Ecology of pathogenic Escherichia coli in water : survival and molecular detection

Recently there has been a surge of interest in the ability of enterohemmorhagic Escherichia coli (EHEC) to survive in aquatic environments. It has been speculated a correlation may exist between the genotype of an E. coli strain and its ability to persist in an aquatic environment. One of the objectives of this study was to determine if any correlation could be made between the persistence of EHEC strains in natural well water and their ERIC-genotype. Sixteen strains of EHEC were monitored, individually, in untreated well water microcosms incubated at 10°C and 22°C for 56 days. The strains were selected from three serogroups (026, 0111 and 0157) and represented six distinct genotypes determined by ERIC-PCR. The microcosms were prepared, in triplicate, each sample having a final cell concentration of approximately 10^ cfu/ml well water. Levine Eosin Methylene Blue agar was used to determine the cell density of culturable E. co//cells. At 10°C, cell density declined depending on the strains by 1.78 to 6.07 log units in 8 weeks, with six strains falling below the detection limit of 0.8 log cfu/ml by day 56. Of the ten persisting strains, four showed superior survival with cell density decreasing to an average of approximately 5 log cfu/ml while the remaining six strains showed moderate levels of survival, decreasing to a average cell density of approximately 3.5 log cfu/ml. At 22°C all strains eventually dropped by 4.87 to 6.52 log in 56 days with 14 strains dropping below the detection limit. The 16 strains demonstrated highly variable levels of survival with no correlation between ERIC-genotype and the strain’s ability to persist being evident. The second objective of the study was to develop a molecular detection method for pathogenic E. coli employing multiplex-PCR and DIG-labeled DNA probes. The molecular detection of EHEC and other pathogenic £. coli of virotypes, entertoxigenic (ETEC), enteropathogenic (EPEC) and enteroinvasive (EIEC), has been the topic of numerous studies. A multiplex PCR-DNA probing assay to detect the four major E. coli virotypes was developed. Six highly specific PCR primer sets and DIG-labeled chemiluminescent probes were designed to target the shiga-like toxin I and II genes {sitl and sitll) of EHEC, heat stable and heat labile toxin genes (hs and hl) of ETEC, EPECadherence factor gene (eaf) of EPEC and the invasiveness plasmid (iai) of EIEC. The primer sets generate amplicons 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers were tested for specificity against 31 pathogenic £. coli strains, various non-pathogenic £. coli strains and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity for strains from their corresponding virotypes and minimal non-specific reaction with the non-target E. coli and control bacterial strains. To improve the specificity of the multiplex-PCR amplification, six DIG-labeled oligonucleotide probes were designed to hybridize with the six multiplex-PCR amplicons. Chemiluminescent detection of probe hybridization against multiplex-PCR dot blots revealed high degrees of specificity of probes for the target amplicons. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major E. coli virotypes

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsa-stained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a co-agglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates

Abundance and Distribution of Enteric Bacteria and Viruses in Coastal and Estuarine Sediments—a Review

The long term survival of fecal indicator organisms (FIOs) and human pathogenic microorganisms in sediments is important from a water quality, human health and ecological perspective. Typically, both bacteria and viruses strongly associate with particulate matter present in freshwater, estuarine and marine environments. This association tends to be stronger in finer textured sediments and is strongly influenced by the type and quantity of clay minerals and organic matter present. Binding to particle surfaces promotes the persistence of bacteria in the environment by offering physical and chemical protection from biotic and abiotic stresses. How bacterial and viral viability and pathogenicity is influenced by surface attachment requires further study. Typically, long-term association with surfaces including sediments induces bacteria to enter a viable-but-non-culturable (VBNC) state. Inherent methodological challenges of quantifying VBNC bacteria may lead to the frequent under-reporting of their abundance in sediments. The implications of this in a quantitative risk assessment context remain unclear. Similarly, sediments can harbor significant amounts of enteric viruses, however, the factors regulating their persistence remains poorly understood. Quantification of viruses in sediment remains problematic due to our poor ability to recover intact viral particles from sediment surfaces (typically <10%), our inability to distinguish between infective and damaged (non-infective) viral particles, aggregation of viral particles, and inhibition during qPCR. This suggests that the true viral titre in sediments may be being vastly underestimated. In turn, this is limiting our ability to understand the fate and transport of viruses in sediments. Model systems (e.g., human cell culture) are also lacking for some key viruses, preventing our ability to evaluate the infectivity of viruses recovered from sediments (e.g., norovirus). The release of particle-bound bacteria and viruses into the water column during sediment resuspension also represents a risk to water quality. In conclusion, our poor process level understanding of viral/bacterial-sediment interactions combined with methodological challenges is limiting the accurate source apportionment and quantitative microbial risk assessment for pathogenic organisms associated with sediments in aquatic environments

Incidence and Tracking of Escherichia coli O157:H7 in a Major Produce Production Region in California

Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995–2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human illness

Alien Registration- Watterworth, Elizabeth F. (Lubec, Washington County)

https://digitalmaine.com/alien_docs/2470/thumbnail.jp

Risk factors for low physical activity levels in preschool-aged children in urban Bangladesh

Physical activity (PA) is a known determinant of health and is important in the development of young children. Risk factors for PA may be different in low- and middle-income countries, such as urban Bangladesh, where the environment is densely populated and nutritional deficits are common. We aimed to describe the PA levels of preschoolers in Dhaka, Bangladesh, as well as estimate the associations between preschooler PA levels and household and nutritional factors. We conducted a cross-sectional, observational study, collecting accelerometry, hemoglobin concentration, socioeconomic status, anthropometry, and data on the home built environment. Linear mixed-effects models were used to assess the associations between PA and the exposures of interest. The entire sample were found to meet Canadian preschooler PA guidelines for daily activity. There were no associations between PA and characteristics of the built environment of the home, nor hemoglobin concentration, suggesting a need for further exploration of potential influential factors.M.Sc.2020-11-15 00:00:0

Alien Registration- Watterworth, Elizabeth F. (Lubec, Washington County)

https://digitalmaine.com/alien_docs/2470/thumbnail.jp

The Power of Partnering: The Cooperative Creation of Digital Collections

The use of consortia and partnerships has increased significantly over the past decade as a result of the increasing complexity of developing projects in a digital world. Funding agencies have acknowledged this by directing support to multi-institutional projects. Some agencies have gone so far as to establish categories that require multi-institutional applicants. Partnerships of disparate institutions provide opportunities for learning and growth. The paper describes one such partnership, Connecticut History Online, and investigates the value and significance that partnering plays in creating a successful digital product

Fitness Measures Among Boy Scouts Completing the Personal Fitness Merit Badge

Introduction: A formative framework for positive lifestyle and health behaviors is established during adolescence. The Boy Scouts of America (BSA) is one organization that promotes healthy lifestyles among adolescent males and includes earning the Personal Fitness Merit Badge (PFMB). Despite the BSA Oath to keep myself physically strong , there is a paucity of evidence describing fitness test (FT) outcomes or correlations between FT and variables including age or body mass index (BMI) among Boy Scouts. Therefore, the purpose of this study is to describe and correlate FT to age and BMI among adolescent Boy Scouts. Methods: A prospective study design recruited Boy Scouts aged 11-17 years from Southeast Michigan using a sample of convenience. After securing physician clearance to participate, FT measures were performed utilizing previously established methodology. Third year doctor of physical therapy students trained in the measurement protocol performed the FT measures. Measures encompassed those required for the PFMB: BMI, pull-ups, push-ups, sit and reach flexibility test (SRF), sit-ups, and the one mile run (1MR). Data was analyzed using descriptive statistics, a Kruskal-Wallis test examined relationships between BMI and FT, and a nonparametric Spearman correlation examined correlations between FT performance and both age and BMI with statistical significance set at less than 0.05. Results: Ninety-nine Boy Scouts, whose mean age was 12.6 years (SD 1.4), met the inclusion criteria. The mean BMI was 21.5 (kg/height (cm)) 2 (SD 5.4) with eight scouts meeting criteria of underweight, 56 normal weight, 14 overweight, and 21 obese. Fitness test results were as follows: pull-ups 1.75 (SD 2.7), push-ups 18.0 (SD 10.6), SRF 21.2 cm (SD 10.5), sit-ups 28.9 (SD 8.7), and the 1MR run 616.5 seconds (SD 156.8). When comparing normal weight scouts to overweight and obese scouts, a significantly higher frequency of pull-ups (p=0.002, p=.001), push-ups (p=0.02, p=0.03), sit-ups (p=0.01, p=0.003,), and decreased time for completion on the 1MR (p=0.001, p=0.001) was identified, respectively. Furthermore, while no correlations were identified by age, a negative correlation was identified between increased sit-up frequency (r=-0.36, p=0.001) and decreased 1MR time performance (r=0.39, p=0.001) and a higher BMI. Discussion: While prior evidence suggests improved FT scores in adolescent males with advancing age, this was not observed. Less favorable PFMB required FT performance with an increased BMI among Boy Scouts is in congruence with prior reports for adolescent males. Conclusion: Variations in FT performance levels were observed among Boy Scouts completing the initial FT requirements of the PFMB. However, correlations between higher BMI and less favorable FT performance were detected