactors in the continuous presence of several milligrams per litre of free chlorine. Repeatability standard deviations for the selected efficacy measures were low, indicating high repeatability between experiments. Log reduction values of viable cell numbers were within ranges observed with standard suspension and hard surface disinfection tests. Biofilm accumulated in laboratory reactors approximately seven times faster than it did in actual toilet bowls. The same ranking was achieved in tests between laboratory biofilms and field-grown biofilms with three of the four measures, using three different concentrations of chlorine. CONCLUSION: This reactor system has been shown to simulate, in a repeatable way, the accumulation of bacterial biofilm that occurs in toilet bowls. The results demonstrate that this system can provide repeatable assays of the efficacy of chlorine against those biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The laboratory biofilm reactor system described herein can be used to evaluate potential antimicrobial and antifouling treatments for control of biofilm formation in toilet bowls.     

Biological properties of salmonellae and local immunity in Salmonella infection

The comparative study of the biological properties of S. enteritidis and S. typhimuruim, isolated from patients and convalescent carriers, was carried out. Factors inactivating the components of the local immunity of the intestine (lysozyme, complement, lactoferrin, IgG, IgM and IgA) were detected in the causative agents of Salmonella infections. The spread and expression degree of properties of a causative agent were serovar-depended: high penetration characteristics and the expression of anti-lactoferrin and anti-immunoglobulin activity were characteristic of S. typhimurium. S. enteritidis strains isolated from patients with carrier state formed in the convalescence period were found to have higher persistence level. In co-profiltrates obtained from carriers at the peak of the disease and during convalescence lower levels of IgM, IgG, sIgA, complement and lactoferrin were determined in comparison with those in coprofiltrates obtained from patients in whom no subsequent carrier state was formed. These results indicate that an increase in the persistence of salmonellae, occurring simultaneously with the local immunodeficiency, contributes to the prolonged survival of bacteria in the intestine.     

In vitro and in vivo pathogenicity of <Emphasis Type="Italic">Salmonella enteritidis</Emphasis> clinical strains isolated from North America

Salmonella enteritidis is a leading cause of food-borne gastroenteritis worldwide. In this study, 48 strains of S. enteritidis isolated from clinical cases of salmonellosis in North America were tested for their virulence-associated traits including cell invasiveness, biofilm, motility, presence of a virulence plasmid, and virulence in orally challenged mice. The majority of strains exhibited high invasiveness (n = 45), whereas only few strains (n = 3) exhibited low invasiveness. All low-invasive strains (100%, 3/3) were biofilm negative, whereas the distribution of biofilm positive and negative phenotypes among high-invasive strains was 53.4% (24/45) and 46.6% (21/45), respectively. The in vitro cell invasiveness was not associated with biofilm formation (Fisher's exact test, P = 0.23) or the presence of a spvB gene, a marker for the virulence-associated plasmid (Fisher's exact test, P = 1). There was no correlation between cell invasiveness and motility (Spearman's rank test, r = -0.15; P = 0.27). Virulence testing in orally challenged mice revealed that the low-invasive strains were as virulent as high-invasive strains, indicating that in vitro cell invasiveness did not correlate with in vivo virulence. In conclusion, we show that despite phenotypic diversity among clinical strains of S. enteritidis, the majority of strains are highly invasive in vitro and in vivo.     

Microbial contamination in kitchens and bathrooms of rural Cambodian village households

Aims: To quantify microbial contamination on kitchen and bathroom surfaces (fomites) in rural Cambodian homes and to compare these concentrations to similar data from the United States and Japan. Methods and Results: This study monitored the numbers of faecal coliforms (i.e. thermotolerant coliforms), total coliforms, Escherichia coli and heterotrophic plate count bacteria on household surfaces in a rural village of Cambodia. Faecal coliform levels in Cambodia were highest on moist locations such as the plastic ladle used for sink water, the toilet seat surface and the cutting board surface with 100‐fold higher levels of faecal coliform bacteria than E. coli and 100‐fold higher levels of faecal coliforms than the US and Japanese studies. Conclusions: A single public health intervention barrier, such as an improved latrine, is only partially effective for household sanitation. For complete sanitation, multiple environmental barriers may be necessary. These barriers occur in a house constructed with easily washable surfaces, a chlorinated water distribution system, house climate control and cleaning product availability. Significance and Impact of the Study: Results of this study can be used to emphasize the importance of increasing household environmental sanitation barriers.     

Salmonella spp. in the working environment of sewage treatment plants in Oslo, Norway.

The presence of Salmonella spp. was investigated at three sewage treatment plants in Oslo, Norway. Salmonella bacteria were isolated from floor surfaces and areas with hand contact in the treatment plant, from floor surfaces and areas with hand contact in the treatment plant, from floor surfaces in the changing rooms, and in one case from floor surfaces in an eating room. The sewage sludge contained from 140 to 140,000 Salmonella spp. per 100 g dry weight. Raw and treated sewage contained an average of 130 and 3 of these bacteria per 100 ml, respectively. There was poor correlation between the pattern of serotypes isolated from the sewage works and the patterns of those which were registered among the population of Oslo during the investigation. Neither enteropathogenic bacteria nor parasite eggs were found in fecal samples from employees at the plant. The health significance of the presence of Salmonella spp. in the environment of sewage workers is discussed.     

Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose

We report here a new screening method based on the fluorescence of colonies on calcofluor agar plates to identify transposon insertion mutants of Salmonella enteritidis that are defective in biofilm development. The results not only confirmed the requirement of genes already described for the modulation of multicellular behaviour in Salmonella typhimurium and other species, but also revealed new aspects of the biofilm formation process, such as two new genetic elements, named as bcsABZC and bcsEFG operons, required for the synthesis of an exopolysaccharide, digestible with cellulase. Non-polar mutations of bcsC and bcsE genes and complementation experiments demonstrated that both operons are responsible for cellulose biosynthesis in both S. enteritidis and S. typhimurium. Using two different growth media, ATM and LB, we showed that the biofilm produced by S. enteritidis is made of different constituents, suggesting that biofilm composition and regulation depends on environmental conditions. Bacterial adherence and invasion assays of eukaryotic cells and in vivo virulence studies of cellulose-deficient mutants indicated that, at least under our experimental conditions, the production of cellulose is not involved in the virulence of S. enteritidis. However, cellulose-deficient mutants were more sensitive to chlorine treatments, suggesting that cellulose production and biofilm formation may be an important factor for the survival of S. enteritidis on surface environments.     

肠炎沙门氏菌鸡源株ompR 基因缺失株的构建及生物学特性与亲本株的比较

【目的】为了探讨ompR基因在肠炎沙门氏菌生物被膜形成及毒力中的作用。【方法】以肠炎沙门氏菌作为母本,运用自杀性载体pGMB151构建了ompR基因缺失株,结晶紫染色法和扫描电镜观察测定缺失株的生物被膜形成能力,细胞的吸附和侵入及小鼠攻毒试验测定缺失株的毒力。【结果】RT-PCR和蛋白表达证明了ompR基因缺失株构建成功;该缺失株不表达纤维素和菌毛,不形成生物被膜;上皮细胞吸附和侵入试验表明缺失株与野生株具有相同的吸附和侵入率;BALB/c鼠腹腔感染性试验表明,缺失株的半数致死量为106.67CFU,而野生株的半数致死量小于2 CFU。【结论】ompR基因既是肠炎沙门氏菌生物膜形成的调控基因,又是重要的毒力基因。     

The relationship between glycogen synthesis, biofilm formation and virulence in salmonella enteritidis

Salmonella enteritidis accumulated large quantities of intracellular polysaccharide when grown in unrestricted nutrient conditions. Dense, abundant cytoplasmic granules were observed by electron microscopy in sections stained by the periodic acid-chlorite technique, indicating that the polysaccharide was of the glycogen type. When biofilm-producing S. enteritidis was pre-incubated in media containing increasing levels of glucose concentration, the levels of both cytoplasmic glycogen and biofilm rose correlatively to a point where a ceiling effect was observed. Studies carried out with activators and inhibitors of glycogen biosynthesis confirmed that biofilm was formed from glycogen cell stores. On the other hand, the virulence of the biofilm-producing strain in infected chickens increased proportionally to the amount of stored glycogen, suggesting a possible role of the glycogen depot in the virulence of S. enteritidis.     

A bacteriological investigation of the effectiveness of cleaning and disinfection procedures for toilet hygiene

The bacterial contamination of hospital and institutional toilets and toilet areas which were cleaned daily was investigated. The effect of daily disinfection with hypochlorite or a quaternary ammonium product, or with a continuous-release hypochlorite disinfectant system, based on the chlorine-releasing agent trichloroisocyanuric acid, was determined. The continuous release system produced substantial and sustained reduction in contamination of the toilet itself (water, toilet bowl and rim) and some reduction in contamination of sites surrounding the toilet (seat, floor, and air). By contrast, although daily disinfection produced some reduction in contamination compared with daily cleaning, the reductions were less than that associated with the continuous release system and indicated the inadequacy of daily disinfection and/or cleaning for toilets where effective procedures are required.     

Virulent strains of Salmonella enteritidis disrupt the epithelial barrier of Caco-2 and HEp-2 cells

To confirm the existence in nature of Salmonella enteritidis strains of different degrees of virulence and to elucidate the mechanisms underlying the effects of such strains on the epithelial barrier function, the consequences of infection of Caco-2 cells and HEp-2 cells with 15 S. enteritidis strains in a chicken infection model were examined. The more virulent strains of S. enteritidis, which are biofilm producers in adherence test medium, were able to disrupt HEp-2 and Caco-2 monolayers, as shown by transmonolayer electrical resistance and lactate dehydrogenase activity. In contrast, the low-virulence strains of S. enteritidis, which do not produce biofilms in adherence test medium, had no effect on the same cells. An avirulent rough mutant of Salmonella minnesota exhibited a pattern of behaviour similar to that of the low virulence strains of S. enteritidis, whilst a clinical Salmonella typhi strain caused rapid injury to the monolayers. The effect of supernatants of Salmonella cultures in adherence test medium on the integrity of Caco-2 cell monolayers indicated that the high-virulence S. enteritidis strains, but not the low-virulence strains, release a soluble factor when incubated under optimum biofilm-forming conditions, which enables the disruption of the integrity of Caco-2 monolayers.     

Bacterial plasmolysis as a physical indicator of viability.

Bacterial plasmolytic response to osmotic stress was evaluated as a physical indicator of membrane integrity and hence cellular viability. Digital image analysis and either low-magnification dark-field, high-magnification phase-contrast, or confocal laser microscopy, in conjunction with pulse application of a 1.5 M NaCl solution, were used as a rapid, growth-independent method for quantifying the viability of attached biofilm bacteria. Bacteria were considered viable if they were capable of plasmolysis, as quantified by changes in cell area or light scattering. When viable Salmonella enteritidis biofilm cells were exposed to 1.5 M NaCl, an approximately 50% reduction in cell protoplast area (as determined by high-magnification phase-contrast microscopy) was observed. In contrast, heat- and formalin-killed S. enteritidis cells were unresponsive to NaCl treatment. Furthermore, the mean dark-field cell area of a viable, sessile population of Pseudomonas fluorescens cells (approximately 1,100 cells) increased by 50% as a result of salt stress, from 1,035 +/- 162 to 1,588 +/- 284 microns2, because of increased light scattering of the condensed, plasmolyzed cell protoplast. Light scattering of ethanol-killed control biofilm cells underwent little change following salt stress. When the results obtained with scanning confocal laser microscopy and a fluorescent viability probe were compared with the accuracy of plasmolysis as a viability indicator, it was found that the two methods were in close agreement. Used alone or in conjunction with fluorochemical probes, physical indicators of membrane integrity provided a rapid, direct, growth-independent method for determining the viability of biofilm bacteria known to undergo plasmolysis, and this method may have value during efficacy testing of biocides and other antimicrobial agents when nondestructive time course analyses are required.     

电解海水的抑菌活性及对食品加工表面材料的消毒效果

为了考察直接电解海水消除细菌污染的可能性,本文将海水及海水稀释成不同浓度后通过氧化电解水装置进行电解不同时间后,所得酸性电解海水、碱性电解海水和中性电解海水对病原菌[埃希氏大肠杆菌(Escherichina coli)、沙门氏菌(Salmonella)、单核细胞增生李斯特菌(Listeria moncytogene)、摩化摩根(Morganella morganii)、副溶血性弧菌(Vibrio parahaemolyticus)]以及食品加工表面接触材料(地板砖、不锈钢板、瓷砖、手套、抹布)的消毒效果进行分析研究.结果表明,酸性电解海水具有良好的杀菌效果,能将107 CFU/mL的病原菌悬液在1 min内几乎全部杀死.碱性电解海水和中性水无明显的杀菌效果.通过模拟食品加工过程,对食品加工表面接触材料人为染菌,研究电解海水对表面材料的消毒效果,结果表明酸性电解海水仍能将表面材料含有的107CFU/cm2病原菌在5 min之内几乎全部杀灭.由此说明电解海水对食品加工表面接触材料具有明显的消毒效果,能取代以淡水为原料的电解水杀菌效果是高效廉价和不浪费淡水资源的一种理想消毒剂.     

Distribution of pathogen inhibition in the Lactobacillus isolates of a commercial probiotic consortium

Pure strains of Lactobacillus ssp. isolated from a commercial probiotic consortium were checked in a double layer solid medium for their inhibition activities against selected pathogenic bacteria including serotypes of Listeria monocytogenes, Escherichia coli and Salmonella. The antagonistic properties of the Lactobacillus strains may be related to the production of bacteriocin-like compounds. All the pathogens tested were inhibited by one or a few strains of Lactobacillus, the best inhibition was observed against L. monocytogenes but the inhibition was also satisfactory against E. coli, Salm. typhimurium and Salm. enteritidis.