Evaluation of the association between cryptosporidiosis outbreaks and two interactive water fountain parks: Epidemiologic analysis and implications for public health

Background. A community-wide outbreak of cryptosporidiosis occurred in Dallas County during the summer of 2008. A subset of cases occurring with onset of illness within a 2 week interval was epidemiologically linked to 2 neighborhood interactive water fountain parks. ^ Methods. A case control study was conducted to evaluate risk factors associated with developing illness with cryptosporidiosis from the fountain parks. Cases were selected from a line list from the epidemiological study. The selection for the controls was either healthy family members or a daycare center nearby. Cases and controls were not matched. ^ Results. Interviews were completed for 44 fountain park attendees who met case definition and 54 community controls. Twenty-seven percent (27.3%) of the cases and 13.0% of the controls were between the ages of 0–4 years. Thirty-nine percent (38.6%) of the cases and 24.1% of the controls were between the ages of 5–13 years. Fourteen percent (13.6%) of the cases and 33.3% of the controls were between the ages of 14–31 years. Twenty percent (20.5%) of the cases and 29.6% of the controls were between the ages of 32–63 years. 47.7% of the cases and 42.6% of the controls were males. Fountain park attendees who reported having been splashed in the face with water were 10 times more likely to become ill than controls (OR = 10.0, 95% CI = 2.8–35.1). Persons who reported having swallowed water from the interactive fountains were 34 times more likely to become ill than controls (OR = 34.3, 95%CI = 9.3–125.7). ^ Conclusion. Prompt reporting of cases, identification of outbreak sources, and immediate implementation of remediation measures were critical in curtailing further transmission from these particular sites through the remainder of the season. This investigation underscores the potential for cryptosporidiosis outbreaks to occur in interactive fountain parks, and the need for enhanced preventive measures in these settings. Education of the public regarding avoidance of behaviors such as drinking water from interactive fountains is also an important component of public health prevention efforts. ^

Viability of enteric bacterial pathogens in transport media

Bacterial pathogens such as enterotoxigenic Escherichia coli, Salmonella, and Campylobacter spp. are associated with up to 80% of diarrheal illness to travelers from developed countries to developing countries. In order to study acute gastrointestinal diseases, researchers from developed countries such as the United States rely on transporting clinical specimens from the developing countries to laboratories in the U.S. in transport media systems. There are few commercially available transport media systems cited in the literature or designated by transport system manufacturers for the transport of enteric bacteria. Therefore a laboratory-based study was conducted to assess three commercial available transport media systems, two gel swabs and one liquid vial, to determine the most appropriate for the maintenance and recovery of common enteric bacterial pathogens. A total of 13 bacterial enteropathogens were recovered from 25°C and 4°C storage temperatures at time points up to 21 days. The results demonstrated that the gel swab and liquid vial transport systems performed similarly for all isolates at both temperatures. All three transport media systems struggled to maintain the isolates at recoverable concentrations when stored at 4°C and it is recommended that isolates be stored at 25°C in transport media systems. Lastly, swab transport systems are recommend for transport since they are small and easy to pack, resist leakage, and are less expensive than similarly performing liquid vial transport media systems.^

Variation of genotype and prevalence of Noro virus in U.S. students in Mexico with Traveler's Diarrhea, summer of 2004 and 2005

Noro virus, a positive single stranded RNA virus has been identified as a major etiologic agent in food borne gastroenteritis and diarrheal diseases. The emergence of this organism as a major non-bacterial cause in such outbreaks is partly due to the improved diagnostic tools like Reverse Transcription Polymerase chain reaction (RTPCR) that enable its detection. Noro virus accounts for nearly 96% of non-bacterial gastroenteritis outbreaks in US (1). Travelers' Diarrhea (TD) has remained a constant public health risk in the developed nations for decades and bacteria like Entero toxigenic Escherichia coli, Entero aggregative Escherichia coli have been described as the main etiologic agents for TD (2-4). A possible viral contribution to TD has been discovered in two studies (5, 6). The current study was designed to determine the prevalence of Noro virus in a population of 107 US students with TD acquired in Mexico in 2005 and to compare the prevalence to the prevalence of Noro virus in a similar study done in 2004. This study involved the testing of clinical stool specimens from 107 subjects in 2005 for the presence of Noro virus using RTPCR. The prevalence of Noro virus in 2004 used for comparison to 2005 data was obtained from published data (5). All subjects were recruited as TD subjects in a randomized, double-blinded clinical trial comparing a standard three day dosing of Rifaximin with and without an anti motility drug Loperamide. The prevalence of Noro virus geno group I was similar in both years, but geno group II prevalence differed across the two years (p = 0.003). This study finding suggests that the prevalence of Noro virus geno groups varies with time even within a specific geographic location. This study emphasizes the need for further systematic epidemiologic studies to determine the molecular epidemiology and the prevalence patterns of different geno groups of this virus. These are essential to planning and implementation of public health measures to lessen the burden of TD due to Noro virus infection among US travelers. ^

RESPONSES OF INDICATOR AND PATHOGENIC BACTERIA TO THE PRESENCE OF CARBARYL IN WATER

Biodegradability is a desirable, if not a necessary characteristic of pesticides. Carbaryl, as Sevin, is one of the more widely used insecticides for the control of agricultural pests and has been reported to be readily degraded by microorganisms. Because of its broad application, the concentration of Sevin in surface waters has been reported to reach nearly four parts per million (PPM) in surface waters, where it has been reported to affect the growth and metabolic rates of aquatic bacterial populations. Following these reports, it is of public health importance to determine the effects of this insecticide on the growth and metabolic rates of bacteria used to indicate water pollution, and on pathogenic organisms which are found in polluted water.^ This study was conducted to determine the effect of carbaryl on the growth and metabolic rates of indicator and pathogenic organisms. Escherichia coli and Streptococcus faecalis were used as indicators, while Staphylococcus aureus and Salmonella typhimurium were the pathogens studied. Pure and mixed cultures of these organisms were exposed to two concentrations of carbaryl (Sevin).^ The study demonstrated that the fecal pollution indicator organisms, E. coli and S. faecalis respond differently to the presence of small concentrations of carbaryl in water as do the two pathogens tested, (S. typhimurium and S. aureus). The growth of all test organisms as measured by spread plate counts, was reduced by the presence of either one mg/l or five mg/l carbaryl within a period of eight days. Survival of the organisms in the presence of five mg/l carbaryl varied dependent upon whether the organism was in pure or mixed culture. In the presence of five mg/l carbaryl, both pure and mixed culture of E. coli showed longer survival. S. faecalis survived for more than eight days in pure culture, neither S. typhimurium nor S. aureus survived for eight days in pure culture.^ The metabolic rate of S. faecalis and S. aureus was reduced by both five mg/l and one mg/l Sevin concentrations, contrary to E. coli and S. typhimurium which had reduced metabolic rate with the introduction of five mg/l Sevin but showed an increase in the metabolic rate with one mg/l Sevin. There was no difference between the test and control when mixed populations were exposed to five mg/l Sevin and the metabolic rate tested. A mixture of E. coli and S. typhimurium populations showed a respiration increase over the control when exposed to one mg/l Sevin concentration. If similar effects occur in polluted surface waters, misleading results from bacteriological water quality testing may occur. ^

Characterization of fecal contamination targeting 16S rRNA bacteroides molecular markers in the Santa Cruz River, Arizona

Understanding the origins, transport and fate of contamination is essential to effective management of water resources and public health. Individuals and organizations with management responsibilities need to understand the risks to ecosystems and to humans from contact with contamination. Managers also need to understand how key contaminants vary over time and space in order to design and prioritize mitigation strategies. Tumacacori National Historic Park (NHP) is responsible for management of its water resources for the benefit of the park and for the health of its visitors. The existence of microbial contaminants in the park poses risks that must be considered in park planning and operations. The water quality laboratory at the Maricopa Agricultural Center (in collaboration with stakeholder groups and individuals located in the ADEQ-targeted watersheds) identified biological changes in surface water quality in impaired reaches rivers to determine the sources of Escherichia coli (E. coli); bacteria utilizing innovative water quality microbial/bacterial source tracking methods. The end goal was to support targeted watershed groups and ADEQ towards E. coli reductions. In the field monitoring was conducted by the selected targeted watershed groups in conjunction with The University of Arizona Maricopa Agricultural Center Water Quality Laboratory. This consisted of collecting samples for Bacteroides testing from multiple locations on select impaired reaches, to determine contamination resulting from cattle, human recreation, and other contributions. Such testing was performed in conjunction with high flow and base flow conditions in order to accurately portray water quality conditions and variations. Microbial monitoring was conducted by The University of Arizona Water Quality Laboratory at the Maricopa Agricultural Center using genetic typing to differentiate among two categories of Bacteroides: human and all (total). Testing used microbial detection methodologies and molecular source tracking techniques.^

PCR-based assay using occult blood detection cards for detection of diarrheagenic Escherichia coli in specimens from U.S. travelers to Mexico with acute diarrhea.

Large field studies of travelers' diarrhea for multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport, and storage of fecal specimens that does not require immediate processing and refrigeration and that is stable for months would be advantageous. This study was designed to determine if enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E. coli (EAEC) DNA could be identified from cards that were processed for the evaluation of fecal occult blood. U.S. students traveling to Mexico during 2005 to 2007 were monitored for the occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard methods. Cards then were stored at room temperature prior to DNA extraction. Fecal PCR was performed to identify ETEC and EAEC in DNA extracted from stools and from occult blood cards. Significantly more EAEC cases were identified by PCR that was performed on DNA that was extracted from cards (49%) or from frozen feces (40%) than from culture methods that used HEp-2 adherence assays (13%) (P < 0.001). Similarly, more ETEC cases were detected from card DNA (38%) than from fecal DNA (30%) or by culture that was followed by hybridization (10%) (P < 0.001). The sensitivity and specificity of the card test were 75 and 62%, respectively, compared to those for EAEC by culture and were 50 and 63%, respectively, compared to those for ETEC. DNA extracted from fecal cards that was used for the detection of occult blood is of use in identifying diarrheagenic E. coli.

A PCR based assay for the detection of enteric pathogens from Hemoccult(RTM) cards

Background. Large field studies in travelers' diarrhea (TD) in multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport and storage of fecal specimens that does not require immediate processing, refrigeration and is stable for months would be advantageous. ^ Objectives. Determine if enteric pathogen bacterial DNA can be identified in cards routinely used for evaluation of fecal occult blood. ^ Methods. U.S. students traveling to Mexico in 2005-07 were followed for occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard method. Cards were then stored at room temperature prior to DNA extraction. A multiplex fecal PCR was performed to identify enterotoxigenic Escherichia coli and enteroaggregative E. coli (EAEC) in DNA extracted from stools and occult blood cards. ^ Results. Significantly more EAEC cases were identified by PCR done in DNA extracted from cards (49%) or from frozen feces (40%) than by culture followed by HEp-2 adherence assays (13%). Similarly more ETEC cases were detected in card DNA (38%) than fecal DNA (30%) or culture followed by hybridization (10%). Sensitivity and specificity of the card test was 75% and 62%, respectively, and 50% and 63%, respectively, when compared to EAEC and ETEC culture, respectively, and 53% and 51%, respectively compared to EAEC multiplex fecal PCR and 56% and 70%, respectively, compared to ETEC multiplex fecal PCR. ^ Conclusions. DNA extracted from fecal cards used for detection of occult blood is of use in detecting enteric pathogens. ^

Household health assessment and associated water sample analysis in the Miches watershed of the Dominican Republic

Background. Diarrhea and malnutrition are the leading causes of mortality for children age one to four in the Dominican Republic. Communities within the Miches watershed lack sanitation infrastructure and water purification systems, which increases the risk of exposure to water-borne pathogens. The purpose of this cross-sectional study was to analyze health information gathered through household interviews and to test water samples for the presence of diarrheagenic pathogens and antibiotic-resistant bacteria within the Miches watershed. Methods. Frequency counts and thematic analysis were used to investigate Human Health Survey responses and Fisher's exact test was used to determine correlation between water source and reported illness. Bacteria cultured from water samples were analyzed by Gram stain, real-time PCR, API® 20E biochemical identification, and for antibiotic resistance. Results. Community members reported concerns about water sources with respect to water quality, availability, and environmental contamination. Pathogenic strains of E. coli were present in the water samples. Drinking aquifer water was positively-correlated with reported stomach aches (p=0.04) while drinking from rivers or creeks was associated with the reported absence of “gripe” (cold or flu) (p=0.01). The lack of association between reported illnesses and water source for the majority of variables suggested that there were multiple vehicles of disease transmission. Antibiotic resistant bacteria were isolated from the water samples tested. Conclusions. The presence of pathogenic E. coli in water samples suggested that water is at least one route of transmission for diarrheagenic pathogens in the Miches watershed. The presence of antibiotic-resistant bacteria in the water samples may indicate the proliferation of resistance plasmids in the environment as a result of antibiotic overuse in human and animal populations and a lack of sanitation infrastructure. An intervention that targets areas of hygiene, sanitation, and water purification is recommended to limit human exposure to diarrheagenic pathogens and antibiotic-resistant organisms. ^

The evidence for groundwater as a source of enteric illness in developing countries: A systematic review of the literature

Groundwater constitutes approximately 30% of freshwater globally and serves as a source of drinking water in many regions. Groundwater sources are subject to contamination with human pathogens (viruses, bacteria and protozoa) from a variety of sources that can cause diarrhea and contribute to the devastating global burden of this disease. To attempt to describe the extent of this public health concern in developing countries, a systematic review of the evidence for groundwater microbially-contaminated at its source as risk factor for enteric illness under endemic (non-outbreak) conditions in these countries was conducted. Epidemiologic studies published in English language journals between January 2000 and January 2011, and meeting certain other criteria, were selected, resulting in eleven studies reviewed. Data were extracted on microbes detected (and their concentrations if reported) and on associations measured between microbial quality of, or consumption of, groundwater and enteric illness; other relevant findings are also reported. In groundwater samples, several studies found bacterial indicators of fecal contamination (total coliforms, fecal coliforms, fecal streptococci, enterococci and E. coli), all in a wide range of concentrations. Rotavirus and a number of enteropathogenic bacteria and parasites were found in stool samples from study subjects who had consumed groundwater, but no concentrations were reported. Consumption of groundwater was associated with increased risk of diarrhea, with odds ratios ranging from 1.9 to 6.1. However, limitations of the selected studies, especially potential confounding factors, limited the conclusions that could be drawn from them. These results support the contention that microbial contamination of groundwater reservoirs—including with human enteropathogens and from a variety of sources—is a reality in developing countries. While microbially-contaminated groundwaters pose risk for diarrhea, other factors are also important, including water treatment, water storage practices, consumption of other water sources, water quantity and access to it, sanitation and hygiene, housing conditions, and socio-economic status. Further understanding of the interrelationships between, and the relative contributions to disease risk of, the various sources of microbial contamination of groundwater can guide the allocation of resources to interventions with the greatest public health benefit. Several recommendations for future research, and for practitioners and policymakers, are presented.^

Development and Characterization of an in vitro Four-species Anaerobic Dental Biofilm Model

Dental caries is the most common chronic disease worldwide. It is characterized by the demineralization of tooth enamel caused by acid produced by cariogenic dental bacteria growing on tooth surfaces, termed bacterial biofilms. Cariogenesis is a complex biological process that is influence by multiple factors and is not attributed to a sole causative agent. Instead, caries is associated with multispecies microbial biofilm communities composed of some bacterial species that directly influence the development of a caries lesion and other species that are seemingly benign but must contribute to the community in an uncharacterized way. Clinical analysis of dental caries and its microbial populations is challenging due to many factors including low sensitivity of clinical measurement tools, variability in saliva chemistry, and variation in the microbiota. Our laboratory has developed an in vitro anaerobic biofilm model for dental carries to facilitate both clinical and basic research-based analyses of the multispecies dynamics and individual factors that contribute to cariogenicity. The rational for development of this system was to improve upon the current models that lack key elements. This model places an emphasis on physiological relevance and ease of maintenance and reproducibility. The uniqueness of the model is based on integrating four critical elements: 1) a biofilm community composed of four distinct and representative species typically associated with dental caries, 2) a semi-defined synthetic growth medium designed to mimic saliva, 3) physiologically relevant biofilm growth substrates, and 4) a novel biofilm reactor device designed to facilitate the maintenance and analysis. Specifically, human tooth sections or hydroxyapatite discs embedded into poly(methyl methacrylate) (PMMA) discs are incubated for an initial 24 hr in a static inverted removable substrate (SIRS) biofilm reactor at 37°C under anaerobic conditions in artificial saliva (CAMM) without sucrose in the presence of 1 X 106 cells/ml of each Actinomyces odontolyticus, Fusobacterium nucleatum, Streptococcus mutans, and Veillonella dispar. During days 2 and 3 the samples are maintained continually in CAMM with various exposures to 0.2% sucrose; all of the discs are transferred into fresh medium every 24 hr. To validate that this model is an appropriate in vitro representation of a caries-associated multispecies biofilm, research aims were designed to test the following overarching hypothesis: an in vitro anaerobic biofilm composed of four species (S. mutans, V. dispar, A. odontolyticus, and F. nucleatum) will form a stable biofilm with a community profile that changes in response to environmental conditions and exhibits a cariogenic potential. For these experiments the biofilms as described above were exposed on days 2 and 3 to either CAMM lacking sucrose (no sucrose), CAMM with 0.2% sucrose (constant sucrose), or were transferred twice a day for 1 hr each time into 0.2% sucrose (intermittent sucrose). Four types of analysis were performed: 1) fluorescence microscopy of biofilms stained with Syto 9 and hexidium idodine to determine the biofilm architecture, 2) quantitative PCR (qPCR) to determine the cell number of each species per cm2, 3) vertical scanning interferometry (VSI) to determine the cariogenic potential of the biofilms, and 4) tomographic pH imaging using radiometric fluorescence microscopy after exposure to pH sensitive nanoparticles to measure the micro-environmental pH. The qualitative and quantitative results reveal the expected dynamics of the community profile when exposed to different sucrose conditions and the cariogenic potential of this in vitro four-species anaerobic biofilm model, thus confirming its usefulness for future analysis of primary and secondary dental caries.

Functional taxonomy of bacterial hyperstructures.

The levels of organization that exist in bacteria extend from macromolecules to populations. Evidence that there is also a level of organization intermediate between the macromolecule and the bacterial cell is accumulating. This is the level of hyperstructures. Here, we review a variety of spatially extended structures, complexes, and assemblies that might be termed hyperstructures. These include ribosomal or "nucleolar" hyperstructures; transertion hyperstructures; putative phosphotransferase system and glycolytic hyperstructures; chemosignaling and flagellar hyperstructures; DNA repair hyperstructures; cytoskeletal hyperstructures based on EF-Tu, FtsZ, and MreB; and cell cycle hyperstructures responsible for DNA replication, sequestration of newly replicated origins, segregation, compaction, and division. We propose principles for classifying these hyperstructures and finally illustrate how thinking in terms of hyperstructures may lead to a different vision of the bacterial cell.

Drinking water quality perception along the U.S.-Mexico border

Drinking water-related exposures within populations living in the United States-Mexico border region, particularly among Hispanics, is an area that is largely unknown. Specifically, perceptions that may affect water source selection is an issue that has not been fully addressed. This study evaluates drinking water quality perceptions in a mostly Hispanic community living along the United States-Mexico border, a community also facing water scarcity issues. Using a survey that was administered during two seasons (winter and summer), data were collected from a total of 608 participants, of which 303 were living in the United States and 305 in Mexico. A (random) convenience sampling technique was used to select households and those interviewed were over 18 years of age. Statistically significant differences were observed involving country of residence (p=0.002). Specifically, those living in Mexico reported a higher use of bottled water than those living in the United States. Perception factors, especially taste, were cited as main reasons for not selecting unfiltered tap water as a primary drinking water source. Understanding what influences drinking water source preference can aid in the development of risk communication strategies regarding water quality. ^

Diffusely adherent Escherichia coli (DAEC) as a cause of acute diarrhea

Acute diarrhea is the most common medical problem in the developing countries. Infectious agents are responsible for a majority of cases of acute diarrhea. Knowing the cause of acute diarrhea is important to developing plans for disease prevention, control and therapy. Acute diarrhea is caused by many viruses, bacteria and parasites. ^ Travelers to developing countries of the world commonly develop diarrhea as a result of eating contaminated food or drinking contaminated water. About 30-50% of travelers who travel from industrialized countries like United States to the developing countries are at risk of developing diarrhea. High risk areas for travelers' diarrhea are Mexico, Latin America and Southeast Asia. Public restaurants are the common sites for exposure to this type of food-borne infectious disease in travelers. Food becomes contaminated when they are handled by people with fecal content on their hands. ^ The importance of Diffusely Adherent Escherichia Coli (DAEC) in travelers to these areas has not been well studied. Some of the studies looking at DAEC have shown the organism to be present in children without symptoms. Other studies have shown a relationship between DAEC infection and presence of symptoms. I have selected this topic because the patho-physiological processes in DAEC infection that allow intestinal and extra-intestinal infections to develop are not fully understood. DAEC related acute diarrhea is a relatively new topic of public health significance. There is a limited number of studies regarding the virulence and pathogenic mechanisms of DAEC. The presumed virulence factor of the organism is diffuse attachment to the intestinal lining of the infected host. However more research needs to be done to identify the pathogenic mechanisms and virulence factors associated with DAEC infection for better treatment planning and diarrhea prevention. ^

Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria

Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. RIF-resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes ≤2 days. However, qPCR identification of resistant isolates, particularly for isolates with mixed RIF-susceptible and RIF-resistant bacteria, is reader dependent and limits its clinical use. The aim of this study was to develop an objective, reader-independent method to define rpoB mutants using beacon qPCR. This would facilitate the transition from a research protocol to the clinical setting, where high-throughput methods with objective interpretation are required. For this, DNAs from 107 M. tuberculosis clinical isolates with known susceptibility to RIF by culture-based methods were obtained from 2 regions where isolates have not previously been subjected to evaluation using molecular beacon qPCR: the Texas–Mexico border and Colombia. Using coded DNA specimens, mutations within an 81-bp hot spot region of rpoB were established by qPCR with 5 beacons spanning this region. Visual and mathematical approaches were used to establish whether the qPCR cycle threshold of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIF-susceptible and RIF-resistant bacteria. Only then had the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF-resistance by culture phenotype and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the mathematical approach has advantages over the visual reading, in that it uses a Microsoft Excel template to eliminate reader bias or inexperience, and allows objective interpretation from high-throughput analyses even in the presence of a mixture of RIF-resistant and RIF-susceptible isolates without the need for reader training.^

TRANSMISSION DYNAMICS OF ENTERIC BACTERIA IN DAY-CARE CENTERS, HOUSTON, TEXAS

An investigation was undertaken to evaluate the role of fomites in the transmission of diarrhea in day-care centers (DCC) and to elucidate the paths by which enteric organisms spread within this setting.^ During a nine-month period (December 1980-August 1981) extensive culturing of inanimate objects, as well as children and staff was done routinely each month and again repeated during diarrhea outbreaks. Air was sampled from the classrooms and toilets using a Single-Stage Sieve Sampler (Ross Industries, Midland, VA.). Stool samples were collected from both ill and well children and staff in the affected rooms only during outbreaks. Environmental samples were processed for Shigella, salmonella and fecal coliforms while stools were screened for miscellaneous enteropathogens.^ A total of 11 outbreaks occurred in the 5 DCC during the study period. Enteric pathogens were recovered in 7 (64%) of the outbreaks. Multiple pathogens were identified in 3 outbreaks. The most frequently identified pathogen in stools was Giardia lamblia which was recovered in 5 (45%) of the outbreaks. Ten of the 11 (91%) outbreaks occurred in children less than 12 months of age.^ Environmental microbiology studies together with epidemiologic information revealed that enteric organisms were transmitted from person-to-person. On routine sampling, fecal coliforms were most frequently isolated from tap handles and diaper change areas. Contamination with fetal coliforms was wide-spread during diarrhea outbreaks. Fecal coliforms were recovered with significantly greater frequency from hands, toys and other classroom objects during outbreaks than during non-outbreak period. Salmonella typhimurium was recovered from a table top during an outbreak of Salmonellosis. There was no association between the level of enteric microbial contamination in the toilet areas and the occurrence of outbreaks. No evidence was found to indicate that enteric organisms were spread by the airborne route via aerosols.^ Toys, other classroom objects and contaminated hands probably play a major role in the transmission of enteropathogens during day-care center outbreaks. The presence of many enteric agents in the environment undoubtedly explains the polymicrobial etiology of the day-care center associated diarrhea outbreaks. ^