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UMD FIRE EP Stream 
http://www.sciencedirect.com/science/article/pii/S0964830515001456
1. What did they do?
2. How did they do it?
3. Why do we care?
In my article researchers investigated antibiotic resistant genes (ARGs) and bacteria (ARB) in water that has travelled from a natural source, to a treatment plant, then to the distribution line for the public.
To do this, triplicate samples were taken from each checkpoint for six months. The samples were first analyzed to determine pH, chemical oxygen demand (COD), total nitrogen, and total phosphate levels. Samples were also cultured and isolated, a BIOLAG method and biochemical tests were used to identify bacteria. Antibiotic resistance was analyzed based on the size of the zone of inhibition surrounding antibiotic discs on lawn plates of the bacteria. Aside from this, DNA was also analyzed; by first extracting it from the water samples with a Fast ID DNA Extraction Kit, then using PCR to amplify the samples to be run on a gel. And finally, chemical data was “subjected to an analysis of variance (ANOVA) test (p ≤ 0.05) followed by a Tukey “post hoc” analysis when needed (SAS)”.
We care for a couple of reasons. Firstly, antibiotic resistance impacts everyone, not just the environment, not just animals, and not just the area it is occurring in. The presence and spread of antibiotic resistance will impact our public health as well as our drinking water industry. Therefore it is important to determine where and how this resistance is occurring, and if it can be determined how to stop it then even better.
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http://web.a.ebscohost.com.proxy-um.researchport.umd.edu/ehost/pdfviewer/pdfviewer?sid=30cb39d3-88aa-4b24-9670-4b8777577aef%40sessionmgr4005&vid=4&hid=4107
The goal of this study was to identify the presence of pathogenic viruses in municipal sewage using a combined method that included cell culture along with microarrays that have multiple targets. The disease potential associated with water pollution on the community was then assessed.
For conducting this experiment, the researchers collected thirteen 6-liters of untreated sewage samples every month over a period of 13 months. Prior to freezing, the samples were concentrated by organic flocculation and centrifugation. African Green Monkey Kidney cell line (BGM) was used for the viral infections which were allowed to proceed until the onset of cytopathic effects. Viral recovery was done by freeze-thawing three times to disrupt cell integrity. Viral particles were then recovered using the Amicon Ultra 100K centrifugation column (Millipore, Billerica, MA). The extraction of viral nucleic acid was carried out by using QIAmp viral RNA mini kit (Qiagen Inc, Valencia, CA). Seven RNA and ten DNA virus groups were identified as targets. The viral genome sequence was obtained from GenBank using the probe design software Oligoarray 2.1. Semi-random primed labeling was used to label viral RNA. This was followed by microarray hybridization and scanned images were analyzed using GenePix Pro 5.0 software. DetectiV software package was used for data analysis. To ensure high degree of specificity in hybridization, stringent wash protocols were implemented. A PCR method was also incorporated to detect viral targets and confirm sequences.
Using the cell culture method in conjunction with microarray and PCR, a panel of different viruses were detected from sewage samples. Of these, enterovirus A and human astrovirus (7/13) were the most predominant followed by adenovirus. Detection of viruses using the microarray was more specific for enteric viruses compared to the PCR method. Another interesting aspect of the study was the difference in circulating titers of viruses during colder months versus warmer months. The prevalence of higher titers of viruses during colder months could be attributed to either higher levels of discharge of virus into sewage or the resilience of viruses to survive during colder climates.
Waterborne disease transmission is a common cause for enteric virus outbreaks in communities. This study is in line with understanding the potential risks of waterborne diseases. The multipronged method of using cell culture, microarray and PCR methods allowed for a broader analysis of viral pathogens. The study is important because it opens up channels of communication between the wastewater industry and health communities to improve the understanding of exposure, disease and prevention strategies. The reason I chose this article was because recently, I had heard about common outbreaks of stomach flu viruses. In addition to this, the fact that viruses have not been studied as extensively as bacteria picked my curiosity and I was interested in learning more about the topic.
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http://www.sciencedirect.com/science/article/pii/S0048969715302813?np=y
In this study, researchers examined the influence of different water treatment procedures on antibiotic resistance, particularly in high microbial environments. This study focused on two water sources, the Huangpu River and Qingcaosha Reservoir, because of their high accumulation of ARG pollutants. The five antibiotics tested were ampicillin (AMP), kanamycin (KAN), rifampicin (RFP), chloramphenicol (CM), and streptomycin (STR). These five were chosen because they are popular in both human health and the environment.
Research was conducted by taking monthly 10 L samples from the two water sources being investigated. Water treatment processes included pre-ozonization, coagulation and sedimentation, sand filtration, post-ozonization, broken activated carbon (BAC) filtration and chloramine disinfection. Antibiotic resistant bacteria were isolated from the water treatment samples by creating lawn plates with R2A agar. The plates were incubated for 7 days at 28 degrees C. The antibiotic rate was calculated as the ratio of the average number of incubated antibiotic resistant bacteria to average heterotrophic plate count. The BAC samples were washed with water three times and soaked in glutaraldehyde followed by being immersed in ethanol. These samples were observed by SEM to measure biomass. DNA extraction from the original samples was performed using a Water DNA Kit. DNA and purity were measured via microspectrophotometry. QIIME data analysis package was used for16S rDNA data analysis.
We care because the widespread use of antibiotics has caused an increase in ARG pollutants among several water sources. The large accumulation of ARGs and ARBs makes it difficult to eliminate antibiotics from drinking water sources, which causes a risk to human health.
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In this study, researchers examined the infection risk of viruses in drinking water in the Netherlands. This requires assessment of the virus concentrations and the efficiency of removing them with treatment processes. They studied 10 locations for 4 years of collecting water and analyzing it for various enteric viruses.
The methods included sampling 200-600 liters from 10 locations in the Netherlands. They were near intake areas, where drinking companies would obtain their water. Temperature, pH, turbidity, and time of year were noted. They were eluated with beef extract solution and neutralized with acetic acid. That was filtered using a 2 phase separation method. The determination of concentration of viruses was determined with a monolayer plaque assay, which involved making cell cultures. The researchers incubated for 2 hours and then added a agar overlay. After they incubated for 9 days at 37 degrees Celsius. For norovirus and rotavirus the researchers used RT-PCR.
This research article is important to our research since we are trying to determine what viruses are contained in the DC-metropolitan area. This article shows that one of the best methods to know the concentration of viruses is to make cell cultures and not just rely on RT-PCR.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935033/
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Click to access 1599.full.pdf
In this paper, the researchers explore how a fluctuating thermal environment, mediated by host thermoregulatory behavior, influences the course of host-pathogen interaction. In order to do so, they used the desert locus, Schistocerca gergaria, and the fungal pathogen M. anisopliae var. acridium as their systems. They examined whether behavioral fever, is adaptive to the host and what the consequences are to the pathogen.
There were 6 different treatments, and 4 duplicates of each treatment, Each treatment and each duplicate had a different cage, for a total of 24 cages. Each cage had 3 lightbulbs, and in each cage were 10 male and 10 female locusts. First, the scientist allowed the pathogen to infect the host through its natural processes: germination and penetration of the cuticle. Each cage had different wattage light-bulbs to simulate 3 different daytime regimes. 40W was optimal, 25W was suboptimal, and 12W was poor. 40W puts the locust in fever range, 25W put the locust in their normal preferred range, and 12W put locusts under their preferred temperature range. Using a barrier, the locusts could not be closer than 2cm to the bulb, limiting the amount the locusts could bask near the bulb to thermoregulate themselves. Thus, the scientist had a controlled and limited environment where they could study how the locusts would attempt to adjust to fever temperatures. Day night cycles were simulated, and the locusts’ temperature was taken using a copper-constantan thermocouple linked to a thermometer. Mortality and moulting were noted daily, including if death was before or after moulting. Adults were examined for defects and haemocyte nodules. If a locust died, it was placed on petri dishes, and analyzed for the colonization of fungi or bacteria. If any mating had been observed, only whether or not hatchlings emerged was noted.
This is important to us because whenever we take water samples, we note different environmental aspects, most relevant to this article being temperature and exposure to sunlight. I honestly don’t know why I write those things down. I just have a vague belief that the temperature probably influences bacterial concentrations somehow, so I jot it down. Geo tracking research takes a hard look at these environmental aspects such as temperature, proximity to bacterial sources, water flow, and weather and tries to correlate that to pathogen concentrations and types. This article focuses on the weather aspects of this goal. While this article is not directly related to MST, understanding how certain organisms interact with pathogens and temperature, could go a long way in our understanding of how bacterial concentrations change over time.
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Potential Human Pathogenic Bacteria in a Mixed Urban Watershed as Revealed by Pyrosequencing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835799/
In this paper, the researchers examined microbial source tracking methods with 454 high throughput pyrosequencing to identify bacterial DNA sequences in a water sample. Through pyrosequencing, a total of 12,959 16S rRNA seqneces are obtained through 19 water sample collection sites.
Water samples were collected from Santa Ana River Watershed in Southern California. The water samples were then filtered to extract DNA via the Power Soil and Water DNA kit. The DNA kit extracted was confirmed with Nanodrop ID-1000 to quantify the amount of DNA in the sample. The samples were then sent to Core for Applied Genomics and Ecology for pyrosequencing. The region of the 16S rRNA gene was amplified and examined for the sequencing. The resulting DNA are then blasted to RDP Classifier databases to identify potential hazardous pathogens. Mycobacteria, Ligionella, Treponema, and Clostridia were commonly found in the water sample sites. Utilizing this method, allows a quicker method to identify potential pathogens rather than using PCR to guess and check different microbial species. Pyrosequencing is a more efficient way compared to that of PCR.
This method can be utilized for our research. A way to implement pyrosequencing would be to run the water samples after DNA extraction to determine different types of pathogens. Using qPCR method is a time and cost consuming method. The amount different of primers and TAQ used to identify potential species can be significantly reduced by knowing exactly which type of species are present in the sample via DNA sequencing. This method can also increase the effectiveness of the Geo Tracking group due to the large sample of DNA extracted. Running a PCR with several samples are prone to high errors and time consumption.
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In this paper the researchers developed a multiplex PCR to identify antibiotic resistances genes in Staphylococcus aureus. The genes that were targeted by the researchers were mecA, aacA-aphD, tetK, tetM, erm(A), erm(C), vat(A), vat(B), and vat(C).
To develop and test their multiplex PCR the researchers started with a strain of MRAS (methicillin resistant Staphylococcus aureus) that was grown in their lab. The researchers performed a broth dilution assay to confirm that the bacterial strain that was being use was actually resistant to antibiotics. To extract DNA in order to perform the PCR the researchers used the DNeassy tissue kit that was supplied by Qiagen. The primers were tested individually first to verify that they worked. The PCR products were run on a gel in addition to being sequenced using the ABI PRISM BigDye Terminator cycle-sequencing kit to test if the primers worked. The multiplex PCR was run and tested using southern blot and hybridizations.
This is relevant to our research because it allows us to use microbiological techniques that may be more accurate to identify bacteria that carry antibiotic resistance genes. It also allows us to identify which genes are carried by bacteria that are antibiotic resistant.
http://jcm.asm.org/content/41/9/4089.full.pdf+html
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“Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8”
By A. Schriewer, A. Wehlmann, and S. Wuertz
http://www.sciencedirect.com/science/article/pii/S0167701211000030
This study focused on improving the sensitivity and reliability of qPCR by removing humic acids from the sample. Humic acids are considered inhibitive compounds that lower the efficiency of PCR and are the most prevalent inhibitors in surface waters. While other methods have been found to remediate the effects inhibitors, they are expensive, unreliable, or prone to affecting the sample negatively as well. With this is in mind, the researchers tested DAX-8 as an absorbent for humic acids in order to make qPCR more reliable for microbial testing in natural water samples.
The team treated the samples with different combinations of DAX-8 and bovine serum albumin (BSA). Every combination with DAX-8 was found to increase amplification efficiency, but the highest efficiency came from the combination of 10% DAX-8 with 50 ng/uL of BSA. BSA alone tends to help in the early cycles of PCR, but is hypothesized to lose effect due to degradation from the temperature cycles or steric inhibition from the humic acid molecules. When DAX-8 is added, it removes the humic acid, allowing an overall increase in amplification efficiency that will more closely mirror the conditions and efficiency of the pure samples used on the standard curve. This makes the resulting calculated concentration for the unknown sample more accurate and reliable. This is important for our lab because we could unknowingly be getting inaccurate results due to the presence of humic acid and other inhibitors. Adding this process to our methodologies would ensure more reliable results especially in samples with low pathogen concentrations and would also increase the product of PCR, allowing for easier detection on gel electrophoresis.
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Article Link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517363/
Environmental contamination with Toxocara eggs: a quantitative approach to estimate the relative contributions of dogs, cats and foxes, and to assess the efficacy of advised interventions in dogs
WHAT DID THEY DO?
In my article, the researchers attempted to detect and quantify the level of Toxocara egg contribution of hosts such as domesticated as well as undomesticated cats, dogs and few other hosts in Netherlands. They relied on using the data provided by the University of Applied Sciences Of Den Bosch and the Council of Animal Affairs in the Hauge and the Dutch Central Bureau of Statistics in order to determine the quantity of various host types and estimated how many toxocara eggs each host releases using a statistical model.
HOW DID THEY DO IT?
In order to do so, the researchers gathered data on overall daily toxocara egg output using Poisson statistical model {Eijz~Poisson(λijz), λijz = Dijz × Pijz × Fi × Iij}. Based on a “Monte Carlo simulation.” The statistical model factored in variances within degree of urbanization and the age ranges of each host demography.
Younger hosts we estimated to yield a minimum of 25,000 plus toxocara eggs per km2. The expected mean for the middle aged hosts was 500-2000 toxocara eggs per km2 and less than 500 toxocara eggs per km2 for the older host generations. In the statistical model, ‘i’ represented the host type, ‘j’ represented the number of individuals per age group and the ‘z’ value represented the degree of urbanization. There was a confidence interval of 95% meaning the confidence value would be 1.96 in the Beta Distribution table.
WHAT DID THEY FIND AND WHY DO WE CARE?
Basically, their data highlights that dogs have a significantly higher output of the toxocara eggs than other hosts. They contribute 39% of total toxocara egg output; even though, the likelihood of their faeces being cleaned up by their owners were factored into the statistical model. Because of the high dog output of toxocara eggs, suggests that there are a significant number of pet owners who do not follow the dog waste cleanup policies. Besides the dogs, stray cats contributed 27%, household cats contributed 19% and the foxes contributed 14.9% to the total toxocara egg output.
We need to be concerned because the toxocara eggs are known to give migraines and asthmatic allergies upon accidental ingestion. The actual data of compliance of pet owners to the pet clean up regulations isn’t available; however, this is a huge matter of concern for regular pet owners and park visitors because on average 84,100 toxocara eggs are released into the atmosphere per km2 in Netherlands. This article suggests that there is a huge gap in the number of pet owners who do clean up after their pets and those who don’t. In order to decrease the degree of output of toxocara eggs, new ordinances and regulations could be proposed and monitored for their impact on the data annually.
This relates to the outside public policy, I am currently working on. My research initiative looks specifically on the presence of ordinances regulating dog fecal contamination and variation on the degree of contamination due to tactics such as sign boards, presence of doggy bags and so on. Fecal contamination usually leads to pathogenic contamination of water bodies surrounding the area (i.e. toxocara egg output).
Dogs are not only highly desirable pets but also, very active ones. Their owners walk them on average of 3-4 times a day to nearby parks or green area. It is my theory, that watering bodies nearby dog parks suffer high quantities of dog fecal contamination; however, other variables mentioned earlier could also significantly impact the degree of the fecal contamination.
Upon researching, I realized that there is a great gap in the research available in this topic. We can fill this gap by conducting a research based on Geo-Tracking method currently used in our lab in combination with conducting qPCR for the toxocara eggs and other references for intensity of contamination.
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http://www.sciencedirect.com/science/article/pii/S0043135407002485
Confirmation of putative stormwater impact on water quality at a Florida beach by microbial source tracking methods and structure of indicator organism populations
M.J. Brownell, V.J. Harwood, R.C. Kurz, S.M. McQuaig, J. Lukasik, T.M. Scott
In their study, Brownell et al. monitored the microbial contamination of water and sediment samples in Siesta Key Beach, located in Sarasota County, Florida, in regards to time elapsed since heavy precipitation. In order to do this, they measured the concentration of two different sets samples collected in duplicate: one within 48 hours of heavy rainfall, and the other after 6 days of no precipitation. In each collection, water samples were collected from various locations, including sites upstream from the beach, the stormpipe that carries runoff to the gulf, the underground concrete retaining vault and the adjacent retaining pond, and finally the gulf itself.
Brownell et al. then filtered the water through .45 µm pore sized filter papers and plated them on mFC agar in search for E. coli, which was being used as an indicator for general microbial contamination. After confirming the colonies were indeed E. coli through the use of EC broth amended with 4-methylumbelliferyl-β-d-glucuronide (MUG) and ultraviolet light, Brownell et al. used BOX-PCR to determine the concentration of the contamination in the sample.
Their results confirmed our belief that precipitation can carry microbial contamination downstream; in comparison to the collection after the dry period, the collection following the rainfall showed a higher concentration of E. coli. In addition to a higher concentration of E. coli, the downstream collection following the rainfall also showed a higher diversity in types of contamination, many of which were more commonly found upstream. While this article mostly only confirms our existing suspicions, it still brings up a new issue that is worth considering in regards to our research: namely, their use of the stormpipes and the underground retention vault. Seeing as how we have a fairly extensive sewage system, it could be helpful to determine where exactly our stormpipes bring water to. This could be especially useful for the Geo Tracking group, as it would give us a more direct idea of where rainfall in one area could affect another area.
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In this study by Hyatt Green and co, they attempt to find a method that can be used to identify and quantify fecal contaminations from birds, in order to better determine the risk of human health in different contaminated waters.
In order to better identify these fecal sources, they retrieved fecal sample from six different locations and collected a couple water samples from five different sites. DNA was filtered and then extracted using the PowerWater DNA Kit. They then made a fecal pool with an equal mixture of the 12 California and 12 Oregon Gull, and a subtracter pool with equal amounts of human, dog, cat, cow, and pig DNA. The DNA samples were then quantified by PicoGreen assay. Subtractive hybridization was used to amplify rRNA genes twice specifically 27F and 1492R. Blast was then used to identify and align the two sequences randomly selected after each amplification, additional blast was used created new Primers which were optimized through PCR. The limit of detection was then found for each of the PCR assay and their GFC and GFD qPCR assays, and any that had the desired fragment where purified using QIAprep spin miniprep kit. Water sample were then analyzed using these qPCR and compared using gull DNA samples.
This issue is of the upmost importance because, tools for identifying the source of fecal contaminates have been created; however, not many tests have been run for different types of birds, other than Chicken. If we are unable to trust our current methods of identifying and quantifying different sources of fecal contamination from birds, we will be incapable of keeping even public bodies of water, used for swimming and fishing, safe from avian fecal contaminants. Being able to run more test on these different birds would help mitigate the risk that they have on human health, by decreasing the contamination due to different birds in different bodies of water.
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Click to access 91.full.pdf
In this study they collected certain samples from the watershed surround Cincinnati. They use both DNA and RNA to test for fecal specific bacteria. they used rRNA-based RT-qPCR assay to demonstrated the increased detection of host-specific marker in water samples from Duck Creek Watershed.
Water samples were collected from 11 different sampling sites over a time of 10 weeks.To extract the DNA they used the AllPrep DNA/RNA minikin following imagine GmbH instructions. They then put the RNA in Ambion Turbo DNA-free DNase kit to further purify the samples. They then determined the samples with Qubit RNA and dsDNA HS assay kits.They used qPCR to measure the target fecal bacterial groups : E.coli, Enterocccus,ENt. faecalis, Ent. facium
and human specific Bacteriodales. Then did a data analysis. 3.
Water borne illness have plagued both human and animal health. There has been little work to investigate the risk of waterborne viruses on health and is not been address to the point it should be. More attention needs to be given to this. With the improvement of of methods for specific detection of the viruses will provide new sources for water quality assessment. Sewage overflowing storm water runoff push high concentrations of bacteria into the watersheds. This posses a serious risk to human health.
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Jasmine Namata
ASN 8: Literature Analysis
Article Link: http://aem.asm.org/content/80/5/1633.short
Title: Prevalence of Virulence Genes Associated with Pathogenic Escherichia coli Strains Isolated from Domestically Harvested Rainwater during Low- and High-Rainfall Periods
Citation: Dobrowsky, P., van Deventer, A., De Kwaadsteniet, M., Ndlovu, T., Khan, S., Cloete, T., and Khan, W. Prevalence of Virulence Genes Associated with Pathogenic Escherichia coli Strains Isolated from Domestically Harvested Rainwater during Low- and High-Rainfall Periods. Applied and Environmental Microbiology. 80, 1633-1638 (2014)
WHAT DID THEY DO IT?
The scientists researched and compared the prevalence of virulence genes in harvested rainfall during dry and wet seasons. Rainfall harvesting is practiced worldwide to collect and harvest rainwater for domestic and agricultural uses. This practice is sustainable and cost effective for developing countries or areas that may not have a big water system nearby. However, depending on the atmospheric pollution, the harvesting method and storage of rainfall, may become more susceptible to pollutants and waterborne pathogens.
HOW DID THEY DO IT?
Countries such as South Africa, Australia, Greece and Bermuda have many villages and towns that participate in rainfall harvesting. South Africa in particular has low income subsidized homes that have DRWH tanks already built and approved by the standards of the Department of Science and Technology. From there the researchers got approval to collect samples from 411 houses and tested each for the presence of E. Coli eight different times. Similar collection and DNA extraction protocol as our lab currently does was conducted. However, 29 homes that came up positive for E. Col. Tanks that were susceptible to E. Coli were then subjected to see how pathogenic the E. Coli were and what can be done to treat it. Samples would be processed over eight times, plated and incubated and further isolated. Once isolated a molecular analysis (IMViC Test) and genomic sequencing were done to trace the phylogeny of the strains. Further statistical analysis with the Statistica version 2 were done to determine a variation in the seasons, pH and temperature for the prevalence of E. Coli. It was shown that during low rainfall, the prevalence for E. Coli was a lot less than humid rainy periods.
WHY DO WE CARE?
Virulence genes are responsible for how pathogenic an organism can be. And with our freshwater supply running low it would be important to look at non-traditional sources for crop irrigation and domestic uses. The push for sustainability is huge, but we also want to ensure that it’s safe for human consumption and uses. Our lab currently works with the School of Public Health and USDA for “Conserve” to look at the various factors in making water sources safe. It’s important to know what’s in the water, and what can be done to treat it where it’s not chemically toxic but the natural pollutants can also be filtered. What’s great about this study is that a lot of the sampling methods could be adopted by my project group “Brackish vs. Freshwater” to see if the various plating methods and dilutions helps increase colony count, and identification of virulence genes in fresh and brackish water around this area.
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Article link: http://www.mdpi.com/1660-4601/11/4/4340/htm
In the United States waste water is being reclaimed and reused, most commonly for landscape irrigation. Although more water is being reused for this purpose, there is very little knowledge of pathogen presence in the water and what kind of health risk it imposes. It is known, however, that some pathogens such as Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) have been detected in wastewater. The authors of this paper wanted to determine the presence of methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus (MSSA), vancomycin-resistant enterococci (VRE), and vancomycin-susceptible enterococci (VSE) among spray irrigation worker that use this reclaimed wastewater to irrigate on a daily basis. The irrigation works have the potential to be exposed to these organisms through dermal contact or inhalation.
This study tested 43 subjects from the Mid-Atlantic region of the U.S., 19 spray irrigation workers and 24 office worker controls who were not exposed to the irrigation water. Subjects were then swiped with nasal swabs and dermal swabs between August 2009 and February 2011 when the irrigation spray heads were in use. The swabs were then streaked on to Baird Parker agar in order to detect S. aureus and Enterococcosel agar in order to detect Enterococcus spp. The isolates were then confirmed using standard biochemical test, such as DNA extraction, and polymerase chain reaction assays.
The results concluded that no MRSA or VRE were detected. MSSA was detected in the 26% of spray irrigators and in 29% of the office worker controls, while VSE was detected in 11% of irrigation workers and not at all in the controls. Of the 97 MSSA isolates, only 32 could be confirmed as phenotypically unique and those confirmed were resistant to a variety of the 18 antibiotics tested. Of the 3 VRE that were phenotypically unique, all three were resistant to rifampicin and quinupristin/dalfopristin. Overall, the results demonstrate that those exposed to irrigation and those that did not had very little, to no difference in MSSA, MRSA, VRE, or VSE colonization. The fact that this paper was one of the first to study the safety of reclaimed water, proves that there is still a lot of research that needs to be done in order to determine that safety of repurposed wastewater before we start spraying in all over the food we eat. As a member of the CONSERVE group I have seen that repurposed water definitely contains pathogens, such as E.Coli, even though they might not be airborne. It demonstrates that we need to determine the source of these contaminants in order to ensure that the water we are using and potentially consuming is safe by determining their source in order to prevent contamination. By running PCR test in our lab we can determine the sources of certain contaminates, such as animals, and see what kind of virulence genes are present in order to better understand the content of the water source, such as that at the UMD Community Learning Garden.
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Article Link: http://aciar.gov.au/files/pr139.pdf#page=46
Presence of the plant pathogen phytophthora in the durian plants of the Philippines has been measured to be as high as 40%, causing problems for famers and the profitability of growing the highly sought after fruit. Phytophthora is a natural soil borne pathogen, and is carried in rainwater running through infested soil in the wet and humid conditions of the Philippines and many other parts of the world. Possible solutions have included using sterilized soil, and growing the plants on raised benches. Researchers set out to determine the most effective prevention method.
In order to do this, 4 different treatments for the plants were set up and analyzed. Each treatment included 10 plants with 5 replicates, all of which were grown for a month in a separate seedbed than transported to their designated treatment condition. The four treatments included growing the durian with sterilized soil on a raised bench, unsterilized soil on a raised bench, sterilized soil on the ground, and unsterilized soil on the ground. Soil was sterilized in a heated iron container for 1-2 hours at 50-60 degrees Celsius. Plants were grown for a total of 235 days, and any infected plants were collected and examined for phytophthora infections. After 60 days, the treatment with sterilized soil on a raised bench had no recorded disease incidence, the treatment with unsterilized soil on a raised bench had .5% disease incidence, the treatment with sterilized soil on the ground had 6% disease incidence, and the treatment with unsterilized soil on the ground had 5% disease incidence. After 235 days, in the same treatment condition order, the disease incidences were 3%, 6%, 24% and 19%.
This study verifies that the most effective condition for preventing the growth and spread of phytophthora plant disease is on a raised bench using sterilized soil. In addition, the results showed that the use of sterile soil did not significantly reduce the disease incidence when the plants were grown on the ground, implying that conditions on the ground, including pooled rainwater and bug presence, are major causes of the spread of phytophthora. Although it is more costly, the use of sterile soil and raised benches in nurseries has proven to be extremely effective in lowering the prevalence of phytophthora plant disease in the Philippines. Although my project group focused our studies mainly on the spread of phytophthora through water systems and not through large nurseries, this study provides insight on how water does play a major role in the spread of plant disease, and possible future research could implement phytophthora testing in the large community learning garden on our campus, and if samples come out positive, the raised bench and sterilized soil treatments could be applied. Overall, I think that more studies need to be done to find more cost effective ways to prevent the growth of phytophthora.
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Diagnostic Strategy for Identifying Avian Pathogenic Escherichia coli Based on Four Patterns of Virulence Genes: http://jcm.asm.org/content/50/5/1673.full.pdf+html
1. What did they do?
a. Because individual virulence factors not indicative of pathogenicity, the authors of this paper developed a methodology for diagnosing avian E Coli strains based on four virulence gene patterns. With the four VG patterns, 70.2% of avian pathogenic E Coli can be identified with a 4.3% margin of error. In comparison, serogroup analysis identified only 56.5% of pathogenic strains with a margin of error of 10.8%.
2. How did they do it?
a. Similarly to our own protocols, E Coli isolates from healthy and sick chicks were tested for a number of virulence genes through multiplex PCR, electrophoresis, and decision tree algrorithms. Strains were identified as either pathogenic or nonpathogenic, and statistical analyses were used to determine correlations between virulence genes and pathogenicity. Surprisingly, some strains isolated from sick chicks were nonpathogenic and some strains isolated from healthy chicks were pathogenic. Pathogenic strains, strains that killed one to five chicks when inoculated, were analyzed and four sets of virulence genes were prominent.
3. Why do we care?
a. Diagnosing infections in baby birds could have highly beneficial economic effects. And as the world’s population is ever-growing, an higher survival rate of baby chicks would also be very beneficial. Those are more immediate effects of effective AEC diagnosis, but long-term effects could include more effective methods of human E Coli diagnosis through virulence genes, a method already widely explored.
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http://aem.asm.org/content/80/16/4814.full
Plant-Pathogenic Oomycetes, Escherichia coli Strains, and Salmonella spp. Frequently Found in Surface Water Used for Irrigation of Fruit and Vegetable Crops in New York State
What Did They Do?
This study evaluated the presence of plant-pathogens as well as human pathogens in surface water sources used for irrigation. The experiment identified correlations with pathogen prevalence and pH, temperature, turbidity, precipitation, and location of surface water sources. One of the most significant results showed that plant-pathogenic oomycete prevalence is greater in creeks than ponds and also greater in more turbid water sources.
How Did They Do It?
210 water samples were collected from 38 sites where surface water was used for irrigation to test for pathogens. Water was collected monthly from May to October of 2010 and again from May to October of 2011. Water conditions were measured and two methods were used for the identification of oomycetes, baiting and polymerase chain reaction (PCR). While PCR is a common practice in the environmental pathogens lab, baiting is a technique more common in plant pathology-based labs and may be unfamiliar to FIRE EP students. Baiting uses pathogen food sources (also known as hosts) to encourage the growth of a pathogen. The hosts in this case were pear, cucumber, and lemon leaves. The hosts are then studied for infection.
Why Do We Care?
Many agriculturists rely on surface water sources for irrigation water. Because of this, it is essential for produce growers to understand plant pathogen infestation in water sources and the environmental conditions associated with them. Introducing infested water into a crop could lead to a dramatic loss in crop production. Further understanding the ecology involving plant-pathogens could lead to more effective methods of prevention of plant disease outbreaks. In addition, understanding the presence of human pathogens in irrigation water can lead to greater prevention of disease exposure to consumers who purchase crops exposed to infested irrigation water.
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Avinaash K Sandhu
Literature Analysis
Article Link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2292937/
Title: Prevalence of Virulence Genes and Cytolethal Distending Toxin Production in Campylobacter jejuni Isolates from Diarrheal Patients in Bangladesh
Talukder, K. A., Aslam, M., Islam, Z., Azmi, I. J., Dutta, D. K., Hossain, S., . . . Endtz, H. P. (2008). Prevalence of Virulence Genes and Cytolethal Distending Toxin Production in Campylobacter jejuni Isolates from Diarrheal Patients in Bangladesh. Journal of Clinical Microbiology, 46(4), 1485-1488. doi:10.1128/jcm.01912-07
What did they do?
In this paper, the scientists isolated 58 Campylobacter (jejuni and coli) strains from over 300 stool samples from diarrheal patients in Bangladesh. The purpose was to identify the presence of different virulence genes present in the samples, and also to examine the role of Cytolethal Distending Toxin in the diarrheal patients. Diarrhea is an extremely common illness/ side effect of illness present in developing country like Bangladesh; examining CDT role is important because its method of pathogenesis is still unclear. However, it is extremely important to the infection and thus must be studied more closely.
How did they do it?
300 stool samples were collected from patients attending the ICDDR,B treatment center in Bangladesh (researchers went on previous studies that had already established role of C.jejuni in diarrhea and went on to isolate that species from the samples). For their research, the scientists selected pathogenic genes responsible for the expression of adherence and colonization ( flaA, cadF, racR, and dnaJ), those responsible for the expression of invasion (virB11, ciaB, and pldA), and then cdtA, cdtB, and cdtC were selected as genes expressing the CDT. The prevalence of these genes was tested using PCR method (with primers created for the specific genes) and action of CDT was tested on HeLa cells. Assays were performed with HeLa cells grown in specific medium with some antibiotic supplement. These cells were incubated twice: once before the toxin was added, and then a second time after introduction of the toxin. The scientists examined the cells for morphological changes for a period of 3 days and found that all C. jejuni strains except one (KC-1375) were positive for cdtA, cdtB, and cdtC genes.
Why do we care?
Diarrhea is an extremely common illness in developing countries and often can even cause death — especially among children. This research shows that CDT activity requires the function of three genes: cdtA, cdtB, and cdtC. Even though nearly all Campylobacter jejuni strains were found positive for these genes, the role of CDT in active pathogenesis is still unclear. Now that there is concrete evidence of its involvement, it is important to conduct further studies and investigate exactly how it functions and what can be done to disable/alleviate its effects.
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Effects of the model PAH phenanthrene on immune function and oxidative stress in the haemolymph of the temperate scallop Pecten maximus
http://www.sciencedirect.com/science/article/pii/S0045653509014726
What did they do?
Phenathrene is one of the most abundant PAHs in the aquatic ecosystem, and is extremely toxic to marine invertabrates. Bivalves have an organ called the haemolymph that acts as a medium through which these PAHs can enter their metabolism. This leaves them in danger of the PAHs deleterious effects on their DNA. This paper studies the effects of PAH on oxidative stress and immune function in the mollusc Pecten maximus.
How did they do it?
They collected samples of similar age, and placed in tanks where they wer treated with PAH for 7 days. Their haemolymphs were then removed and subjected to further study. They counted the number of haemocytes in a microscope. They tested the plasma membrane protein concentrations by conducting a Bradford assay. They tested membrane stability by checking its ability to retain Neutral Red.They measured phagocytic activity by measuring uptake of zymogen granules tagged with Neutral Red. Last, they measured oxidative damage in the form of lipid peroxidation by using a modified method of thiobarbituric acid reacting substances.
Why do we care?
They discovered that PAH causes this bivalve to become immunocompromised causing significant reductions in cell stability and phadocytosis, and significant increase in haemocytes. Oxidative stress was observed due to increased levels of lipid peroxidation. This is very important, because phenathrene is a relatively weak PAH, and if it causes major problems then if they are subjected to worse PAHs they could have worse reactions. If bivalves get sick and/or die, the health of the entire ecosystem waivers and worsens, since bilvalves filter water and keep the water clean and healthy enough for other organisms to survive.
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http://www.sciencedirect.com/science/article/pii/S1319610315000575
Anti-microbial activity of cobalt doped zinc oxide nanoparticles: Targeting water borne bacteria.
What did they do?
This Lab developed a nano-particle which was effective as an anti-microbial and can be used to clean water bodies of pathogenic bacteria, including those we are very familiar with; Escherichia coli and Vibrio cholerae. As the article name suggests this nano-particle is made from zinc and cobalt, both of which are safe in small amounts to humans and animals and these same particles have been used in hospital wallpaper to keep bacterial growth down. This lab found that by treating their particles with cobalt to form a crystalline structure they could increase the effectiveness of the particle, and make it effective against a broad range of different pathogens.
How did they do it?
Metal oxide particles had been used as an antimicrobial before such as copper, aluminum, and tin, but this lab took it a step further by incorporating another known antimicrobial of cobalt. they did this by first forming zinc oxide and binding cobalt around it to form a crystalline structure, what this accomplished was a safe particle which could reduce growth in a broad range of different bacterial water-borne pathogens.
Why do we care?
While our lab focuses primarily on identifying pathogens present in the water it is also important to think about what can be done to remove the pathogens from the water. This is especially important in countries where clean water is scarce and the required facilities and funds to clean water are nonexistent. Nano-particles like these offer an effective way to reduce pathogen growth cheaply and safely, and further research could definitely help those in need of clean water.
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Small-Molecule Inhibitor of Vibrio cholerae Virulence and Intestinal Colonization
http://science.sciencemag.org/content/310/5748/670.full
In an effort to combat the rapid development of antibiotic resistance, Hung et al. has developed a novel approach to preventing the spread of infectious bacteria. Through examination of the pathogen Vibrio cholerae, a molecule known as virstatin was identified to be an inhibitor of the bacteria’s ToxT transcriptional regulator. It was found that inhibiting the ToxT regulator led to the prevention of the expression of both the diarrheagenic cholera toxin (CT) as well as the toxin coregulated pilus (TCP), a protein which allows for the adhesion and colonization of V. cholerae in the GI tract. This study aims to further investigate the mechanism for which virstatin inhibits the ToxT coregulator. By directly targeting the regulation of virulence factors, it is proposed that a new class of antibiotics can be produced.
50,000 compounds recorded in the Chembridge Research Laboratories library were screened for V. cholerae virulence factor inhibitors. Virstatin was selected for the study due to its inhibitory effects and low bacterial toxicity. After the administration of 50μM virstatin on 600 and 1200 μM strains of O395 and C6706, CT production was undetectable using a CT-ELISA. Growth inhibition was not observed. TCP expression was also investigated since it is coregulated with CT. As predicted, Western blotting revealed that 50 μM virstatin inhibited the production of TCP in both strains. Total gene expression was assessed using a genomic microarray to determine that 11 out of 15 and 21 out of 22 genes within the ctx and tcp loci of the O395 and C6706 strains, respectively, were repressed by virstatin. The same effects were observed using an E. coli strain containing the ToxT plasmid. When a virstatin-resistant mutant ToxT gene was implemented in V. cholerae and E. coli, both strains continued to produce CT and TCP even when virstatin concentrations were increased to 60 μM. Finally, the effects were examined in-vivo by administering V. cholerae and virstatin orally in infant mice. After 18-24 hours, small intestine homogenate was plated on streptomycin LB for bacterial counts. Results showed that virstatin demonstrated a reduction in TCP using C6706 but not for the TCP-independent strain S533.
It has been widely recognized that antibiotic resistance poses a great threat to public health. Finding alternate sources for antibiotics is key for fighting constantly evolving environmental pathogens such as V. cholerae and E. coli. Studying these genes in detail in addition to their inhibitors may be valuable to our lab when understanding antibiotic resistance for the pathogens we work with in EP such as E. coli and V. cholerae.
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In the academic research paper titled “A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences”, a group of researchers used genome and other sequence databases to identify over 225 possible genetic markers for phylogenetic analyses. This was done in order to produce a genus-wide phylogeny for 82 Phytophthora species. This allowed the researchers in the study to confidently divide the genus into ten well-supported clades.
In order to achieve the results in this study, the researchers analyzed 234 isolates from 82 species of Phytophthora and 2 species of phythium from the World Phytophthora Genetic Resources Collection at the University of California. Working cultures were maintained on either clarified or non-clarified V8 agar or ryeseed B agar with the species. The cultures were checked for bacterial contamination and DNA extractions were done on actively growing cultures in either clarified, V8 broth or pea broth every 4-10 days. DNA concentrations was determined using 26/280 ratio with a Beckman DU 64 UV spectrophotometer. All DNA samples were stored at -86 degrees Celsius. This process was repeated with samples from other databases and other markers and primers.
We care about what was done by the researchers in this study because by establishing a better phylogeny of Phytophthora species the overall evolutionary history of the Phytophthora genus can be better understood. This is an important advantage because clues to Phytophthora evolutionary history can help in finding ways to combat its destruction. Phytophthora is responsible for failed cash crops around the world annually and it has led to mass famine in the past as well as economic depression. By better understanding Phytophthora and on an evolutionary level, experiments and studies can be done to find more efficient was to combat this destructive plant pathogen.
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http://search.proquest.com/docview/222637623?pq-origsite=gscholar
“Contamination of Potable Water Distribution Systems by Multiantimicrobial-Resistant Enterohemorrhagic
Escherichia coli”
What did they do: The authors of this paper sought to test potable water samples along the River Gomti in India for the presence of enterohemorrhagic E. coli (EHEC). To accomplish this, they tested for virulence determinants in the water. Additionally, they tested these virulence determinants to assess their sensitivity/resistance to anti-microbials. India has a high incidence of waterborne diseases, but not much attention has been paid to EHEC. While testing of surface waters for EHEC presence occurs, drinking waters have yet to be assessed in a meaningful capacity.
How did they do it: Six samples were taken from sites in Lucknow, which is situated on the River Gomti. These sites were essentially straight from the tap, and they contained water that had been through purification processes. The samples were collected on the same day and in triplicate. Much of the methods followed by the authors mirror our practices in lab. To isolate the E. coli, 100mL from each sample was filtered in triplicate. The filter paper was then cut up and placed in MacConkey broth, and after incubation, E. coli cultures were gathered and streaked on agar plates. Upon growth of E. coli on the plates, 15 isolates were taken from each sampling site. Each isolate was tested for the presence of various virulence genes that are specific to EHEC (stxl, stx2, eaeA, hlyA, and chuA). To determine the presence of virulence genes, PCR was used. Isolates that tested positive for virulence genes were then further analyzed to determine their sensitivity to 15 antimicrobials from six classes: aminoglycosides, β-lactams, cephalosporins, folate inhibitors, fluoroquinolones, phenicols, quinolones, and tetracyclines. Tests for antimicrobial sensitivity were performed using an agar-diffusion method and antimicrobial-impregnated paper discs. Data for antimicrobial susceptibility was recorded as resistant, intermediate, or sensitive. The researchers found that five of the six sites were contaminated by E. coli, showing that the drinking water distribution system as a whole is susceptible to fecal bacterial contamination. Only 30% of the E. coli isolates were virulent, however. Each E. coli isolate studied was resistant to at least one antimicrobial.
Why do we care: It is concerning that E. coli was found to have contaminated the water distribution systems in Lucknow. With India’s ever-growing population, it is highly important that water is distributed in a safe manner. Determining contamination levels and factors that may contribute to fecal bacteria entering the water purification systems of India is critical to avoiding potential public health crises. Additionally, it is important to determine the resistance of these bacterial strains to antimicrobials because resistant, virulent E. coli strains pose risks to the health of Lucknow’s inhabitants.
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https://vtechworks.lib.vt.edu/bitstream/handle/10919/48392/404-233_pdf.pdf?sequence=1
What did they do?
The scientists in my research journal analyze how mastitis adversely impacts the quality and quantity of dairy products. Mastitis describes an inflammation of cow’s udders due to infectious bacteria that enter the teat canal and release toxins. These pathogens reduce the milk-producing ability of dairy cows, and also make it painful for general milking processes to be performed on these cows. The article seeks to analyze what environmental conditions make dairy cows the most susceptible to mastitis, as well as observe how modern-day automated milking processes contribute to growing rates of mastitis infections.
How did they do it?
The researchers used an AfiMilk Milk Meter (AMM), which collected milk from 26 non-mastitis and 26 healthy cows from the same dairy farm in Virginia. The tests the conductivity of the milk to see the differences between the milk produced by cows with and without mastitis. Cows with mastitis have higher somatic cell counts (SCC) than cows without mastitis, and these high SCC counts occur because of how leukocytes in the cow aggregate along the bacterially infected teat canal of the cow. By the end of the three month long study of the 52 cows, there was a lot of collected data. For one thing, the cows with mastitis ended up having milk with less fat, less total protein, less lactose, and more sodium than that of the healthy cows. Some qualitative analysis was also conducted, with the researchers assessing that mastitis spreads more easily in dirty and damp conditions. They also explained how faulty automatic milking machinery maintenance and operation can harm the dairy cows udders, damaging the mammary tissue and making the area more susceptible to infection. Lastly, it was observed that two hours after milking a cow was the time when cows are most vulnerable to contracting mastitis, because that’s how long the teat canal remains dilated after milking.
Why do we care?
It is important to understand mastitis and it’s adverse effects on milk production because mastitis reduces the typical yield of milk produced by cows, making the milking process less efficient than it could be. This milking inefficiency hurts all parties involved in the milking process. On the cows end, mastitis causes discomfort due to clogged milk ducts and higher concentrations of blood clots. As a result of the cows reduced performance, people are unable to get as much milk as they could from the cows, which could raise dairy prices if more and more cows develop mastitis. The cheese and dairy industry in the U.S. alone is a multi-billion industry so when cows are happy, we’re happy. Dairy milk products are also a great source of protein, which is especially important for children’s bone development.
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Understanding the Basics of Mastitis
https://vtechworks.lib.vt.edu/bitstream/handle/10919/48392/404-233_pdf.pdf?sequence=1
What did they do?
The scientists in my research journal analyze how mastitis adversely impacts the quality and quantity of dairy products. Mastitis describes an inflammation of cow’s udders due to infectious bacteria that enter the teat canal and release toxins. These pathogens reduce the milk-producing ability of dairy cows, and also make it painful for general milking processes to be performed on these cows. The article seeks to analyze what environmental conditions make dairy cows the most susceptible to mastitis, as well as observe how modern-day automated milking processes contribute to growing rates of mastitis infections.
How did they do it?
The researchers used an AfiMilk Milk Meter (AMM), which collected milk from 26 non-mastitis and 26 healthy cows from the same dairy farm in Virginia. The tests the conductivity of the milk to see the differences between the milk produced by cows with and without mastitis. Cows with mastitis have higher somatic cell counts (SCC) than cows without mastitis, and these high SCC counts occur because of how leukocytes in the cow aggregate along the bacterially infected teat canal of the cow. By the end of the three month long study of the 52 cows, there was a lot of collected data. For one thing, the cows with mastitis ended up having milk with less fat, less total protein, less lactose, and more sodium than that of the healthy cows. Some qualitative analysis was also conducted, with the researchers assessing that mastitis spreads more easily in dirty and damp conditions. They also explained how faulty automatic milking machinery maintenance and operation can harm the dairy cows udders, damaging the mammary tissue and making the area more susceptible to infection. Lastly, it was observed that two hours after milking a cow was the time when cows are most vulnerable to contracting mastitis, because that’s how long the teat canal remains dilated after milking.
Why do we care?
It is important to understand mastitis and it’s adverse effects on milk production because mastitis reduces the typical yield of milk produced by cows, making the milking process less efficient than it could be. This milking inefficiency hurts all parties involved in the milking process. On the cows end, mastitis causes discomfort due to clogged milk ducts and higher concentrations of blood clots. As a result of the cows reduced performance, people are unable to get as much milk as they could from the cows, which could raise dairy prices if more and more cows develop mastitis. The cheese and dairy industry in the U.S. alone is a multi-billion industry so when cows are happy, we’re happy. Dairy milk products are also a great source of protein, which is especially important for children’s bone development.
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David Cricchi
Antibiotic Resistance of Enterococci and Related Bacteria in an Urban Wastewater Treatment Plant
Click to access 322.full.pdf
What did they do?
To study the ecology of enterococci and related bacteria, samples were collected from a wastewater treatment plant in Portugal. The water was tested for the presence of antibiotic resistance phenotypes in the ecosystem. The antibiotic-resistant strains of enterococci were determined to not be removed by the wastewater treatment plant. Enterococcus bacteria are common opportunistic pathogens that are associated with certain infections. Local waste water treatment plants are important parts of the water cycles in urban areas, and these scientists aim to gain more insights on the water’s microbial biology. They evaluated the microorganisms present in the water based on their resistance to 6 different types of antibiotics.
What did they do?
For wastewater treatment sampling, raw water samples and treated samples were collected in each season of the year from the waste water treatment plant. Water samples were filtered through a .45 ul membrane. The water samples were then tested for biochemical oxygen demand (BOD) and chemical oxygen demand (COD). The colonies formed from the waste water analysis had an abundance of 20-80 colonies. They were presented on m-Enterococcus agar. Antibiotic resistance was tested on the Kirby-bauer method. The prevalence of antibiotic resistance was compared using the chi-squared test. Random amplified polymorphic DNA (RAPD) is used to compare and cluster the enterococci bacteria and related bacteria. DNA and PCR amplification were carried out to determine rRNA sequence analysis.
Why do we care?
Enterococci is a very common pathogen found in fecal matter and microorganisms in waste water. Antibiotic resistance prevents the successful elimination of the enterococci bacteria. It was found that strains of antibiotic resistant bacteria was not removed from the water treated by the water treatment plant. However, the results were too varied to determine that there was a positive concentration of enterococci and related bacteria. In an urban areas like the one we live in, it is important that we understand the impacts of antibiotic-resistant pathogens in our waste water and the part that waste water treatment plants play in our urban water cycle.
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http://ac.els-cdn.com/S1050464801903730/1-s2.0-S1050464801903730-main.pdf?_tid=e61a0302-b908-11e6-b3a3-00000aacb361&acdnat=1480736035_72c1052a944ab1d7d514bc6fa45e77c8
1. What did they do?
2. How did they do it?
3. Why do we care?
In this study, the impact of temperature on developing sockeye salmon was studied to determine if there was a significant impact on the developing immune systems and other immune responses. Specifically, they used a T-dependent antibody response to measure specific immune response. Fish were measured at both 8 and 12 degrees Celsius and differences were compared between the two. It is currently believed that water temperature may be one of the most important factors in fish development and growth, so various assays were created to test long term changes in fish health.
This study was done by growing fish in captivity from Broodyear sockeye salmon of the Lake Wenatchee at 10 degrees Celsius in conditions modeling lake water, then transferring the group into two separate temperatures, 8 and 12 degrees Celsius. Fish were sampled periodically to test basal immune function Aeromonas salmonicida and Staphylococcus aureus was used to induce immune response in fish and was grown under culturing procedures and washed with PBS pH 7.4. Actual tests for infection was taken from the fish blood using caudal vein puncture. The collected blood was allowed to clot overnight and then stored at -80 Celsius. Kindy tissue was also sampled and fixed in absolute methanol. The protein concentration of each plasma sample was determined by its refractive index using a clinical refractometer and a variety of other assays were created to test for various chemical and immune responses. The fish were then analyzed further upon basis of gender and tank temperature in order to obtain results about specific response from each group.
We care because rarely is aquatic bacterial invasion of immune systems is studied, and in this case we can look at it in better detail, and see how temperature has an effect on fish responses. Also, this study parallels the fish project in some ways that we are currently doing in the lab. As part of our current fish project, we analyze water samples taken from the Anacostia River, and we record various data such as water pH and outdoor and water temperature. Based on the findings of this study, we can better draw results from our own findings, as we can equate some of the infection rates of fish caught from the Anacostia to the temperature at the time we received them. Furthermore based on the findings from this study, we have learned that fish at higher temperatures may exhibit symptoms of some common aquatic diseases, and we can be more observant before equating phenotypic anomalies to pathogens.
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Should We Put Our Feet In the Water? Use of a Survey to Assess Recreational Exposures to Contaminants in the Anacostia River
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476373/
What did they do?
The Anacostia River (a tributary of the Potomac River) is heavily contaminated with raw sewage, heavy metals, trash, and other harmful ingredients. Many individuals use the land area in and around the river for recreation. Up until 2015, there had been no information available on the potential health risks faced by the users of the Anacostia. In this study, 197 recreational users were selected and asked to first give demographic information. Second, the users were asked about their recreational activities in the Anacostia watershed (behavior around the river, frequency and duration of exposure to the water, etc.). The researchers found that 84.1% of respondents who engaged in canoeing, kayaking, rowing, rafting, or paddling were exposed to river water during their recreation. 27.2% of these respondents reported getting water in their mouths and of both groups combined, 60.7% reported swallowing the water.
How did they do it?
The methods for this experiment were broken down into three categories: The Study Population and Recruitment, Surveying, and Exposure Assessment. Recruitment came from two major recreation population centers along the Anacostia River: one at Bladensburg Waterfront Park (Maryland) and another at the Anacostia Community Boathouse Association (Washington, D.C.). To promote the study, the researchers utilized flyers, social media, created a dedicated webpage, spread information through word of mouth, among other methods. The surveying was completed by test subjects either on a personal computer with Internet access or in-person with a researcher on a tablet device. The survey platform was adapted from the NEEAR and CHEERS studies (click the hyperlink above for more information). Self exposure to water was assessed by the test subjects by reporting affected tissues of the body (head, face, torso, upper extremities, etc.). Cases of exposure versus non-exposure were handled separately through the Qualtrics Research Suite (Version 44586).
Why do we care?
Over the last few decades, there have been a few experiments conducted on the health of the Anacostia River. In many of these, the indicator was the health of the fish living in the river (other animal and plant species have been thoroughly researched as well). With the more recent experiment conducted in 2015, research has transformed and grown to include humans as well. Although this study is only in the preliminary stages, scientists in the District and surrounding communities will continue to test the plant and fish populations of the Anacostia watershed, but may now add humans to the mix noting the similarities and differences in disease and pathogens between the three subjects…it is research building upon research.
“This study should be viewed as the foundation for future work with this population, and many possibilities exist for taking this investigation forward, particularly determining true associations of exposure and health outcomes and improving risk surveillance and communication efforts”.
CHECK OUT some of the authors of this work!
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