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INVESTIGATOR: Parveen, S.

PERFORMING INSTITUTION:
University of Maryland Eastern Shore
11868 College Backborne Road
Princess Anne, MARYLAND 21853

SHEWANELLA SPECIES AS POTENTIAL EMERGING PATHOGENS IN OYSTERS AND SEAWATER FROM APALACHICOLA, CHESAPEAKE AND MARYLAND COASTAL BAYS

NON-TECHNICAL SUMMARY: Shewanella species are important flesh-eating bacteria which have been transmitted through contaminated seawater and seafood. Shewanella may emerge as significant pathogens toward immunocompromised individuals who consume raw or lightly cooked oysters or through exposure of cuts in the skin in contaminated marine environments. Recently, cases of Shewanella infections were reported around the Chesapeake Bay (CB). Adequate information is not available on the levels of pathogenic Shewanella species in oysters and seawater from the Apalachicola Bay (AB), CB and the Maryland Coastal Bays (MCBs). The overall goal of this project is to address this data gap through the following objectives: 1) to investigate the prevalence of potentially pathogenic Shewanella species in AB, CB and MCBs; 2) to study the effect of environmental factors such as temperature, salinity, pH, and chlorophyll a on the prevalence of Shewanella in oysters and seawater; 3) to characterize Shewanella recovered from oyster and seawater using phenotypic (antimicrobial susceptibility testing) and genotypic (Pulsed-Field Gel Electrophoresis) methods; 4) to share the research findings with the public, seafood industry and regulatory agencies; and 5) to increase capacity and enhance student educational and leadership opportunities. The proposed research will provide, for the first time, a simple, rapid and reliable tool for monitoring Shewanella concentrations in shellfish that can be employed by commercial growers, state resource managers, and educational institutions. The unique combination of multi-institutional research and outreach activities will enhance the research capacity of the University of Maryland Eastern Shore (UMES) and Florida A & M University (FAMU) in environmental, food and agricultural sciences.

OBJECTIVES: The overall goal of this project is to investigate the prevalence of potentially pathogenic Shewanella spp. in oysters and seawater samples collected from the Apalachicola Bay (AB), Florida, the Chesapeake Bay (CB) and the Maryland Coastal Bays (MCBs), Maryland to enhance seafood safety and water quality research, outreach, and extension at two HBCUs. The specific objectives are:1) To investigate the prevalence of potentially pathogenic Shewanella species in AB, CB and MCBs; 2) To study the effect of environmental factors such as temperature, salinity, pH, and chlorophyll a on the prevalence of Shewanella in oysters and seawater; 3) To characterize Shewanella recovered from oyster and seawater using phenotypic (antimicrobial susceptibility testing) and genotypic (Pulsed-Field Gel Electrophoresis) methods; 4) To share the research findings with the public, seafood industry and regulatory agencies; and 5) To increase capacity and enhance student educational and leadership opportunities.

APPROACH: In Years 1 & 2, we will monitor and characterize Shewanella spp. in oyster and water samples from coastal bays in MD and FL using microbiological and molecular methods (objectives 1, 2 & 3). These studies will provide valuable information about the distribution of pathogenic Shewanella spp. in oysters and seawater. We will also train students and initiate extension and outreach activities (objectives 4 & 5).In Year 3, we will analyze data, prepare manuscripts, and share the research findings with the seafood industry, state and federal regulatory agencies (objectives 2, 3, 4 & 5).A total of 168 collections of oysters, with 12 oysters per collection, and 168 samplings of water (12 months x 2 years x 7 sites) will be made from FL and MD sites. During the collection of samples, seawater temperature, salinity, dissolved oxygen, turbidity, chlorophyll a, and pH will be measured in the upper 0.5 m of the surface water with a YSI pro plus multiparameter meter (Yellow Springs Instrument Co., Yellow Springs, OH). All samples will be analyzed using established methods. In addition, we will use antibiotic susceptibility testing and Pulsed Field Gel Electrophoresis (PFGE) to understand the distribution of antibiotic resistant profiles and clonal diversity of Shewanella spp. recovered from oyster and water samples from two geographical regions.Repeated-measure Analysis of Variance (ANOVA) will be used to compare variations in physico-chemical characteristics of the environment and densities within and between sampling locations. If there are statistically significant differences, a Post-Hoc Tukey Test will be used to determine which samples and sample locations are statistically different. Regression and correlation analyses will be used to determine the relationships between Shewanella concentration and pathogenic Shewanella spp. and with physico-chemical parameters. All calculations will be performed using the Statistical Analysis System (SAS Institute, Cary, NC). An alpha level of 0.05 will be considered the minimum level for statistical significance.Project results will be disseminated via classroom discussions, presentations at professional and industry meetings, and regulatory conferences. Workshops will be conducted to demonstrate the most practical alternative methods for the detection of pathogenic Shewanella in oysters and seawater. One or more PowerPoint presentations will be made available for trainer and students and will be posted online for download. Building capacity for this project will enable UMES and FAMU to offer similar outreach opportunities to other food industries and audiences.Students involvement and leadership in this project will include developing schedules for conducting their portions of the research; planning field trips to collect samples; participating in collecting and summarizing data; leading, to the extent practicable, in the organization and presentation of information at workshops; training of other students (training of students by postdoc; training of undergraduate students by graduate students) in analytical methodologies; participating in organizing the annual UMES Graduate School symposium, presenting talks and posters at meetings; preparing thesis/dissertation and participating in the writing and publication of manuscripts and reports.An evaluation committee comprised of investigators, collaborators, and external evaluators of the project will meet at least four times annually to evaluate the progress of the projects and determine if modifications are needed. In addition, the project directors will regularly conduct meetings with the involved students and postdoctoral associate. Their needed skills and knowledge for the projects will be tested on a regular basis in order to maintain a consistent and competent level of research expertise. Samples will be promptly processed and data will be tightly scrutinized so that timely adjustments can be made to procedures, if needed.

PROGRESS: 2019/03 TO 2020/02
Target Audience:Target audiences are the oyster harvesting and processing industries in Florida (FL) and Maryland (MD), regulatory agencies, risk assessors, risk managers, students and the scientific community. The efforts to reach the target audiences include presentations of the research findings at professional meetings, publications in peer-reviewed journals, classroom discussion, workshops, personal communication with the seafood industry, state, and regulatory agencies, extension and outreach. Changes/Problems:Due to nonavailablity of oysters, we collected samples from two sites in Florida instead of three sites. What opportunities for training and professional development has the project provided?One postdoctoral associate, one technician, two graduate students, and two undergraduate students had the opportunities to learn state-of-the-art microbiology and molecular biology techniques. One undergraduate student graduated with experience in food and environmental microbiology. PIs and students were able to attend the regional, national and international professional meetings to share the project findings with the scientific community and update the knowledge in molecular biology, water quality, food microbiology and safety. How have the results been disseminated to communities of interest?Two presentations were made and one undergraduate thesis was published. Findings of this project were also shared with the students of independent studies, advanced food microbiology, food microbiology laboratory, and seminar in food science and technology courses at the University of Maryland Eastern Shore; the seafood industry; and regulatory agencies through classroom discussion, personal communication, workshops, outreach and extension activities. What do you plan to do during the next reporting period to accomplish the goals?To accomplish the goal/objectives, we are planning to conduct the following analyses/experiments during the next reporting period: 1) analysis of samples collected in spring 2020; 2) sequencing and analysis of presumptive isolates for confirmation at species level; 3) investigating the prevalence of potentially pathogenic Shewanella species in AB, CB and MCBs; 4) determining the effect of environmental factors such as temperature, salinity, pH, dissolved oxygen, turbidity and chlorophyll a on the prevalence of Shewanella in oysters and seawater; 5) characterizing Shewanella recovered from oyster and seawater using phenotypic (antimicrobial susceptibility testing) and genotypic (Pulsed-Field Gel Electrophoresis) methods; 6) to share the research findings with the public, seafood industry and regulatory agencies; and 7) to increase capacity and enhance student educational and leadership opportunities.

IMPACT: 2019/03 TO 2020/02
What was accomplished under these goals? The overall goal of this project is to investigate the prevalence of potentially pathogenic Shewanella species (spp.) in oyster and seawater samples collected from the Apalachicola Bay (AB), Florida, the Chesapeake Bay and the Maryland Coastal Bays (MCBs), Maryland to enhance seafood safety and water quality research, outreach, and extension at two HBCUs. To accomplish this goal, we conducted several trial and error experiments to determine the optimum medium and incubation conditions for the growth of Shewanella from March through June 2019. We compared TSA with added salt and Iron agar. We also compared growth at 30 and 37 degree C and incubation times of 24 and 48 hrs. We determined the optimum medium is Iron agar and the best incubation conditions are 48 h at 37 degree C. In summer 2019, one graduate and one undergraduate students were hired and trained in microbiology and molecular biology to collect and analyze oyster and water samples. In fall 2019, one M.S. student was hired and trained in microbiology and molecular biology to conduct research for her Master's thesis in partial fulfillment of the requirements for her degree program. Florida A and M University also trained one undergraduate student in microbiology and molecular biology through direct involvement in this project to collect data for her undergraduate thesis. We also identified a biotech company to sequence presumptive isolates for confirmation using 16s RNA sequencing. During this period, the postdoctoral associate tried developing Shewanella-specific PCR and qPCR primers for easy detection and enumeration, targeting 16S rRNA genes, and found that targeting SSU ribosomal genes for this purpose will likely not be free of biases as this gene is highly conserved among the bacterial domain. She found a Shewanella-specific primer was previously designed and published, but it will amplify non-pathogenic strains as well, which is not our target. For this study, oyster and water samples were collected monthly from three sites [Honga River (HR), Tangier Sounds (TA), and Horns Point (HP)] in the Chesapeake Bay and one site in the MCBs from March 2019 through February 2020, and two sites [Cat point (CA)] and Oyster farm (OF)] in the AB. In addition, physiochemical parameters, such as water temperature, salinity, turbidity, dissolved oxygen, chlorophyll a, and pH were recorded during collection of samples. All samples were analyzed for Shewanella spp. using the standardized methods. As our preliminary experiments indicates that plating of samples on Iron agar plate and incubation of inoculated plates at 37 degree C for 48 hours provide optimum results, all samples were analyzed using Iron agar plates at 35 degree C for 48 hr. Oyster homogenates and seawater samples were serially diluted using 10-fold dilutions and spread plated onto Iron agar. Initially, we used both Iron agar and TSA, until we determined that Iron agar would be our medium of choice. After incubation, plates of 20 to 200 colonies (or fewer if no plate had 20 colonies) were counted and at least 10 percent of suspect Shewanella colonies (black colonies on Iron agar or orange colonies on TSA) were picked and grown in broth for freezing. During the months of June and July 2019, 53 suspect colonies were isolated from Honga River and Maryland Coastal Bay sites. These colonies were tested using the API 20E system for biochemical identification of Enterobacteriaceae. Of the samples that underwent biochemical analysis, 25% of the samples from MCBs and 83% from HR were confirmed to be S. putrefaciens. Overall, 92% and 88.8% of samples were positive for presumptive Shewanella spp. in Maryland and Florida, respectively. The counts ranged from <1 (March 2020) to 5x104 CFU/g (August 2019) in oysters in Maryland and <1 (December 2020) to 3.5x102 CFU/g (October) in oysters in Florida, respectively. In water samples, the counts ranged from <1 (November 2019) to 6.7 x 10 CFU/mL (October 2019) and <1 (December) to 4.7 x10 CFU/mL (October) except for the month of October (2.2x102 CFU/mL) in Maryland and Florida, respectively. The results indicate that the counts of presumptive Shewanella spp. were lower in water than in oyster samples in both regions. A total of 671 and 718 presumptive Shewanella isolates collected from Maryland and Florida were tested for beta hemolytic activity. In Maryland, 42% of isolates were positive for beta hemolysin. In the warmer months (June through August 2019), more than 25% isolate recovered from the Chesapeake Bay exhibited beta hemolytic activity. In contrast, only <1% isolate were positive for beta hemolytic activity in the MCBs. Approximately, 30% of isolates were beta-hemolytic in Florida. Currently, experiments are underway to confirm and characterize these isolates using genotypic and phenotypic methods. The pH ranged from 7.2 (July 2019) to 10.7 (November 2019). Salinity ranged from 6.26 ppt. (July 2019) at Horn Point to 31.82 ppt (December 2019 at MCBs). Horn Point and HR consistently had the lowest salinities and MCBs consistently had the highest salinity. Temperature ranged from 3.0°C (January 2020) to 31.3°C (July 2019). Turbidity ranged from 0.6 Formazin Nephelometric Units (FNU) (January 2020) to 48.1 FNU (July 2019), with the exception of one reading of 500.4 at HR in October. Dissolved oxygen ranged from 3.3 mg/L (July 2019) to 12.0 mg/L (December 2019). Chlorophyll ranged from 1.1 µg/L (December 2019) to 77.4 µg/L (August 2019). Interestingly, all chlorophyll values ≥ to 10 µg/L were from the HP or HR sites during both warm and cold months. In Florida, temperature, salinity, dissolved oxygen and pH were recorded during collection of samples only from CP. The water temperature ranged between 13.8 (February 2020) and 30.4? (August 2019). Salinity ranged between 0.31 (February 2020) and 32.89 ppt. (November 2019). pH ranged between 7.94 (November 2019) and 8.49 (August 2019), while dissolved oxygen ranged between 6.04 (October 2019) and 10.33 mg/L (February 2020). The results of this study indicate that Shewanella spp., especially beta hemolytic isolates were more prevalent in oyster and water samples during the warmer months in both geographical regions. High numbers of Shewanella isolates in oysters and seawater during the summer months suggest that monitoring the levels of pathogenic spp. should be continued in the Bays in Maryland, Florida and other geographical locations. Pathogenic Shewanella spp. could pose a health threat through the ingestion of contaminated seafood, by cuts or abrasions acquired in the marine environment, or by swimming and other recreational activities. Therefore, cautions should be taken during harvesting of oysters and recreational activities, especially, in the warmer months. The investigators anticipate that the project will motivate minority graduate and undergraduate students to pursue higher education in molecular biology, food microbiology, and food safety as well as careers in these disciplines in academia, industry, and federal and state agencies. In addition, successful development of project objectives for enumeration of Shewanella species in oyster and seawater samples will provide a simple, rapid and reliable tool for monitoring this emerging pathogen in shellfish that can be employed by commercial growers, state resource managers, regulators and educational institutions. In addition, the project's objectives will augment our understanding of the ecology, antibiotic resistance and genetic diversity of Shewanella and provide valuable information pertaining to seafood safety and monitoring strategies as well as aquatic and human health.

PUBLICATIONS (not previously reported): 2019/03 TO 2020/02
1. Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Hernandez, R. Meredith, J., and Parveen, S. 2020. Prevalence of Shewanella in oysters and seawater in the Chesapeake Bay and the Maryland Coastal Bays. REU presentation. August 2019. UMES, Paul S. Sarbanes Coastal Ecology Center, MD.
2. Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Hernandez, R. and Parveen, S. 2020. Prevalence of Shewanella in oysters and seawater in Maryland. 2020. Emerging Researchers National (ERN) Conference in STEM. Washington, D.C. February 6-8, 2020.
3. Type: Theses/Dissertations Status: Accepted Year Published: 2019 Citation: Gleason, M. 2019. The prevalence of Vibrio and Shewanella spp. in oyster and seawater in Apalachicola Bay. Undergraduate thesis. Florida A and M University.