Progress 12/01/20 to 11/30/24

Outputs
Target Audience:Target Audiences The proposed study addresses criticalquestions about multidrug-resistant plasmids in bacterial pathogens from U.S. catfish farms, targeting key stakeholders in aquaculture and fish health. This project provides valuable insights for catfish producers seeking to mitigate AMR risks, researchers developing new disease control strategies, veterinarians managing fish health, microbiologists studying resistance mechanisms, and extension specialists advising on best practices in aquaculture. Efforts Results from the project have been discussed in various fish health-related courses and meetings, highlighting the growing concern over antimicrobial resistance (AMR) in aquaculture. The project participated in several key events: The American Society for Microbiology (ASM) Hill's Day of Antimicrobial Resistance in Washington D.C., where microbiologists and policymakers addressed AMR as a global public health threat. The MSU Aquatic Antimicrobial Resistance Workshop, held in August 2024, which brought together faculty, staff, and students from different departments to discuss the global challenge of AMR in agriculture and aquaculture. The FAO Reference Center hands-on workshop at Nitte University, India, which provided practical training on utilizing microbiome and genomic resources for understanding and mitigating AMR in aquaculture within a One Health context. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The research project successfully supported the professional development oftwo graduate students and three research scientists. How have the results been disseminated to communities of interest?The results from this project have been disseminated to various communities of interest through multiple channels. The findings were presented at a Fish Health Update seminar for East Mississippi catfish growers on June 29, 2022, in Macon, Mississippi. This seminar aimed to provide updates on current activities, research findings, and treatment recommendations for major fish health issues to local aquaculture practitioners. Additionally, the research gained broader recognition when it was presented at ASM Microbe 2023, a major conference focusing on microbiology and infectious diseases.Following this presentation, the American Society for Microbiology (ASM) selected the research to participate in ASM Hill's Day of Antimicrobial Resistance in Washington D.C.This event offered a unique opportunity for policymakers to collaborate with researchers and experts, highlighting the importance of innovative research in reducing the risks of AMR emergence and spread. The research findings have indeed been disseminated through two significant international workshops: the Mississippi State University (MSU) Aquatic Antimicrobial Resistance Workshop held in August 2024 and the FAO Reference Center which took place from November 18-22, 2024. These workshops demonstrate ongoing efforts to address AMR in aquaculture and related fields through international collaboration and knowledge sharing. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Activities completed: Aim One: The maintenance and fate of pEIMS-171561 were evaluated using mobilization experiments and plasmid stability assays. Conjugation experiments were performedon filter paper to assesswhether pEIMS-171561 can transfer from E. ictaluri to Escherichia coli J53. These experiments involved mixing equal amounts of donor and recipient bacteria, incubating them overnight, and then spreading the bacterial suspensions on selective agar plates to identify transconjugants. The stability of pEIMS-171561 in its original host was evaluated by inoculating a single colony of E. ictaluriMS-17-156 in broth with and without florfenicol, followed by overnight incubation at 30°C. The copy number of pEIMS-171561 in E. ictaluri strain MS-17-156 was determined using real-time PCR. Real-time PCR was used to measure the expression ofthe floR in the presence and absence of selection pressure Aim two: We investigated the impact of florfenicol selective pressure on the dissemination of pEIMS-171561. In vivo transfer of pEIMS-171561 from E. ictaluri to E. coli J53: This experiment assessed transfer of pEIMS-171561 from E. ictaluri to E. coli J53. Specific pathogen free (SPF) catfish were stocked into 40 L tanks and divided into three groups: a negative control (uninfected group), an infected group and fed a regular feed, and an infected group and fed florfenicol-medicated feed. E. ictaluri strain MS-17-156 and E. coli J53 were introduced into the tanks via immersion. The entire intestine was collected at regular intervals over 27 days post-infection, homogenized, and cultured on selective media to quantify transconjugant E. coli carrying pEIMS-171561. In vivo conjugal transfer of pEIMS-171561 to endogenous catfish microbiota: This experiment evaluated in vivo conjugal transfer of pEIMS-171561 from E. ictaluri to endogenous catfish microbiota. Three groups of channel catfish were established: a negative control, an E. ictaluri-challenged group with regular feed to determine whether the pEIMS-171561 can transfer in the absence of selective pressure, and an E. ictaluri-challenged group with florfenicol-medicated feed to determine pEIMS-171561 transfer in the presence of selective pressure. Post-infection, catfish were sampled and euthanized, and theintestine was collected. A portion of the intestinal tract was used for RNA extraction to evaluate floR expression, while theremaining intestinal samples were spread on plates supplemented with florfenicol to determine the presence of microbiota transconjugants and to enumerate donor bacteria. Characterization of Plesiomonas shigelloides strain MS-17-188 plasmids: The whole genome sequencing of P. shigelloides strain MS-17-188 revealed three plasmids named pPSMS-171881, pPSMS-171882, and pPSMS-171883. Of particular interest was pPSMS-171883,an 18,970 bp plasmid carrying tetracycline resistance gene (tetA), and florfenicol/chloramphenicol resistance gene (floR). We investigated the maintenance and stability of pPSMS-171883 to understand its role in the dissemination of AMR. Pathogenicity assessment of P. shigelloides strain MS-17-188: We evaluated the virulence of P. shigelloides strain MS-17-188 using a dose-response challenge model in catfish. Fish were intraperitoneally injected with varying concentrations of the P. shigelloides,ranging from 106 to 109 CFU/fish. Mortality was monitored for two weeks to determine the strain's pathogenicity. The specific objective of examining the transmission dynamics and fate of MDR-plasmids in catfish microbiota was successfully met through a comprehensive experimental approach. Mobilization experiments were conducted to assess the plasmid's transfer capabilities, while plasmid stability assays were performed to evaluate its persistence within host bacteria. In vivo transfer assays were carried out to examine the plasmid's behavior in live catfish environments. Real-time PCR was successfully employed to quantify the presence of pEIMS-171561 in total DNA extracted from catfish microbiota. Furthermore, the genome of P. shigelloides strain MS-17-188 was fully sequenced. These accomplishments demonstrate successful completion of the objectives related to investigating the persistence and transmission ofMDR-plasmids. Results achieved. Successful mating experiments demonstrated that the transfer ofpEIMS-171561 from E. ictaluri to E. coli with an average efficiency of 6.86 × 10−5 CFU/recipient. Thisindicated the plasmid's ability to move between different bacterial species, which is consistent with the behavior of IncA/C plasmids known for their broad host range. Plasmid stability assessments revealed that pEIMS-171561 remained stable in its original host for 33 days without selection pressure. This stability suggests that the plasmid can be maintained in the bacterial population even in the absence of antibiotics, potentially contributing to long-term resistance in aquaculture settings. Real-time PCR analysis showed similar cycle threshold (Ct) values obtained for the floR gene (representing the pEIMS-171561) and the L-asparaginase gene (representing the strain MS-18-199) chromosome, suggesting a single copy of pEIMS-171561 per cell. Real-time PCR showed a 60-fold upregulation of the floR gene when E. ictaluri MS-17-156 was grown with florfenicol, indicating the plasmid's response to antibiotic pressure. No transconjugants were detected in the intestine of catfish after infection with E. ictaluri MS-17-156 strain. These results suggest that pEIMS-171561 may be not acquired by the catfish microbiota species or unstable in these species. Real-time PCR revealed the upregulation of floR gene in catfish intestine when fed florfenicol-medicated feed compared to regular feed. Despite both E. ictaluri and E. coli strains colonizing the gut in high numbers, in vivo transfer of pEIMS-171561 from E. ictaluri to E. coli was not detected. Channel catfish fingerlings showed no mortalities when injected with P. shigelloides at doses ranging from 106 to 109 CFU/fish. Conjugation experiments indicated that pPSMS-171883successfully transferred from?P. shigelloides to E. coli. The stability of pPSMS-171883 in P. shigelloides decreased over time without antibiotic selection.Temperature significantly affected pPSMS-171883 stability, with greater stability at 30°C and 37°C compared to 40°C and 43°C. The plasmid was completely lost after 144 hours at 40°C and 48 hours at 43°C. ?Key outcomes. The conjugation experiment suggested that pEIMS-171561 can transfer from E. ictaluri to E. coli. The stability assay revealed high stability of the plasmid in the E. ictaluri host strain. In vivo experiments using catfish as a model showed no detectable transfer of pEIMS-171561 from E. ictaluri strain MS-17-156 to endogenous microbiota or E. coli in catfish intestines, both in the presence and absence of florfenicol selection. These results suggest a limited role of catfish microbiota as a reservoir for pEIMS-171561 and its associated resistance. Florfenicol-medicated feed exhibited an inhibitory effect on E. ictaluri MS-17-156 in the catfish microbiome, as evidenced by significant decreases in E. ictaluri quantities in the intestinal tract following treatment. This significant increase in expression demonstrates the plasmid's ability to confer resistance in response to selective pressure, potentially reducing the efficacy of florfenicol treatment in aquaculture. The whole genome sequencing of P. shigelloides strain MS-17-188 revealed a novel 18 kb plasmid, pPSMS-171883, belonging to the IncP-6 incompatibility group. Stability assays demonstrated that pPSMS-171883 gradually lost overtime in the absence of selective pressure, indicating a fitness cost on its host in non-selective environments. The elevated temperatures promote pPSMS-171883 instability and loss, potentially due to increased metabolic stress or altered replication dynamics.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Ogunleye S, Lawrence ML, Abdelhamed H. Identification of genetic mechanisms of antimicrobial resistant in aquaculture pathogens. Conference of Research Workers in Animal Diseases (CRAWD. 2021), Chicago, IL. (Oral presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Lawrence ML and Abdelhamed H. Genetic Mechanisms of AMR in Aquaculture Pathogens. Understanding Antimicrobial resistance in aquaculture. Food and Agriculture Organization of the United Nation. 13-14 April. 2021. (Oral presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Lawrence ML and Abdelhamed H. The genetic basis for antimicrobial resistance in aquaculture. Food and Agriculture Organization of the United Nation. AMR virtual meeting. 20-22 December 2021. (Oral presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Islam S, Munshi R, Mannan S, Lawrence ML, Abdelhamed H. Characterization and mobilization of an IncA/C plasmid-mediated antibiotic resistance in Edwardsiella ictaluri. American Society of Microbiology, South-Central Branch Annual Meeting. October 27-29, 2022. Hilton Shreveport (Poster presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Hanson L and Abdelhamed H. Sustainable research program on AMR. Fish Health Seminar in Macon, MS on June 29, 2022 (Oral presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Hanson L and Abdelhamed H. Examples of Global, National, and Regional AMR and Biosecurity Activities at Mississippi State University. FAO Reference Center meeting on AMR and Aquaculture Biosecurity. Virtual meeting. November 20, 2022. (Oral presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Abdelhamed H, Islam S, Riman M, Mannan S, Lawrence ML. Characterization of genetic mechanisms of antimicrobial resistance identified in Edwardsiella ictaluri. Conference of Research Workers in Animal Diseases (CRAWD. 2023), Chicago, IL. (Abstract accepted, oral presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Abdelhamed H, Riman MM, Lawrence ML. Characterization of genetic mechanisms of antimicrobial resistance in catfish pathogens. 87th Annual Mississippi Academy of Sciences Meeting February 23-24, 2023, Biloxi Convention Center Biloxi, MS. (Oral presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Abdelhamed H, Islam S, Riman M, Mannan S, Lawrence ML. Characterization of genetic mechanisms of antimicrobial resistance identified in Edwardsiella ictaluri. Conference of Research Workers in Animal Diseases (CRAWD), January 20-24, 2023, Chicago, IL. (Oral presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Abdelhamed H, Riman MM, Lawrence ML. Characterization of genetic mechanisms of antimicrobial resistance in catfish pathogens. 87th Annual Mississippi Academy of Sciences Meeting February 23-24, 2023, Biloxi Convention Center Biloxi, MS. Oral presentation.
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Mannan SB, Tekedar HC, Lawrence ML, Abdelhamed H. Comparative genomics of three new plasmids in multidrug-resistant Plesiomonas shigelloides strain MS-17-188 isolated from catfish. The American Society for Microbiology Conference (ASM Microbe) in Houston on June 15-19, 2023.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Rostami S, Mannan SB, Tekedar C, Lawrence ML, Abdelhamed H. Genomics analysis of multidrug-resistant Plesiomonas shigelloides strain MS-17-188. Conference of Research Workers in Animal Diseases (CRAWD. 2024), Chicago, IL. (Poster presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Abdelhamed H. Transfer and Persistence of Multi-drug Resistance Plasmids in the Intestinal Microbiota of Catfish. Project Directors Meeting. Saturday, July 13, 2024. Poster presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Abdelhamed H. Transfer and Persistence of Multi-drug Resistance Plasmids in the Intestinal Microbiota of Catfish. MSU Aquatic AMR Workshop. August 5, 2024. (Oral presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Abdelhamed H. Mechanisms of Antimicrobial Resistance (AMR) in Aquatic Bacteria in the USA. FAO reference center hands on workshop on utilization of microbiome and genomic resources for understanding and mitigation of antimicrobial resistance in one health context. Nitte University Enclave, Medical Sciences Complex, Mangalore-575018, India. November 18-22, 2024. (Oral presentation).
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2020 Citation: Tekedar, H. C., Arick 2nd, M. A., Hsu, C. Y., Thrash, A., Blom, J., Lawrence, M. L., and Abdelhamed, H. (2020). Identification of Antimicrobial Resistance Determinants in Aeromonas veronii Strain MS-17-88 Recovered from Channel Catfish (Ictalurus punctatus). Frontiers in Cellular and Infection Microbiology, 10, 348-348. PMID: 32766165. PMCID: PMC7379393. DOI: 10.3389/fcimb.2020.00348
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Abdelhamed H, Islam S, Munshi R, Mannan S, Lawrence ML. 2023. Characterization and mobilization of an IncA/C plasmid-mediated antibiotic resistance in Edwardsiella ictaluri. Submitted to Journal of Global Antimicrobial Resistance. Journal of Global Antimicrobial Resistance. 33. P 177-185. PMID: 36944411. https://doi.org/10.1016/j.jgar.2023.03.005
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Abdelhamed, H., Mannan, S. B., Riman, M. M., Tekedar, H. C., & Lawrence, M. L. 2024. Comparative analysis of three plasmids from Plesiomonas shigelloides strain MS-17-188 and their role in antimicrobial resistance. JAC-antimicrobial resistance, 6(4), dlae109. PMID: 39035015. PMCID: PMC11258559. https://doi.org/10.1093/jacamr/dlae109

Progress 12/01/22 to 11/30/23

Outputs
Target Audience:-Target Audiences The proposed study addresses questions about the transfer and stability of multidrug-resistant plasmids reported in bacterial pathogens isolated from catfish farms in the United States. Thus, our primary target audiences are catfish producers, fish disease researchers, veterinarians, microbiologists, diagnostic and extension specialists seeking alternatives to existing antimicrobials. -Efforts Results have been discussed in fish health-related courses and fish health related meetings. Participate in the American Society for Microbiology (ASM) Hill's Day of Antimicrobial Resistance held in Washington D.C. Changes/Problems:We requested one year no cost extension due to state and university safety guidelines during the COVID-19 pandemic. What opportunities for training and professional development has the project provided?Two graduate students and one research scientist have been trained on this project. How have the results been disseminated to communities of interest?During the presentation at ASM Microbe 2023, this research was chosen by the American Society for Microbiology (ASM) to participate in ASM Hill's Day of Antimicrobial Resistance held in Washington D.C. The event provided a way for policymakers to collaborate with researchers and experts on the importance of innovative research to reduce the risks of antimicrobial resistance emerging and spreading in the United States and around the world. What do you plan to do during the next reporting period to accomplish the goals?We will identify the environmental factors that drive the conjugational transfer of plasmid-mediated antibiotic resistance.

Impacts
What was accomplished under these goals? The whole genome sequencing of Plesiomonas shigelloides strain MS-17-188 MS17-188 isolated from diseased catfish revealed three plasmids named pPSMS-171881 (GenBank accession no. CP027853), pPSMS-171882 (CP027854), and pPSMS-171883 (CP027855). pPSMS-171883 is 18,970 bp and carries tetracycline resistance gene (tetA), and florfenicol/chloramphenicol resistance gene (floR), which were flanked by two transposable elements (IS15 and ISVsa3) and mobilization proteins, suggesting that there is a conjugative mechanism by which this plasmid can be mobilized. In the third year of the current project, we investigated the maintenance and stability of pPSMS-171883. The goal of this experiment was to assess the relative pathogenicity of P. shigelloides strain MS-17-188 using a channel catfish challenge model. Specific pathogen free (SPF) catfish were stocked into 40 L tanks at a rate of 20 fish per tank. The tanks were divided into five groups of four replicates. Four groups were injected intraperitoneally (IP) with 0.1 ml of 1×106, 1×107, 1×108, and 1×109 CFU/fish of P. shigelloides strain MS-17-188 strain. One group was a negative control that was injected with sterile phosphate buffer saline. Fish were monitored twice daily, and mortalities were recorded for a total of two weeks. Conjugation experiments were performed on filter paper to determine whether pPSMS-171883 can transfer from P. shigelloides (donor) to Escherichia coli J53 (as recipients). Conjugation mating experiments were performed by mixing an equal amount of donor and recipient bacteria before incubating them overnight on agar plates. After incubation, bacterial suspensions were spread on agar plates for selecting transconjugants. The stability of pPSMS-171883 in the original host was evaluated by inoculating a single colony of P. shigelloides in broth with and without florfenicol. Significant results achieved. Results from fish challenge revealed no mortalities in channel catfish fingerlings following injection with P. shigelloides at four tested doses (106, 107, 108, or 109 CFU/fish). Conjugation mating experiments indicated that pPSMS-171883 was capable of being transferred from P. shigelloides to E. coli. The stability of pPSMS-171883 was assessed by inoculating and propagating of P. shigelloides in the presence and absence of antibiotic selection. Our results show that the plasmid pPSMS-171883 is lost over time in the absence of selection pressure compared to presence of selective pressure. The frequency of plasmid-carrying population under nonselective pressure was 77.5%, 68.3 %, 64.1%, 61.6%, 66.6%, 70%, 64.1%, and 53.3% on days 1, 2, 3, 4, 5, 6, 7, and 8, respectively. We compared the plasmid loss during growth of P. shigelloides at 30°C, 37°C, 40°C, and 43°C. The result demonstrated that pPSMS-171883 is more stable in P. shigelloides at 30 and 37 °C compared to 40°C and 43°C. P. shigelloides grew at 40°C and 43°C had lost the plasmid after 144 hours and 48 hours, respectively. Key outcomes or other accomplishments realized.? The whole genome sequencing demonstrated that the resistance to florfenicol and tetracycline in P. shigelloides strain MS-17-188 is driven by an 18 kb-sized plasmid (pPSMS-171883) from the IncP-6 incompatibility group. Comparative analysis revealed that pPSMS-171883 was not reported among any previously published P. shigelloides genomes, indicating it is a novel plasmid. The stability assay revealed that the frequency of pPSMS-171883-free cells was lower in the P. shigelloides populations cultured in presence of selective pressure in comparison to those cultured in the absence of selective pressure. Interestingly, P. shigelloides lost pPSMS-171883 at 40°C and 43°C, indicating that high growth temperatures appear favorable for plasmid loss and instability compared to the optimal or normal growth temperatures (30°C and 37°C).

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Abdelhamed H, Islam S, Munshi R, Mannan S, Lawrence ML. 2023 Characterization and mobilization of an IncA/C plasmid-mediated antibiotic resistance in Edwardsiella ictaluri. Submitted to Journal of Global Antimicrobial Resistance. Journal of Global Antimicrobial Resistance. 33. P 177-185
• Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Abdelhamed H, Mannan SB, Munshi R, Tekedar H, and Lawrence ML. Characterization and comparative analysis of three plasmids in Plesiomonas shigelloides strain MS-17-188 isolated from catfish. Frontiers in Microbiology (under review).
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Abdelhamed H, Islam S, Riman M, Mannan S, Lawrence ML. Characterization of genetic mechanisms of antimicrobial resistance identified in Edwardsiella ictaluri. Conference of Research Workers in Animal Diseases (CRAWD), January 20-24, 2023, Chicago, IL. Oral presentation.
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Abdelhamed H, Riman MM, Lawrence ML. Characterization of genetic mechanisms of antimicrobial resistance in catfish pathogens. 87th Annual Mississippi Academy of Sciences Meeting February 23-24, 2023, Biloxi Convention Center Biloxi, MS. Oral presentation.
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Mannan SB, Tekedar HC, Lawrence ML, Abdelhamed H. Comparative genomics of three new plasmids in multidrug-resistant Plesiomonas shigelloides strain MS-17-188 isolated from catfish. The American Society for Microbiology Conference (ASM Microbe) in Houston on June 15-19, 2023.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Rostami S, Mannan SB, Tekedar C, Lawrence ML, Abdelhamed H. Genomics analysis of multidrug-resistant Plesiomonas shigelloides strain MS-17-188. Conference of Research Workers in Animal Diseases (CRAWD. 2024), Chicago, IL. (Abstract accepted, poster presentation).

Progress 12/01/21 to 11/30/22

Outputs
Target Audience:The proposed study address questions about the transfer and stability of multidrug-resistant plasmid reported in pathogenic Edwardsiella ictaluri isolated from catfish farms in the United States. Thus, our primary target audiences are catfish producers, fish disease researchers, veterinarians, microbiologists, diagnostic and extension specialists seeking alternatives to existing antimicrobial. Changes/Problems:We requested one year no cost extension due to state and university safety guidelines during the COVID-19 pandemic. What opportunities for training and professional development has the project provided?Two graduate students and one research scientists have been trained on this project. How have the results been disseminated to communities of interest?The results from this project were presented at a Fish Health Update seminar for East Mississippi catfish growers in Macon, Mississippi, on June 29, 2022. The seminar was facilitated by Mississippi State University Extension Service, and its purpose was to provide an update on current activities, research findings, and treatment recommendations for major fish health issues. What do you plan to do during the next reporting period to accomplish the goals?We will investigate the resistance determinants and transposable elements of three plasmids reported in Plesiomonas shigelloides strain MS-17-188 isolated from diseased catfish.

Impacts
What was accomplished under these goals? Aim one: The maintenance and fate of pEIMS-171561 were evaluated using mobilization experiments, plasmid stability assays, and in vivo transfer assay. Conjugation experiments were performed on filter paper to determine whether pEIMS-171561 can transfer from E. ictaluri (donor) to Escherichia coli J53 (as recipients). Conjugation mating experiments were performed by mixing an equal amount of donor and recipient bacteria before incubating them overnight on agar plates. After incubation, bacterial suspensions were spread on agar plates for selecting transconjugants. The stability of pEIMS-171561 in the original host was evaluated by inoculating a single colony of E. ictaluri strain MS-17-156 in broth with and without florfenicol and grown overnight at 30?C. The pEIMS-171561 copy number in E. ictaluri strain MS-17-156 host was determined using real-time PCR.Our result suggests that only a single copy of pEIMS-171561 is carried in E. ictaluri strain MS-17-156. Real-time PCR was used to measure the expression offloR in the presence and absence of selection pressure. Florfenicol selection pressure caused 60-fold upregulation of floR gene when E. ictaluri MS-17-156 strain was grown with florfenicol. Aim two: Determine the impact of selective pressure on the transfer of MDR-plasmids. In year 2 of our current project, we determined the impact of florfenicol selective pressure on the dissemination of pEIMS-171561. The goal of this experiment was to assess the in vivo transfer of pEIMS-171561 from E. ictaluri to E. coli J53. Specific pathogen free (SPF) catfish were stocked into 40 L tanks at a rate of 20 fish per tank. The tanks were divided into three groups of six replicates. The first group was a negative control (uninfected group). The second and third groups were experimentally infected with E. ictaluri strain MS-17-156 and E. coli J53 by immersion. Catfish in the second group received a regular feed. The fish in third group received florfenicol medicated feed starting 24 hours post-infection for 10 consecutive days. At 1, 2, 3, 4, 6, 7-, 9, 11, 13, 16, 20, and 27-day post-infection, the entire intestine was collected and homogenized. Homogenates were serially diluted and spread on media containing florfenicol and sodium azide to quantify E. coli carrying pEIMS-171561 (transconjugant strain). The goal of this experiment was to assess in vivo conjugal transfer of pEIMS-171561 from E. ictaluri to endogenous catfish microbiota. Channel catfish were stocked into 40 L tanks at a rate of 10 fish per tank. The tanks were divided into three groups of six replicates. The first group was a negative control (uninfected group). The second group was challenged with E. ictaluri strain MS-17-156 and received a regular feed to determine whether the pEIMS-171561 can transfer in the absence of selective pressure. The third group was challenged with E. ictaluri strain MS-17-156 and received florfenicol medicated feed to determine pEIMS-171561 transfer in the presence of selective pressure. At several time points post-infection, catfish were randomly sampled and euthanized, and the entire intestine was collected. A portion of intestinal tract was used for RNA extraction to assess floR expression. The remaining intestinal samples were spread on plates supplemented with florfenicol to determine the presence of microbiota transconjugants and to enumerate donor bacteria. Specific objectives met -Aim 1. In vivo conjugal transfer experiment was performed to evaluate the potential in vivo transfer of pEIMS-171561 from E. ictaluri MS 17-156 to intestinal microbiota of catfish in absence of selective pressure. In this experiment, catfish were infected with E. ictaluri strain MS-17-156 via immersion. The catfish microbiota was subsequently screened for the acquisition of pEIMS-171561. -Aim 2. We investigated the ability of pEIMS-171561 to transfer from E. ictaluri to E. coli using catfish in vivo model. In this experiment, catfish were infected by both E. ictaluri and E. coli strains through immersion and received either florfenicol medicated feed or regular feed. Catfish intestines were collected, and intestinal homogenates were spread on media containing florfenicol and sodium azide to quantify E. coli carrying pEIMS-171561 (transconjugant strain). We also evaluated the impact of florfenicol selective pressure on the dissemination of pEIMS-171561 in intestinal microbiota. In this experiment, catfish were infected with E. ictaluri strain MS-17-156 and feed either florfenicol medicated feed or regular feed. The catfish microbiota was subsequently screened for the acquisition of pEIMS-171561. Real-time PCR assays were performed to quantify the presence of pEIMS-171561 in the total DNA extracted from catfish microbiota. Significant Results Achieved Successful mating experiments indicated that pEIMS-171561 can transfer from Edwardsiella ictaluri to E. coli. The stability of pEIMS-171561 was assessed by inoculating and propagating E. ictaluri in the absence of antibiotic selection. E. ictaluri MS-17-156 strain retains the pEIMS-171561 until 33 days of continuous culture in even in the absence of the selective pressure. The floR gene encoded on pEIMS-171561 was induced by about 60-fold when E. ictaluri MS-17-156 strain when grown in florfenicol (selection pressure) compared to absence of pressure. No transconjugants were detected in the intestine of catfish after infection with E. ictaluri MS-17-156 strain harboring pEIMS-171561 and subsequently received either florfenicol medicated feed or regular feed. These results suggest that pEIMS-171561 may be not acquired by the catfish microbiota species. Results from real-time PCR revealed upregulation of floR gene in catfish intestine when receiving florfenicol medicated feed compared to fish receiving a regular feed. Both E. ictaluri and E. coli strains were able to proliferate and colonize the gut in high numbers within a few days after infection but in vivo transfer of pEIMS-171561 from E. ictaluri to E. coli was not detected. ?Key outcomes or other accomplishments realized Result from in vitro conjugation experiment suggested that pEIMS-171561 can transfer from E. ictaluri to E. coli. The stability assay revealed that pEIMS-171561 is highly stable in the E. ictaluri host strain. In vivo, using E. ictaluri strain MS-17-156 as donor, no transconjugants were detected from catfish intestine in absence and presence of florfenicol selection. Furthermore, we were unable to detect any evidence of conjugal transfer of the pEIMS-171561 from E. ictaluri to E. coli in the catfish intestine. Thus, results suggest that conjugal transfer may not have a significant role in pEIMS-171561 dissemination between bacterial species in the catfish intestine. At several time points, E. ictaluri MS-17-156 quantities in the digestive tract significantly decreased following treatment with florfenicol. These suggest that florfenicol-medicated feed could have an inhibitory effect on E. ictaluri MS-17-156 in the catfish microbiome under experimental conditions.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Abdelhamed H, Riman MM, Lawrence ML. Characterization of genetic mechanisms of antimicrobial resistance in catfish pathogens. 87th Annual Mississippi Academy of Sciences Meeting February 23-24, 2023, Biloxi Convention Center Biloxi, MS. (Abstract accepted, oral presentation).
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Abdelhamed H, Islam S, Munshi R, Mannan S, Lawrence ML. Characterization and mobilization of an IncA/C plasmid-mediated antibiotic resistance in Edwardsiella ictaluri. Submitted to Journal of Global Antimicrobial Resistance (under review).
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Islam S, Munshi R, Mannan S, Lawrence ML, Abdelhamed H. Characterization and mobilization of an IncA/C plasmid-mediated antibiotic resistance in Edwardsiella ictaluri. American Society of Microbiology, South-Central Branch Annual Meeting. October 27-29, 2022. Hilton Shreveport (Poster presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Hanson L and Abdelhamed H. Sustainable research program on AMR. Fish Health Seminar in Macon, MS on June 29, 2022 (Oral presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Hanson L and Abdelhamed H. Examples of Global, National, and Regional AMR and Biosecurity Activities at Mississippi State University. FAO Reference Center meeting on AMR and Aquaculture Biosecurity. Virtual meeting. November 20, 2022. (Oral presentation).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Abdelhamed H, Islam S, Riman M, Mannan S, Lawrence ML. Characterization of genetic mechanisms of antimicrobial resistance identified in Edwardsiella ictaluri. Conference of Research Workers in Animal Diseases (CRAWD. 2023), Chicago, IL. (Abstract accepted, oral presentation).

Progress 12/01/20 to 11/30/21

Outputs
Target Audience:The proposed study address questions about the transfer and stability of multidrug-resistant plasmid reported in pathogenic Edwardsiella ictaluri isolated from catfish farms in the United States. Thus, our primary target audiences are catfish producers, fish disease researchers, veterinarians, microbiologists, and extension specialists seeking alternatives to existing antimicrobial. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two research scientists have been trained on this project and a postdoctoral scientist (Dr. Shamima Islam). How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The impact of oxytetracycline selective pressure on dissemination of MDR-plasmids will be determined in vivo.

Impacts
What was accomplished under these goals? Aim one: The maintenance and fate of pEIMS-171561 were evaluated using conjugation mating experiments, plasmid stability assays, and in vivo transfer assay. Conjugation experiments were performed on filter papers to determine if pEIMS-171561 plasmid can transfer from E. ictaluri (donor) to Escherichia coli J53 and Aeromonas hydrophila ML09-119 (as recipients). Conjugation mating experiments were performed by mixing an equal amount of donor and recipient bacteria before incubating them overnight on agar plates. After incubation, cells were resuspended in sterile PBS, diluted, and plated on agar plates with antibiotics for selecting transconjugants. The stability of pEIMS-171561 in the host E. ictaluri strain MS-17-156 was evaluated by inoculating a single colony of E. ictaluri strain MS-17-156 in broth with and without florfenicol and grown overnight at 30?C. The pEIMS-171561 plasmid copy number in theE. ictaluri strain MS-17-156 host was determined using the quantitative real-time PCR (qRT-PCR). The threshold cycle (CT) value of the single-copy chromosomally carried gene(DBV23) entero hemolysinwas compared to theCTof a pEIMS-171561 gene,floR(coding for antibiotic efflux pump).Our result suggests that only a single copy of pEIMS-171561 plasmid was carried in E. ictaluri strain MS-17-156. Real-time PCR was used to measure the expression offloR in the presence and absence of selection pressure. The drug selection pressure MS-17-156 showed 60-fold upregulation of floR gene when MS-17-156 strain was grown in BHI plus florfenicol compared to BHI without florfenicol. To demonstratein vivotransfer of plasmid pEIMS-171561 in the intestine in the absence of tetracycline, catfish one-year old were infected with E. ictaluri strain MS-17-156 donor strain via immersion in water containing.The catfish microbiota was subsequently screened for the acquisition of pEIMS-171561.At 2, 3, 4, 7-, 11-, 14- and 17-days post-infection, intestinal were collected and plated on agar plates with appropriate selective plates. Bacteria colonies that appeared on the agar plates were probed by PCR for the presence of pEIMS-171561-specific genes. ?Aim two: Determine the impact of selective pressure on the transfer of MDR-plasmids. The impact of florfenicol selective pressure on the dissemination of pEIMS-171561 was determined. Specific objectives met -Aim 1. In vivo conjugal transfer of pEIMS-171561 plasmid was performed to examine the transmission dynamics and fate of MDR-plasmids in catfish microbiota. For objective 1, specific pathogen-free (SPF) catfish fingerlings were stocked in tanks. The treatment groups (eight replicate tanks per group) included: a non-treated control group and Edwardsiella ictaluri-inoculated group. The challenged fish were euthanized on 3, 7, 14-, 28-, 42-, and 60-days post-infection, and their intestines were harvested for tissue culture assay and real-time PCR analysis. Real-time PCR (qPCR) assays were performed to detect and quantify the presence of pEIMS-171561 plasmid in the total DNA extracted from catfish microbiota. For objective two, a similar setup experiment was conducted to find the impact of AM selection pressure on the pEIMS-171561 transfer. Significant Results Achieved Successful mating experiments indicated that the pEIMS-171561 plasmid was capable of being transferred from E. ictaluri to E. coli with an average efficiency of 6.86 × 10−5 CFU/recipient, respectively. Conversely, pEIMS-171561 could not be transferred from E. ictaluri to Aeromonas hydrophila ML09-119 strain. The stability of pEIMS-171561 plasmid was assessed by inoculating and propagating E. ictaluri in the absence of antibiotic selection. Following 33 serial subcultures without any antibiotic pressure, no colonies (among 100 colonies each subculture) had lost the plasmid as indicated by no change in the antimicrobial-resistant profiles of the cultures and was further confirmed by PCR. E. ictaluri MS-17-156 strain retains the pEIMS-171561 plasmid until 33 days of continuous culture in even in the absence of the selective pressure. Following qRT-PCR, cycle threshold (Ct) values obtained for the floR gene representing the pEIMS-171561 were similar to that of the L-asparaginase gene representing the strain MS-18-199 chromosome. The floR gene encoded on pEIMS-171561 was induced by about 60-fold when Edwardsiella ictaluri MS-17-156 strain was grown in florfenicol (selection pressure) compared to absence of pressure. No transconjugants were detected in the intestine of catfish after infection with E. ictaluri MS-17-156 strain. These results suggest that pEIMS-171561 may be not acquired by the catfish microbiota species or maybe it is transiently acquired but probably unstable and incapable of replicating. ?Key outcomes or other accomplishments realized Result from mobilization experiment suggested that pEIMS-171561 plasmid can transfer among other bacterial species within Enterobacteracea family. The stability assay revealed that pEIMS-171561 is highly stable in the host cell. In vivo, using E. ictaluri strain MS-17-156 as donor, no transconjugants were detected from catfish intestine in absence and presence of antibiotic selection. These results suggested that pEIMS-171561 plasmid is incapable of transferring in the gut microbiota of catfish.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Ogunleye S, Lawrence ML, Abdelhamed H. Identification of genetic mechanisms of antimicrobial resistant in aquaculture pathogens. Conference of Research Workers in Animal Diseases (CRAWD. 2021), Chicago, IL.
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Lawrence ML and Abdelhamed H. Genetic Mechanisms of AMR in Aquaculture Pathogens. Understanding Antimicrobial resistance in aquaculture. Food and Agriculture Organization of the United Nation. 13-14 April. 2021. (Oral presentation).
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Lawrence ML and Abdelhamed H. The genetic basis for antimicrobial resistance in aquaculture. Food and Agriculture Organization of the United Nation. AMR virtual meeting. 20-22 December 2021. (Oral presentation).