Running head: Two Rivers Wastewater Treatment Project

Wastewater Treatment Project
Zoe French
Biology 111

Abstract
Municipal wastewater treatment plants were suspected to be reservoirs for antibiotic resistant bacteria so a study was conducted. In the study, two samples were collected from a local Wastewater Treatment Plant. The samples were taken at different stages of treatment so one of the samples in the study is untreated, and should contain a greater amount of antibiotic resistant bacteria and the sample that is further along in treatment phase should contain a smaller number of antibiotic resistant samples. The study should demonstrate that, as the wastewater moves through the stages of treatment, less antibiotic resistant bacteria should be detected. The samples should demonstrate that even with treatment, there are still antibiotic resistant bacteria present. Two samples were collected from a local Wastewater Treatment Plant and were taken back to the lab where further study could be done on the types of bacteria that were present. The samples were taken at different stages of treatment. The purpose of this study was to determine what, if any types of antibiotic resistant bacteria were present in the wastewater. The samples taken were processed in the lab, and then agar plated to produce colonies. The colonies were separated using the quadrant streak method to grow individual colonies. These individual colonies were sampled with inoculating needles to determine if there were any single bacterial colony present that was antibiotic resistant. The concentration of ARB was way greater in the semi-treated wastewater than in the untreated wastewater. 86% were resistant to ampicillin in the untreated and 63% in the semi-treated this showed that there was only a 23% reduction. There was only a 26% out of 50% reduction in erythromycin, 13% out of 44% reduction in streptomycin, and 22% out of 30% reduction in tetracycline. The results did show that the level of ARB did decrease after the treatment however, high concentration of ARB was found in the wastewater treatment plants. Two Rivers Wastewater treatment, even though the concentration of ARB was still high in the semi-treated sample, with the continuation of treatment the percentage of ARB should be controllable and decline further in number.

Two Rivers Wastewater Treatment
Antibiotic Resistant Bacteria (ARB) is the main topic being looked into as it relates to the ever increasing decline in public health and how it is linked to the environmental contaminates (Czekalski, Nadine; Gascón Díez, Elena; Bürgmann, Helmut. Jul 2014). There is a growing problem in our communities today with the treatment of infectious diseases. The number of bacteria strains that are becoming untreatable with antibiotics is rapidly increasing (Aali, R. Nikaeen, M. Khanahmad, H. Hassanzadeh, A. 2014). Human health is at greater risk with these new bacteria’s. Infections from these bacteria are difficult to treat, sometimes require hospitalization, and they can even cause death. “Clinically relevant bacteria refers to bacteria that, due to the presence of specific virulence factors, the ability to colonize humans, in particular, immunocompromised hosts and/or the capacity to resist antibiotic therapy may be harmful to humans” (Varela & Manaia, 2013). Many studies have been conducted to get to the bottom of how this problem occurred and reports revealed that half of the antibiotics released into the environment ends up in water, while the other half found its way into the soil, giving antibiotics a chance to enter into the human food chain(Beijing review, 2015). Antibiotics found in our food is bad for us humans by itself but because it's small quantity it gives bacteria a chance to develop a resistance to the antibiotics whenever too little antibiotics is used. If a harmful bacteria survives the antibiotic doses it can develop a resistance to the antibiotic making it hard to treat because of the threat of antibiotic resistant found in waste and water researchers are trying their best to put restrictions on the use antibiotics. It was noted that municipal and hospital wastewaters were a direct contaminate cause to the fresh water bodies. This find, however, created a new issue. How are ARB’s making their way into the communities and eventually into human consumption and drinking water(Aali, R. Nikaeen, M. Khanahmad, H. Hassanzadeh, A. 2014). One answer seemed to lie in our water systems. Municipal wastewater treatment plants were suspected to be reservoirs for antibiotic resistant bacteria so a study was conducted. In the study, two samples were collected from a local Wastewater Treatment Plant. The samples were taken at different stages of treatment so one of the samples in the study is untreated, and should contain a greater amount of antibiotic resistant bacteria and the sample that is further along in treatment phase should contain a smaller number of antibiotic resistant samples. The study should demonstrate that, as the wastewater moves through the stages of treatment, less antibiotic resistant bacteria should be detected. The samples should demonstrate that even with treatment, there are still antibiotic resistant bacteria present.
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Materials and Methods Section The first sample that was taken from the untreated water and the second sample that was taken from treated water not yet completed were taken back to the lab where further study could be done on whether there was ARB in the treated and untreated wastewater. The purpose of this study was to determine what, if any, types of antibiotic resistant bacteria were present in the treated and untreated wastewater.
Labeling
Samples were pulled from local CR wastewater treatment plant. 2mL was then pulled from CR and put into primary; primary dub; secondary; and secondary dub beakers. Twelve 15mL test tubes were then labeled with the following: CR1 10°; CR1 10ˉ¹; CR1 10ˉ²; CR1 dud 10°; CR1 dud 10ˉ¹; CR1 dud 10ˉ²; CR2 10°; CR2 10ˉ¹; CR2 10ˉ²; CR2 dud 10°; CR2 dud 10ˉ¹; CR2 dud 10ˉ². Twelve petri dishes were labeled with the following: : CR1 10°; CR1 10ˉ¹; CR1 10ˉ²; CR1 dud 10°; CR1 dud 10ˉ¹; CR1 dud 10ˉ²; CR2 10°; CR2 10ˉ¹; CR2 10ˉ²; CR2 dud 10°; CR2 dud 10ˉ¹; CR2 dud 10ˉ² and all was dated. All the petri dishes were poured with agar mixed with cycloheximide.
Serial Dilation of wastewater Samples
900 mL of PBS solution was pipetted into each 15mL tube. Then 100 mL of sample was pipetted into the 10° tube and capped. 100 mL of the sample 10° was pipetted into the 10ˉ¹ tube and capped. 100 mL of the 10ˉ¹ sample was pipetted into the 10ˉ² tube and capped. Then three drops from 10°, 10ˉ¹, and 10ˉ² were placed on their corresponding petri dishes and spread evenly on the petri dish using L-spreaders. All plates were then incubated at 37°c for 18 hours.
Aseptic Transfer and Quadrant Streak The colonies were then sub-cultured using the quadrant streak method, a common isolation technique. After obtaining inoculum on the loop, the loop was streaked back and forth across the agar on the first quadrant. The loop was then flamed. Rotating the plate about 90° the petri dish was then streaked again. As the loop was moved back and forth, the first streak was crossed and the inoculum was pulled into the second quadrant. Then the loop was flamed. The plate was rotated 90° again and streaked. The second streak was crossed and the inoculum was pulled into the third quadrant. Then the loop was flamed. The plate was rotated one last time and streaked, crossing the third. The fourth streak was ended by streaking into the center of the plate without touching quadrant one, two, or three.

Inoculating Slant Tubes Twelve 15mL agar slant tubes were then labeled with the following: CR1 10°; CR1 10ˉ¹; CR1 10ˉ²; CR1 dud 10°; CR1 dud 10ˉ¹; CR1 dud 10ˉ²; CR2 10°; CR2 10ˉ¹; CR2 10ˉ²; CR2 dud 10°; CR2 dud 10ˉ¹; CR2 dud 10ˉ². Inoculating needles were flamed and an isolated colony was pulled from a corresponding petri dish and streaked into the corresponding agar slant tube. The tube was then sealed and the inoculating needle was flamed again. This process was used for each corresponding petri dish with its corresponding agar slated tube.
Inoculating Petri Dishes
Twelve petri dishes were labeled with the following: : CR1 10°; CR1 10ˉ¹; CR1 10ˉ²; CR1 dud 10°; CR1 dud 10ˉ¹; CR1 dud 10ˉ²; CR2 10°; CR2 10ˉ¹; CR2 10ˉ²; CR2 dud 10°; CR2 dud 10ˉ¹; CR2 dud 10ˉ² . Then the petri dishes were inoculated with four swabs, each with a bacterium from its corresponding slanted tubes. Antibiotic discs ( ampicillin, tetracycline, erythromycin, streptomycin) were then placed in the agar of inoculated petri dishes.
Measuring and Recording Inhibition Zones
Illustrations of Tuesday October 27, 2015 class petri dishes were drawn and recorded. The size of inhibition zones were measured and recorded. The sample location and whether a bacterium was resistant, intermediate, or susceptible to the antibiotic discs were also recorded.

Results Section
CR Primary: Antibiotic | Resistant | Intermediate | Susceptible | Ampicillin | 50 | 5 | 3 | Erythromycin| 33 | 18 | 7 | Streptomycin| 16 | 13 | 29 | Tetracycline | 22 | 12 | 24 |

CR Secondary Antibiotic | Resistant | Intermediate | Susceptible | Ampicillin | 53 | 4 | 23 | Erythromycin| 26 | 41 | 13 | Streptomycin| 15 | 16 | 49 | Tetracycline | 17 | 19 | 44 |

Discussion
The samples that was untreated should have contained a greater amount of antibiotic resistant bacteria than the sample that was further along in treatment phase that should have contain a smaller number of antibiotic resistant samples. The study should have demonstrated that as the wastewater moves through the stages of treatment 50% less of antibiotic resistant bacteria should be detected. The samples should have demonstrate that even with treatment, there were still low concentrations of antibiotic resistant bacteria present. The concentration of ARB was way greater in the semi-treated wastewater than in the untreated wastewater. 86% were resistant to ampicillin in the untreated and 63% in the semi-treated this showed that there was only a 23% reduction. There was only a 26% out of 50% reduction in erythromycin, 13% out of 44% reduction in streptomycin, and 22% out of 30% reduction in tetracycline. The results did show that the level of ARB did decrease after the treatment however, high concentration of ARB was found in the wastewater treatment plants. these results goes hand in hand with the study results done on three wastewater plants that had different bio treatment processes and were compared towards each other. one of the wastewater treatment plant was from the hospital and another was from wastewater near industrial plants and the third one was from natural raw wastewater; the study was on the raw inflow and treated effluents of antibiotics resistant. The study showed that longer hydraulic residence periods resulted in higher bacterial removal rates than shorter periods but this did not significantly decrease the antibiotic resistant bacteria. the end results of the study shows that antibiotic resistant bacteria could be controlled according to the treatment efficiency such as inflow volume, the type of biological treatment, and hydraulic residence time (Novo, Ana, and Cé Manaia. 2010).The same end conclusion goes for the Two Rivers Wastewater treatment, even though the concentration of ARB was still high in the semi-treated sample, with the continuation of treatment the percentage of ARB should be controllable and decline further in number.

References 1. Aali R, Nikaeen M, KhanahmadH, Hassanzadeh A. Monitoring and Comparison of Antibiotic Resistant Bacteria and Their Resistance Genes in Municipal and Hospital Wastewaters. Int J Prev Med 2014;5:887-94. 2. "overdosing on antibiotics new research yields insights into China's antibiotics abuse and its impact on the environment." Beijing review July 23rd 2015 ProQuest web 17 September 2015. 3. Novo, Ana, and Cé Manaia. "Factors Influencing Antibiotic Resistance Burden in Municipal Wastewater Treatment Plants." Applied Microbiology and Biotechnology 87.3 (2010): 1157-66. ProQuest. Web. 17 Sep. 2015. 4. Czekalski, Nadine, Elena Gascón Díez, and Helmut Bürgmann. "Wastewater as a Point Source of Antibiotic-Resistance Genes in the Sediment of a Freshwater Lake." The ISME Journal 8.7 (2014): 1381-90. ProQuest. Web. 17 Sep. 2015.