Tetracycline Stress Research Articles

Laccase surface-display for environmental tetracycline removal: From structure to function

Facing the increasingly prominent tetracycline pollution and the resulting environmental problems, how to find environmental and efficient treatment means is one of the current research hotspots. In this study, the laccase surface-display technology for tetracycline treatment was investigated. Via study, the type of anchoring protein had a minor influence on the laccase ability, while the type of laccase showed a major impact. Bacillus subtilis spore coat protein (CotA) exhibited higher laccase activity, stability, and efficiency in degrading tetracycline than Pleurotus ostreatus laccase 6 (Lacc6). The superiority of bacterial laccase over fungal laccase was elucidated from the perspective of crystal structure. Besides, a variety of technical means were used to verify the success of surface-display. pGSA-CotA surface-displayed bacteria exhibited good tolerance to high temperature, pH, and various heavy metals. Importantly, surface-displayed bacteria showed faster degradation efficiency and better treatment effects than the intracellular expression bacteria in tetracycline degradation. This implies that surface display technology has greater potential for laccase-mediated environmental remediation. Due to the adverse impacts of tetracycline on soil enzyme activity and microorganisms, our study found that pGSA-CotA surface-displayed bacteria can alleviate tetracycline stress in soil and partially activate the soil, thereby increasing soil enzyme activity and certain nitrogen cycling genes.

Characteristics, Performance and Microbial Response of Aerobic Granular Sludge for Treating Tetracycline Hypersaline Pharmaceutical Wastewater.

Salt-tolerant aerobic granular sludge(AGS) was successfully cultivated under the dual stress of tetracycline and 2.5% salinity, resulting in an average particle size of 435.0 ± 0.5 and exhibiting a chemical oxygen demand(COD) removal rate exceeding 80%, as well as excellent sedimentation performance. The analysis of metagenomics technology revealed a significant pattern of succession in the development of AGS. The proportion of Oleiagrimonas, a type of salt-tolerant bacteria, exhibited a gradual increase and reached 38.07% after 42 days, which indicated that an AGS system based on moderate halophilic bacteria was successfully constructed. The expression levels of targeted genes were found to be reduced across the entire AGS process and formation, as evidenced by qPCR analysis. The presence of int1 (7.67 log10 gene copies g-1 in 0 d sludge sample) enabled microbes to horizontally transfer ARGs genes along the AGS formation under the double pressure of TC and 2.5% salinity. These findings will enhance our understanding of ARG profiles and the development in AGS under tetracycline pressure, providing a foundation for guiding the use of AGS to treat hypersaline pharmaceutical wastewater.

Deciphering survival strategies: Oxidative stress and microbial interplay in Eisenia fetida under tetracycline contamination

Soil contamination resulting from residual antibiotics presents a pressing need to understand the survival mechanisms of soil organisms in polluted environments. This study focused on Eisenia fetida, and tetracycline stress experiments were conducted in a controlled environment using sterile artificial soil. The stress concentration ranged from 0 to 600 mg/kg, and stress cycles lasted either 10 or 30 days. The objective of this study was to assess the effects of oxidative stress and the changes in microbial communities both within and outside the earthworms. A comprehensive mathematical model was developed to elucidate the responses of organisms following exposure to stress utilizing factor analysis, grey relational analysis, and hierarchical entropy weight analysis. Under tetracycline stress, the initial stage (1–3 days) exhibited a coordinated regulation of oxidative stress and microbial communities in the soil with the assistance of CAT and GPX enzymes. The subsequent stage (4–5 days) further emphasized the influence of soil microbial communities. A notable “feedback regulation” of soil microbial communities on oxidative stress was observed during the third stage (6–8 days). Earthworms maintained a metabolic balance in the fourth stage (9–10 days). In the long term, the stress-induced a self-detoxification mechanism within soil microbial communities, which collaborated with GPX to respond to oxidative stress.

Integrated transcriptomic, proteomic and metabolomic analysis provides new insights into tetracycline stress tolerance in pumpkin

The aim of this study was to conduct transcriptomic, proteomic, and metabolomic analysis to provide a comprehensive view of plant response to tetracycline stress. Pumpkin seeds were cultured for 7 days without or with tetracycline at 10 mg/L. Pumpkin roots showed excessive growth inhibition, but not yet strong growth restraining in cotyledons. Tetracycline affected the abundance of metabolites related to flavonoid biosynthesis and amino acid metabolism. Main changes of metabolites in flavonoid biosynthesis were consistent with mRNA changes. Amino acid changes are mainly mediated by proteins or mRNAs. To be specific, tetracycline treatment increased the levels of rutin, caffeate, cinnamaldehyde, 4-hydroxycinnamic acid, ferulic acid, naringenin, apigenin, luteolin, (−)-epigallocatechin, astragalin, L-serine, and glutathione and the transcript levels related to these compounds; and decreased the levels of indole pyruvate, indole acetaldehyde, L-arginine, S-adenosylhomocysteine, L-glutamine, and gamma-glutamylcysteine and the transcript levels related to these compounds. Tetracycline treatment also increased the levels of oxoglutaric acid, L-glutamic acid, gamma-aminobutyric acid, and gamma-glutamylalanine and enzymes involved in their production; and decreased the levels of L-isoleucine, L-valine, and L-leucine and enzymes involved in their production. We elucidated several biological processes (e.g. phenylpropanoid/flavonoid biosynthesis pathways, amino acid metabolic pathways) that were altered by tetracycline, and provided a multi-omic perspective on the mechanisms underlying the response to tetracycline stress in pumpkin roots. We provide a useful reference for the development of environmental quality management methods.

Metagenomic insights into the nitrogen metabolism, antioxidant pathway, and antibiotic resistance genes of activated sludge from a sequencing batch reactor under tetracycline stress

Tetracycline is prevalent in wastewater treatment plants and poses a potential threat to biological nitrogen removal under long-term exposure. In the present study, the influence of different tetracycline concentrations on the nitrogen removal, bioactivity response, and the spread of antibiotic resistance genes (ARGs) was assessed in sequencing batch reactor (SBR). The nitrogen removal efficiency, nitrification rate, and denitrification rate and their corresponding enzymatic activities gradually decreased with an increase in tetracycline concentration from 0.5 to 15 mg/L. The remarkable toxicity induced by tetracycline led to a significant increase in the peroxidation and the response of antioxidant system, as evidenced by strengthened antioxidant enzymatic activity and abundant genes (SOD12, katG, PXDN, gpx, and apx). Tetracycline addition significantly inhibited the ammonia-oxidizing bacterium Nitrosomonas and functional genes (amoA, amoB, and amoC). The presence of tetracycline decreased the abundance of citrate synthase and genes (CS, IDH3, and acnA) and interfered with carbon source metabolism, leading to impaired bioactivity and treatment performance. In addition, the presence of tetracycline induces diversity and differences in ARGs. The results provide reliable basic data for a deeper understanding of the effects of tetracycline on the nitrogen removal performance of bioreactors and provide a theoretical basis to build a promising strategy for relieving antibiotic-caused process fluctuations.

Treatment of antibiotic-containing wastewater with self-suspended algae-bacteria symbiotic particles: Removal performance and reciprocal mechanism

Emerging contaminants such as antibiotics in wastewater have posed a challenge on conventional biological treatment processes. Algae-bacteria symbiotic mode could improve the performance of biological treatment processes. Self-suspended algae-bacteria symbiotic particles (ABSPs) were prepared with Chlorella vulgaris and Bacillus subtilis using the sol-gel method and hollow glass microspheres in this study. The removal effect of nitrogen and phosphorus as well as the feedback mechanism of ABSPs under tetracycline stress were investigated through three-cycles wastewater treatment experiments. The antioxidant enzyme activity and phycosphere extracellular polymeric substance (EPS) content were identified as well. The results indicated that the removal rates of NH4+-N, TP, COD, and tetracycline in the ABSPs group finally reached 96.18%, 95.44%, 81.36%, and 74.20%, respectively, which were higher than the single algae group apparently. The phycosphere EPS content increased by 20.41% and algae cell structure maintained integrity in ABSPs group as compared with that in single algae group. This study demonstrates that the self-suspended ABSPs can improve contaminants removal performance and alleviate the antioxidant stress response of algae through algal-bacterial reciprocity mechanism.

Tea polyphenols and catechins postpone evolution of antibiotic resistance genes and alter microbial community under stress of tetracycline

Relying on the high mobility of water flow, the dissemination of antibiotic resistance genes (ARGs) in the water tends to be exacerbated and enlarged. It caused negative impacts on a wider scope of the environment. The ARGs dissemination monitoring and the methods efficiently reducing their concentration in water became the focus of interest. Green chemicals with antibacterial effects such as tea polyphenols (TPs) and catechins (CA) have been considered as auxiliary disinfectants for ARGs removal in the water environment. However, the antibacterial performance of TPs and CA under the stress of external antibiotics still lacks sufficient research. The results show that more operational taxonomic units can be observed in water samples with TPs and CA than in those without the ingredients under pressure of tetracycline. An unexpected increase along with the increase of ARGs concentrations and the diversity of microbial communities under the low-concentration TPs or CA (1 mg/L). Besides, under the stress of tetracycline, the inhibition of TPs was detected to be strengthened for increase of inti1 and tetC but weakened towards for the increase of tetA. Whilst CA substantially diminished abundances of tetC and tetA under tetracycline pressure. This research demonstrated that TPs and CA are able to assuage development of ARGs under the pressure of antibiotic in water system.

The exploration of hydrogen production promoting mechanism of Rhodobacter sphaeroides by expressing tetA under tetracycline stress

Photo-fermentative hydrogen production rate improving has been a key limiting link in the field of biological hydrogen production by photosynthetic bacterium (PSB) in recent years. In our previous study, apparent hydrogen production enhancement was observed in R. sphaeroides HY01 with tetA expressed under tetracycline (Tc), which attracted our interests. It was found that Tc-resistant strains always presented stable hydrogen production promotion on the maximum hydrogen production rate (Rm), which was approximately 30% with Tc addition. Figuring out the underlying connections between Tc resistance and hydrogen production of R. sphaeroides may provide new methods for obtaining engineered PSB with high hydrogen production performance. In this study, basing on experimental tests, hydrogen promotion of resistant strain was found to be Tc dose-dependent. Some relevant gene expression differences on hydrogen production of Rodobacter sphaeroides induced by the expression of TetA and the addition of Tc were also tested and discussed.

Stress-responses of microbes in oil reservoir under high tetracycline exposure and their environmental risks

As the groundwater ecosystem is connected with surface, antibiotics and antibiotic resistance genes (ARGs) in aquatic environments will gradually infiltrate into the deep environment, posing a potential threat to groundwater ecosystem. However, knowledge on the environmental risk of antibiotics and ARGs in groundwater ecosystem and their ecological process still remains unexplored. In this study, lab-scale oil reservoirs under high tetracycline stress were performed to evaluate the dynamics of microbial communities, ARGs and potential functions by using 16S rRNA gene sequencing and metagenomics analysis. Although the presence of antibiotics remarkably reduced the microbial abundance and diversity in a short term, but remain stable or even increased after a long-term incubation. Antibiotic stress caused a greater diversity and abundance of ARGs, and higher numbers of ARGs-related species with the capacity to transfer ARGs to other microbes through horizontal gene transfer. Thus, a much more frequent associations of microbial community at both node- and network-level and a selective pressure on enrichment of antibiotic resistant bacteria related to “anaerobic n-alkane degradation” and “methylotrophic methanogenesis” were observed. It is important to emphasize that high antibiotic stress could also prevent some microbes related to “Sulfate reduction”, “Fe(II) oxidation”, “Nitrate reduction”, and “Xylene and Toluene degradation”. This study provides an insight into the long-term stress-responses of microbial communities and functions in oil reservoir under tetracycline exposure, which may help to elucidate the effect of antibiotic stress on biogeochemical cycling with microbial involvement in groundwater ecosystem.

Role of iron(II) sulfide in autotrophic denitrification under tetracycline stress: Substrate and detoxification effect

Autotrophic denitrification using inorganic compounds as electron donors has gained increasing attention in the field of wastewater treatment due to its numerous advantages, such as no need for exogenous organic carbon, low energy input, and low sludge production. Tetracycline (TC), a refractory contaminant, is often found coexisting with nutrients (NO3− and PO43−) in wastewater, which can negatively affect the biological nutrient removal process because of its biological toxicity. However, the performance of autotrophic denitrification under TC stress has rarely been reported. In this study, the effects of TC on autotrophic denitrification with thiosulfate (Na2S2O3) and iron (II) sulfide (FeS) as the electron donors were investigated. With Na2S2O3 as the electron donor, TC slowed down the nitrate removal rate, which decreased from 1.32 to 0.18 d−1, when TC concentration increased from 0 mg/L to 50 mg/L. When TC concentration was higher than 2 mg/L, nitrite reduction was seriously inhibited, leading to nitrite accumulation. With FeS as the electron donor, nitrate removal was much more efficient under TC-stressed conditions, and no distinct nitrite accumulation was observed when the initial TC concentration was as high as 10 mg/L, indicating the effective detoxification of FeS. The detoxification effects in the FeS autotrophic denitrification system mainly resulted from the rapid adsorption of TC by FeS and effective degradation of TC, as proven by a relatively higher living biomass area. This study offers new insights into the response of sulfur-based autotrophic denitrifiers to TC stress and demonstrates that the FeS-based autotrophic denitrification process is a promising technology for the treatment of wastewater containing emerging contaminants and nutrients.

Multi-omics analysis reveals the influence of tetracycline on the growth of ryegrass root

Environmental factors, including antibiotics such as tetracycline, can alter biological processes in plants. To ascertain how cell/tissue response to tetracycline, a multi-omic analysis was implemented to explore the molecular mechanism of tetracycline influencing the growth of ryegrass root. Tetracycline induced extensive changes in the root metabolome in plants, particularly impacting metabolites of flavonoid metabolic pathways, which were supported through consistent differences between transcriptome and proteome. Cross-comparison between mRNA and protein contents considered the authentication of congruence with related metabolites and revealed changes of several biological processes under tetracycline stress. Overall, we present an undemanding multi-omic strategy to survey the significant influence on the root under tetracycline stress.

Antibiotic resistance gene profile in aerobic granular reactor under antibiotic stress: Can eukaryotic microalgae act as inhibiting factor?

Antibiotic resistance gene (ARG) pollution is critical environmental problem, and horizontal gene transfer acts as a driving evolutionary force. In theory, due to the phylogenetic distance between eukaryotes and prokaryotes, eukaryotic microalgae can be a natural barrier that plays a negative role in ARG transfer among the symbiotic bacteria to decrease ARG abundance in sludge during wastewater treatment. However, this hypothesis is far from proven and needs to be tested experimentally, so this study investigated the influence of eukaryote microalgae (Scenedesmus) on the ARG profile of symbiotic bacteria based on aerobic granular reactor. The results indicated that Scenedesmus symbiosis could affect ARG diversity of bacteria, and the detected numbers of ARG in aerobic granular sludge (AG) group and algae-bacteria granular consortia (AAG) group were 45–53 and 44–47, respectively. In terms of relative abundance, after target microalgae symbiosis, the total abundance of ARGs significantly decreased from 1.17 × 10°, 2.69 × 10° and 1.36 × 10−1 to 6.53 × 10−1, 9.64 × 10−1 and 1.04 × 10−1 in the systems with the addition of streptomycin, azithromycin and vancomycin, respectively (P < 0.05), yet there was no significant difference between AG and AAG under the stress of ampicillin, sulfamethazine and tetracycline (P > 0.05). Redundancy analysis showed that the eukaryotic microalgae were significant factor explaining the change in ARG relative abundance (P < 0.05), which contributed 15.3% of ARG variation. Furthermore, the results show that, except for the tetracycline treatment system, the total relative abundances of MGEs in the AAG under the stress of the other five antibiotics were 3.54 × 10−2-7.13 × 10−1, which were all significantly lower than those in the AG (8.38 × 10−2-1.59 × 10°). There was a more significant positive correlation relationship between ARGs and mobile genetic elements (MGEs) than that between ARGs and dominated bacteria.

Evolution of the earthworm (Eisenia fetida) microbial community in vitro and in vivo under tetracycline stress

Bioremediation of contaminated soil has received increasing attention, and the effects of antibiotic residues on the soil ecological environment are a current research hotspot. Earthworms are the first choice of soil organisms to indicate the degree of soil pollution, and their detoxification mechanism after antibiotic stress must be further explored. Taking Eisenia fetida as the research object, an antibiotic (tetracycline) stress test was carried out in sterile artificial soil. The stress concentrations were set at 0, 0.3, 3, 30, 300 and 600 mg/kg. The ECO method was used to cultivate microbes in earthworms and soil. The carbon source utilization intensity algorithm developed by our team was used for data statistics, and a factor analysis model was constructed to explore the succession process of microbes of earthworms in vivo and in vitro under tetracycline stress. The results showed that there were four processes in the evolution of microbes under short-term tetracycline stress: at 1–3 days, the microbes in worms played a leading role; at 4–5 days, the microbes in the worms and the soil microbes jointly resisted TET stress; after 6–8 days of stress, the microbes in worms still played the main role, but their role was weakened; and after 9–10 days, soil microbes played a leading role, and tolerant microbes appeared. Under long-term stress, the microbes of earthworms in vivo and in vitro were obvious different, and there may be no regulatory relationship. And the factor analysis model is suitable for the analyse of the changes in microbial communities in vivo and in vitro under TET stress. The research results provide a reference method and model basis for the bioremediation of antibiotic-contaminated soil and the study of earthworm detoxification mechanisms, and help agricultural development.

Role of Iron(Ii) Sulfide in Autotrophic Denitrification Under Tetracycline Stress: Substrate and Detoxification Effect

Role of Iron(Ii) Sulfide in Autotrophic Denitrification Under Tetracycline Stress: Substrate and Detoxification Effect

Adaptability of enhanced bioretention cell for nitrogen and phosphorus removal under two antibiotics stress

The overuse of antibiotics in the medical and aquaculture industries has led to the frequent detection of antibiotics in wastewater. Considering antibiotics would have an unknown impact on wastewater treatment in the future, the long-term effects of sulfamethoxazole (SMX) and tetracycline (TC) stress on the performance, functional genes and microbial community in three bioretention cells were investigated. The results showed that during the experiment, 0.8–1.2 mg/L of SMX would not destroy the water treatment capacity of the bioretention cells, and had a promoting effect on total nitrogen and ammonia nitrogen. 1.6 mg/L of SMX would cause the reduction of nitrogen removal efficiency and the phenomenon of phosphorus release, but it could be restored after a period of operation. TC of 0.8–1.2 mg/L did not have a significant impact on the removal of nutrients in AC-BRC (activated carbon-bioretention cell) and ACI-BRC (activated carbon and iron-bioretention cell), but TC of 1.2 mg/L caused the phenomenon of phosphorus release in BRC and the decrease of total nitrogen removal rate, 1.6 mg/L TC could make the bioretention cell lose its water treatment capacity. qPCR analysis of denitrification genes showed that the abundance of nirS, nirK, nosZ, and hzo had varying degrees of decrease before and after antibiotic stress, which meant the two antibiotics significantly inhibited the reduction of nitrite and nitrous oxide. But for the total number of bacteria, the relative abundance of the four genes has increased. The results of microbial community analysis also found that Proteobacteria, Bacteroidetes, Chloroflexi, and BIrii41, Denitratisoma, Ferritrophicum, Thiobacillus occupied the dominant species at the phylum level and the genus level respectively, which included most of the denitrifying bacteria. During the experiment, the nitrogen and phosphorus removal efficiency of AC-BRC and ACI-BRC were enhanced obviously, but ammonia nitrogen accumulated in ACI-BRC in the early stage of the reaction.

Application of electric fields to mitigate inhibition on anammox consortia under long-term tetracycline stress

The feasibility of applying electric fields to mitigate inhibition of tetracycline (TC) on anammox process and improve system stability was evaluated in this study. Three electric field intensities of 1, 3 and a variable intensity of 1–6 V (VEF) were used to optimize electric field intensity under gradually increasing addition of TC (0.5, 2 and 10 mg L−1). Results showed that the application of electric fields (3 V and VEF) could improve TC tolerance and keep relatively high-efficiency nitrogen removal performance, especially at TC ≥ 2 mg L−1. Furthermore, applying electric fields contributed to mitigate irreversible inhibition and improve the stability of community structure. Underlying mechanism analysis indicated that the main mechanism of applying electric fields to mitigate inhibition relies on sludge structure strengthening. This study explored a novel strategy to reduce the inhibition of antibiotics on microbial denitrification and broaden the application of anammox in industrial water treatment.

Incubation trial indicated the earthworm intestinal bacteria as promising biodigestor for mitigating tetracycline resistance risk in anthropogenic disturbed forest soil

The continuous input of antibiotics due to frequent anthropogenic activities have increased the dissemination risk of antibiotic resistance genes (ARGs) in forest soil. As soil engineers, it remains unclear whether earthworm intestinal microbial communities might play a role in controlling the ARG proliferation in forest soil. This study collected forest soil in the Yangtze River Delta, China, and its resident Metaphire guillelmi to investigate the interaction between tetracycline (50 μg kg−1) and the bacteria in worm gut and soil. Metagenome sequencing analysis indicated that the abundance of the total ARGs in both the soil (S2) and the worm gut (E2) was 1.3 (p < 0.001) and 1.2 (p < 0.001) times higher than the soil (S1) and (E1) without tetracycline exposure; and under tetracycline stress, the relative abundance of 36 and 20 bacterial genera in forest soil and worm gut were significantly increased respectively. However, the ARGs/ARB abundance decreased in the soil with the worm addition than that without, which may be related to the fact that earthworm intestinal bacteria harbored more tetracycline-degrading genes, i.e. dehydrogenase genes adh, ETFDH, and gpr, etc. Structural equation model analysis indicated that bacteria in worm intestinal has stronger ability to degrade tetracycline than in soil, and the main dissipate way was dehydrogenation. Together, the results contributed to understanding the promising role of worm intestinal bacteria in controlling the ARG risk caused by antibiotic disturbed forest soil.

Adaption strategy of up-flow anaerobic sludge blanket reactor on tetracycline stress during tetracycline antibiotic wastewater treatment

Tetracycline antibiotic wastewater with high chemical oxygen demand (COD) concentration and containing bio-refractory compounds was investigated in a lab-scale up-flow anaerobic sludge blanket (UASB) and anoxic-oxic (A/O) reactor. Specific methanogenic activity (SMA) was determined to reveal the inhibition levels of methane production and volatile fatty acid (VFA) accumulation. The results showed that microorganisms can adapt to tetracycline or oxytetracycline when the concentration of antibiotics is less than 400 mg/L. Acetic acid, propanoic acid, and n-butyric acid accounted for more than 90% of VFA with increasing antibiotic concentration from 50 mg/L to 600 mg/L. During the 215 d of operation, the concentration of tetracycline antibiotic increased from 0 to 95 mg/L, and the COD concentration from 3,500 to 6,500 mg/L. The average COD and tetracycline antibiotic removal rates of the reactors were maintained above 90%. The methane production of the UASB reactor achieved 0.32 L/g COD. VFA accumulation was observed when tetracycline antibiotic concentration increased to 47.5 mg/L. The sludge granule diameter increased from 0–300 μm to 300–1,500 μm during the operation. Rod-shaped and coccus-shaped organisms with pores and internal channels on the granule surface were observed using scanning electron microscopy. In the internal space, methanogenic filamentous bacteria constitute a rock-steady structure.

The survival and removal mechanism of Sphingobacterium changzhouense TC931 under tetracycline stress and its’ ecological safety after application

Sphingobacterium changzhouense TC931 was isolated as a novel TC (tetracycline) removal bacterium through adsorption on extracellular polymerase substances (EPS) and cellular surface and biodegradation. TC biodegradation efficiency by strain TC931 was affected by solution initial pH and carbon source. Polysaccharides and hydrocarbons in EPS and cellular surface were responsible for TC biosorption. Eight possible biodegradation products were identified and the biodegradation pathway was proposed. Strain TC931 was rich in antibiotic resistance genes, and tetX-TC931 and antibiotics resistance genome island (GI) may be acquired via horizontal gene transfer in early evolutionary history. The GI was incomplete and may stable in strain TC931, but it could develop into an intact and transferability GI with help of other mobile genetic elements. This work offers a theoretical basis for understanding the survival and biodegradation mechanisms of S. changzhouense TC931 under TC stress, and offers an ecological safety assessment for its application in environmental bioremediation.

Histological and molecular characterization of Edwardsiella tarda infection in Siamese crocodile (Crocodylus siamensis) hatchlings

Edwardsiellosis is an important bacterial disease of fish, birds, reptiles and mammals. This study was conducted to describe the histopathology and etiology of the disease caused by Edwardsiella tarda in farmed hatchling Siamese crocodiles (Crocodylus siamensis) in Hainan, China. The main macroscopic lesions were congestion and hemorrhage of various internal organs, pericardial fluid and subcutaneous edema. Main histopathological lesions included necrotic hepatitis, hemorrhagic necrotizing nephritis, fibrinous pneumonia, and necrotic splenitis. E. tarda isolates were isolated from blood and various internal organs. Genotyping showed that isolates were positive for eight of ten investigated virulence genes (80%) and eleven of twenty antibiotic-resistance genes (55%), which were largely correlated phenotypically, except for erythromycin resistance. Moreover, tetracycline resistance genes were detected after two years of oxytetracycline use on the farm, but only tetA-harboring strains yielded high resistance. All isolates formed biofilm that increased under low concentration tetracycline stress. Zebrafish experimental infections revealed that all isolates were virulent, and that the most virulent isolate formed the highest amount of biofilm, rather than harboring the highest number of virulence and resistance genes. This study is the first to demonstrate the histopathology of edwardsiellosis among hatchling Siamese crocodiles, and characterize virulence factors and tetracycline resistance in E. tarda isolates from a farm that employed long-term prophylactic use of oxytetracycline. Biofilm was suggested as a key virulent factor in the pathogenesis of E. tarda.