ABSTRACT
Balancing selection is an evolutionary process that maintains genetic polymorphisms at selected loci and strongly reduces the likelihood of allele fixation. When allelic polymorphisms that predate speciation events are maintained independently in the resulting lineages, a pattern of trans-species polymorphisms may occur. Trans-species polymorphisms have been identified for loci related to mating systems and the MHC, but they are generally rare. Trans-species polymorphisms in disease loci are believed to be a consequence of long-term host-parasite coevolution by balancing selection, the so-called Red Queen dynamics. Here we scan the genomes of three crustaceans with a divergence of over 15 million years and identify 11 genes containing identical-by-descent trans-species polymorphisms with the same polymorphisms in all three species. Four of these genes display molecular footprints of balancing selection and have a function related to immunity. Three of them are located in or close to loci involved in resistance to a virulent bacterial pathogen, Pasteuria, with which the Daphnia host is known to coevolve. This provides rare evidence of trans-species polymorphisms for loci known to be functionally relevant in interactions with a widespread and highly specific parasite. These findings support the theory that specific antagonistic coevolution is able to maintain genetic diversity over millions of years.
Subject(s)
Daphnia , Polymorphism, Genetic , Selection, Genetic , Animals , Daphnia/genetics , Daphnia/microbiology , Daphnia/immunology , Pasteuria/genetics , Pasteuria/pathogenicity , Disease Resistance/genetics , Crustacea/genetics , Crustacea/microbiology , Crustacea/immunology , Evolution, Molecular , Genome/genetics , Phylogeny , AllelesABSTRACT
Enterococci are emerging nosocomial pathogens. Their widespread distribution causes them to be food contaminants. Furthermore, Enterococci can colonize various ecological niches and diffuse into the food chain via contaminated animals and foods because of their remarkable tolerance to unfavorable environmental circumstances. Due to their potential dissemination to humans, antimicrobial-resistant Enterococci in fish are a worldwide health issue. This study characterized AMR, ARGs, VAGs, gelatinase activity, and biofilm formation in Enterococcus spp. recovered from fish and seafood and evaluated potential correlations. 54 Enterococcus spp. strains(32.73 %)were isolated from 165 samples (75 Oreochromis niloticus, 30 Argyrosomus regius, and 60 Shrimp), comprising 30 Enterococcus faecalis (55.6 %) and 24 Enterococcus faecium (44.4 %) with total 32.73 % (54/165), The maximum prevalence rate of Enterococcus spp. was observed in Nile tilapia (34/54; 63 %), followed by shrimp (14/54; 25.9 %) and Argyrosomus regius (6/54; 11.1 %). The maximum prevalence rate of E. faecalis was observed in Nile tilapia (22/30; 73.3 %), followed by shrimp (8/30; 26.7 %) with significant differences. The prevalence rate of E. faecium was observed in Nile tilapia (12/24; 50 %), followed by shrimp (6/24,25 %). E. faecium is only isolated from Argyrosomus regius (6/24,25 %). Isolates exhibited high resistance against both tetracycline (90.7 %) and erythromycin(88.9 %), followed by gentamycin (77.8 %), ciprofloxacin (74.1 %), levofloxacin (72.2 %), penicillin (44.4 %), vancomycin (37 %), and linezolid (20.4 %). 50 strains (92.6 %) exhibited resistance to more than two antibiotics, 5 strains (10 %) were XDR, and the remaining 45 strains (90 %) were classified as MDR. 92.6 % of the isolates had MARindices >0.2, indicating they originated in settings with a high risk of contamination. Additionally, ten ARGs were identified, with tet(M) 92.6 %, followed by erm(B) (88.9 %), aac(6')-Ie-aph(2â³)-Ia(77.8 %), tet(K) (75.9 %), gyrA (74.1 %), blaZ (48.1 %), vanA (37 %), vanB (31.5 %), optrA (20.4 %), and catA(3.7 %). Biofilm formation and gelatinase activity were observed in 85.2 %, and 61.1 % of the isolates, respectively. A total of 11 VAGs were detected, with gelE as the most prevalent (83.3 %) followed by agg(79.6 %), pil (74.1 %), both sprE and asa1 (72.2 %), hyl (70.4 %), eps(68.5 %), EF3314 (57.4 %), ace (50 %), and cylA (35.2 %) with no detection of cylB. In conclusion, the emergence of linezolid-resistant -vancomycin-resistant enterococci recovered from Egyptian fish and shrimp, suggests that fish and seafood might participate a fundamental part in the emergence of antimicrobial resistance among humans.
Subject(s)
Anti-Bacterial Agents , Linezolid , Animals , Anti-Bacterial Agents/pharmacology , Linezolid/pharmacology , Virulence , Fishes/microbiology , Microbial Sensitivity Tests , Enterococcus/drug effects , Enterococcus/isolation & purification , Drug Resistance, Bacterial , Crustacea/microbiology , Seafood/microbiology , Vancomycin-Resistant Enterococci/drug effects , Vancomycin-Resistant Enterococci/isolation & purification , Biofilms/drug effects , Biofilms/growth & developmentABSTRACT
Ferroptosis, characterized by iron-dependent cell death, has recently emerged as a critical defense mechanism against microbial infections. The present study aims to investigate the involvement of exosomes in the induction of ferroptosis and the inhibition of bacterial infection in crustaceans. Our findings provide compelling evidence for the pivotal role of exosomes in the immune response of crustaceans, wherein they facilitate intracellular iron accumulation and activate the ferroptotic pathways. Using RNA-seq and bioinformatic analysis, we demonstrate that cytochrome P450 (CYP) can effectively trigger ferroptosis. Moreover, by conducting an analysis of exosome cargo proteins, we have identified the participation of six-transmembrane epithelial antigen of prostate 4 in the regulation of hemocyte ferroptotic sensitivity. Subsequent functional investigations unveil that six-transmembrane epithelial antigen of prostate 4 enhances cellular Fe2+ levels, thereby triggering Fenton reactions and accelerating CYP-mediated lipid peroxidation, ultimately culminating in ferroptotic cell death. Additionally, the Fe2+-dependent CYP catalyzes the conversion of arachidonic acid into 20-hydroxyeicosatetraenoic acid, which activates the peroxisome proliferator-activated receptor. Consequently, the downstream target of peroxisome proliferator-activated receptor, cluster of differentiation 36, promotes intracellular fatty acid accumulation, lipid peroxidation, and ferroptosis. These significant findings shed light on the immune defense mechanisms employed by crustaceans and provide potential strategies for combating bacterial infections in this species.
Subject(s)
Bacteria , Crustacea , Exosomes , Ferroptosis , Iron , Cytochrome P-450 Enzyme System/metabolism , Exosomes/metabolism , Ferroptosis/physiology , Iron/metabolism , Lipid Peroxidation , Peroxisome Proliferator-Activated Receptors/metabolism , Oxidoreductases/metabolism , Membrane Proteins/metabolism , CD36 Antigens/metabolism , RNA-Seq , Ferrous Compounds/metabolism , Crustacea/cytology , Crustacea/genetics , Crustacea/metabolism , Crustacea/microbiology , Hydroxyeicosatetraenoic Acids , Arachidonic Acid/metabolism , Fatty Acids/metabolism , Bacteria/metabolismABSTRACT
Ample evidence shows dysbiosis in the gut microbiota when comparing healthy shrimp with those affected by severe acute hepatopancreatic necrosis disease (AHPND). However, the static comparison used in available studies leads to the uncertainties regarding how and to what extent the gut microbiota responds to the progressive severity of AHPND. In addition, shrimp AHPND is featured by rapid and massive mortality, thus the initiation of AHPND must be diagnosed for preemptive therapy. For these reasons, we explored the ecological assembly of gut microbiota over shrimp AHPND progression. Increasing AHPND severity was associated with linear increase in the copies of pirAB genes, relative abundance of gut Vibrio and potentially pathogenic, and reduction in the gut bacterial diversity, stability, and relative abundance of Bdellovibrio. Negative and significant association between gut Vibrio and Bdellovibrio were noted, indicating that compromised predation exerts a role in AHPND progression. Notably, the extents of departure to the healthy shrimp gut microbiota were positively coupled with the increasing severity of AHPND. After controlling the temporal variation in the gut microbiota as healthy shrimp age, we constructed a diagnosis model that accurately diagnosed the initial, progressed or moribund stages of AHPND, with an overall accuracy of 86.5%. Shrimp AHPND induced more stochastic gut microbiotas as a consequence of the attenuated ability of diseased shrimp to select their commensals, resulting in convergent bacterial communities between gut and rearing water over AHPND progression. Collectively, our findings provide important step toward the ecological assembly of gut microbiota implicating in AHPND etiology and in diagnosing AHPND stages. KEY POINTS: ⢠The departure of shrimp gut microbiota positively linked with AHPND severity. ⢠The diagnosis model accurately diagnosed the stages of AHPND. ⢠Shrimp AHPND induced more stochastic gut microbiota.
Subject(s)
Bdellovibrio , Gastrointestinal Microbiome , Penaeidae , Vibrio parahaemolyticus , Vibrio , Animals , Humans , Hepatopancreas/microbiology , Hepatopancreas/pathology , Disease Progression , Acute Disease , Crustacea/microbiology , Necrosis/pathology , Penaeidae/microbiology , Vibrio parahaemolyticus/geneticsABSTRACT
The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.
Subject(s)
Crustacea , Animals , Crustacea/genetics , Crustacea/immunology , Crustacea/metabolism , Crustacea/microbiology , Drosophila melanogaster , Lipopolysaccharides , Receptors, Pattern Recognition/genetics , Receptors, Pattern Recognition/metabolism , Up-Regulation , Vibrio , Signal Transduction , HumansABSTRACT
Intensive case study has established dysbiosis in the gut microbiota-shrimp disease relationship; however, variability in experimental design and the diversity of diseases arise the question of whether some gut indicators are robust and universal in response to shrimp health status, irrespective of causal agents. Through an unbiased subject-level meta-analysis framework, we re-analysed 10 studies, including 261 samples, four lifestages and six different diseases (the causal agents are virus, bacterial, eukaryotic pathogens, or unknown). Results showed that shrimp diseases reproducibly altered the structure of gut bacterial community, but not diversity. After ruling out the lifestage- and disease specific- discriminatory taxa (different diseases dependent indicators), we identify 18 common disease-discriminatory taxa (indicative of health status, irrespective of causal agents) that accurately diagnosed (90.0% accuracy) shrimp health status, regardless of different diseases. These optimizations substantially improved the performance (62.6% vs. 90.0%) diagnosing model. The robustness and universality of model were validated for effectiveness via leave-one-dataset-out validation and independent cohorts. Interspecies interaction and stability of the gut microbiotas were consistently compromised in diseased shrimp compared with corresponding healthy cohorts, while stochasticity and beta-dispersion exhibited the opposite trend. Collectively, our findings exemplify the utility of microbiome meta-analyses in identifying robust and reproducible features for quantitatively diagnosing disease incidence, and the downstream consequences for shrimp pathogenesis from an ecological prospective.
Subject(s)
Gastrointestinal Microbiome , Animals , Bacteria/genetics , Crustacea/microbiology , Dysbiosis , Gastrointestinal Microbiome/physiology , Prospective StudiesABSTRACT
Toll and Toll-like receptors (TLRs) play essential roles in the innate immunity of Drosophila and mammals. Recent studies have revealed the presence of Toll-mediated immune signaling pathways in shrimp. However, the recognition and activation mechanism of Toll signaling pathways in crustaceans remain poorly understood due to the absence of key recognition molecules, such as peptidoglycan recognition proteins. Here, a novel MD2-related lipid-recognition (ML) member named PvML1 was characterized in Penaeus vannamei. We found that PvML1 shared a similar 3D structure with human MD2 that could specifically recognize lipopolysaccharides (LPS) participating in LPS-mediated TLR4 signaling. PvML1 was highly expressed in hemocytes and remarkably upregulated after Vibrio parahemolyticus challenge. Furthermore, the binding and agglutinating assays showed that PvML1 possessed strong binding activities to LPS and its key portion lipid A as well as Vibrio cells, and the binding of PvML1 with bacterial cells led to the agglutination of bacteria, suggesting PvML1 may act as a potential pathogen recognition protein upon interaction with LPS. Besides, coating V. parahemolyticus with recombinant PvML1 promoted bacterial clearance in vivo and increased the survival rate of bacterium-challenged shrimp. This result was further confirmed by RNAi experiments. The knockdown of PvML1 remarkably suppressed the clearance of bacteria in hemolymph and decreased the survival rate of infected shrimp. Meanwhile, the silencing of PvML1 severely impaired the expression of a few antimicrobial peptides (AMPs). These results demonstrated the significant correlation of bacterial clearance mediated by PvML1 with the AMP expression. Interestingly, we found that PvML1 interacted with the extracellular region of PvToll2, which had been previously shown to participate in bacterial clearance by regulating AMP expression. Taken together, the proposed antibacterial model mediated by PvML1 might be described as follows. PvML1 acted as a potential recognition receptor for Gram-negative bacteria by binding to LPS, and then it activated PvToll2-mediated signaling pathway by interacting with PvToll2 to eliminate invading bacteria through producing specific AMPs. This study provided new insights into the recognition and activation mechanism of Toll signaling pathways of invertebrates and the defense functions of ML members.
Subject(s)
Bacterial Infections , Crustacea , Vibrio parahaemolyticus , Animals , Humans , Bacterial Infections/veterinary , Crustacea/immunology , Crustacea/microbiology , Immunity, Innate , Invertebrates , Lipopolysaccharides , Toll-Like Receptors/metabolismABSTRACT
The hepatopancreas is an important digestive and immune organ in crustacean. There were low but stable numbers of microbes living in the hemolymph of crustacean, whereas the organs (including hepatopancreas) of crustacean were immersed in the hemolymph. It is very important to study the immune mechanism of the hepatopancreas against bacteria. In this study, a novel CTL (HepCL) with two CRDs, which was mainly expressed in the hepatopancreas, was identified in red swamp crayfish (Procambarus clarkii). HepCL binds to bacteria in vitro and could enhance bacterial clearance in vivo. Compared with the C-terminal CRD of HepCL (HepCL-C), the N-terminal CRD (HepCL-N) showed weaker bacterial binding ability in vitro and stronger bacterial clearance activity in vivo. The expression of some antimicrobial proteins, such as FLP, ALF1 and ALF5, was downregulated under knockdown of HepCL or blocked with Anti-HepCL after challenge with Vibrio in crayfish. These results demonstrated that HepCL might be involved in the antibacterial immune response by regulating the expression of antimicrobial proteins.
Subject(s)
Crustacea/immunology , Crustacea/metabolism , Disease Resistance/immunology , Hepatopancreas/metabolism , Host-Pathogen Interactions/immunology , Immunity, Innate , Lectins/metabolism , Animals , Bacterial Infections/veterinary , Crustacea/genetics , Crustacea/microbiology , Disease Resistance/genetics , Gene Expression , Host-Pathogen Interactions/genetics , Organ Specificity/genetics , Organ Specificity/immunology , Vibrio/immunologyABSTRACT
BACKGROUND: Vibrio species display variable and plastic fitness strategies to survive and interact with multiple hosts, including marine aquaculture species that are severely affected by pathogenic Vibrios. The culturable Vibrio sp. strain ArtGut-C1, the focus of this study, provides new evidence of such phenotypic plasticity as it accumulates polyhydroxybutyrate (PHB), a biodegradable polymer with anti-pathogen activity, particularly in the marine larviculture phase. The strain was isolated from the gut of laboratory-reared Artemia individuals, the live diet and PHB carrier used in larviculture. Its main phenotypic properties, taxonomic status and genomic properties are reported based on the whole-genome sequencing. RESULTS: Vibrio sp. ArtGut-C1 yielded 72.6% PHB of cells' dry weight at 25 C. The genomic average nucleotide identity (ANI) shows it is closely related to V. diabolicus (ANI: 88.6%). Its genome contains 5,236,997- bp with 44.8% GC content, 3,710 protein-coding sequences, 96 RNA, 9 PHB genes functionally related to PHB metabolic pathways, and several genes linked to competing and colonizing abilities. CONCLUSIONS: This culturable PHB-accumulating Vibrio strain shows high genomic and phenotypic variability. It may be used as a natural pathogen biocontrol in the marine hatchery and as a potential cell factory for PHB production.
Subject(s)
Animals , Artemia/microbiology , Vibrio/metabolism , Polyhydroxyalkanoates/metabolism , Hydroxybutyrates/metabolism , Genetic Variation , Vibrio/isolation & purification , Vibrio/classification , Aquaculture , Probiotics , Crustacea/microbiology , Gastrointestinal Microbiome , Biological Variation, PopulationABSTRACT
Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1 pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5 pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.
Subject(s)
Aeromonas hydrophila/isolation & purification , Densovirinae/isolation & purification , Edwardsiella tarda/isolation & purification , Iridoviridae/isolation & purification , Microfluidics/methods , Molecular Diagnostic Techniques/veterinary , Nucleic Acid Amplification Techniques/veterinary , Vibrio/isolation & purification , White spot syndrome virus 1/isolation & purification , Animals , Crustacea/microbiology , Crustacea/virology , DNA Virus Infections/diagnosis , DNA Virus Infections/veterinary , DNA Virus Infections/virology , Fish Diseases/diagnosis , Fish Diseases/microbiology , Fish Diseases/virology , Fishes/microbiology , Fishes/virology , Gram-Negative Bacterial Infections/diagnosis , Gram-Negative Bacterial Infections/microbiology , Gram-Negative Bacterial Infections/veterinary , Limit of Detection , Molecular Diagnostic Techniques/methods , Mollusca/microbiology , Mollusca/virology , Nucleic Acid Amplification Techniques/methods , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Chitinases or chitinolytic enzymes have different applications in the field of medicine, agriculture, and industry. The present study is aimed at developing an effective hyperchitinase-producing mutant strain of novel Bacillus licheniformis. A simple and rapid methodology was used for screening potential chitinolytic microbiota by chemical mutagenesis with ethylmethane sulfonate and irradiation with UV. There were 16 mutant strains exhibiting chitinase activity. Out of the chitinase-producing strains, the strain with maximum chitinase activity was selected, the protein was partially purified by SDS-PAGE, and the strain was identified as Bacillus licheniformis (SSCL-10) with the highest specific activity of 3.4 U/mL. The induced mutation model has been successfully implemented in the mutant EMS-13 (20.2 U/mL) that produces 5-6-fold higher yield of chitinase, whereas the mutant UV-11 (13.3 U/mL) has 3-4-fold greater chitinase activity compared to the wild strain. The partially purified chitinase has a molecular weight of 66 kDa. The wild strain (SSCL-10) was identified as Bacillus licheniformis using 16S rRNA sequence analysis. This study explores the potential applications of hyperchitinase-producing bacteria in recycling and processing chitin wastes from crustaceans and shrimp, thereby adding value to the crustacean industry.
Subject(s)
Bacillus licheniformis/isolation & purification , Bacillus licheniformis/metabolism , Chitin/metabolism , Chitinases/metabolism , Animals , Artemia/microbiology , Bacillus licheniformis/genetics , Chitin/genetics , Chitinases/genetics , Crustacea/microbiology , DNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Seafood/microbiologyABSTRACT
Vibrio cholerae is a waterborne bacterium and can cause epidemic cholera disease worldwide. Continuous monitoring of V. cholerae contamination in aquatic products is imperative for assuring food safety. In this study, we determined virulence, antimicrobial susceptibility, heavy metal tolerance, and genomic fingerprints of 370 V. cholerae isolates recovered from 12 species of commonly consumed aquatic products collected from July to September of 2018 in Shanghai, China. Among the species, Leiocassis longirostris, Ictalurus punetaus, Ophiocephalus argus Cantor, and Pelteobagrus fulvidraco were for the first time detected for V. cholerae. Toxin genes ctxAB, tcpA, ace, and zot were absent from all the V. cholerae isolates. However, high occurrence of virulence-associated genes was detected, such as hapA (82.7%), hlyA (81.4%), rtxCABD (81.4%, 24.3%, 80.3%, and 80.8%, respectively), and tlh (80.5%). Approximately 62.2% of the 370 V. cholerae isolates exhibited resistance to streptomycin, followed by ampicillin (60.3%), rifampicin (53.8%), trimethoprim (38.4%), and sulfamethoxazole-trimethoprim (37.0%). Moreover, â¼57.6% of the isolates showed multidrug resistant phenotypes with 57 resistance profiles, which was significantly different among the 12 species (multiple antimicrobial resistance index, p < 0.001). Meanwhile, high incidence of tolerance to heavy metals Hg2+ (69.5%), Ni2+ (32.4%), and Cd2+ (30.8%) was observed among the isolates. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR)-based fingerprinting profiles classified the 370 V. cholerae isolates into 239 different ERIC-genotypes, which demonstrated diverse genomic variation among the isolates. Overall, the results in this study meet the increasing need of food safety risk assessment of aquatic products.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/genetics , Seafood/microbiology , Vibrio cholerae/genetics , Virulence Factors/genetics , Ampicillin/pharmacology , Animals , Bacterial Proteins/classification , Bacterial Proteins/metabolism , Bacterial Typing Techniques , Catfishes/microbiology , China , Crustacea/microbiology , DNA Fingerprinting , Food Contamination/analysis , Gene Expression , Genotype , Humans , Metals, Heavy/pharmacology , Microbial Sensitivity Tests , Rifampin/pharmacology , Shellfish/microbiology , Streptomycin/pharmacology , Trimethoprim/pharmacology , Trimethoprim, Sulfamethoxazole Drug Combination/pharmacology , Vibrio cholerae/classification , Vibrio cholerae/drug effects , Vibrio cholerae/pathogenicity , Virulence Factors/classification , Virulence Factors/metabolismABSTRACT
Marine pollution is a significant issue in recent decades, with the increase in industries and their waste harming the environment and ecosystems. Notably, the rise in shellfish industries contributes to tons of shellfish waste composed of up to 58% chitin. Chitin, the second most ample polymer next to cellulose, is insoluble and resistant to degradation. It requires chemical-based treatment or enzymatic hydrolysis to cleave the chitin polymers. The chemical-based treatment can lead to environmental pollution, so to solve this problem, enzymatic hydrolysis is the best option. Moreover, the resulting biopolymer by-products can be used to boost the fish immune system and also as drug delivery agents. Many marine microbial strains have chitinase producing ability. Nevertheless, we still lack an economical and highly stable chitinase enzyme for use in the industrial sector. So we isolate a novel marine bacterial strain Achromobacter xylosoxidans from the shrimp waste disposal site using chitin minimal medium. Placket-Burman and central composite design statistical models for culture condition optimisation predicted a 464.2 U/ml of chitinase production. The culture conditions were optimised for maximum chitinase production recording up to 467 U/ml. This chitinase from the A. xylosoxidans was 100% active at an optimum temperature of 45 °C (withstand up to 55 °C) and pH 8 with 80% stability. The HPLC analysis of chitinase degraded shellfish waste reveals a major amino acid profile composition-arginine, lysine, aspartic acid, alanine, threonine and low levels of isoleucine and methionine. These chitinase degraded products and by-products can be used as supplements in the aquaculture industry.
Subject(s)
Achromobacter denitrificans/enzymology , Achromobacter denitrificans/isolation & purification , Chitin/metabolism , Chitinases/biosynthesis , Crustacea/microbiology , Refuse Disposal , Amino Acids/analysis , Animals , Chitin/chemistry , Chitinases/isolation & purification , Enzyme Stability , Hydrogen-Ion Concentration , Phylogeny , TemperatureABSTRACT
The increasing prevalence of antibiotic resistance genes (ARGs) is a challenge to the health of humans, animals and the environments. Human activities and aquatic environments can increase ARGs. Few studies have focused on the temporal variation of aquatic bacteria with multiple ARGs in aquatic environments affected by human production activity. We studied culturable bacteria (CB) carrying ARGs, including sul1, sul2, floR, strA and gyrA in the shrimp hepatopancreas (HP) and in pond water during shrimp culture. The relative abundance of ARGs carried by CB in HP was higher than that in water (P < 0.05). However, CB carrying ARGs generally varied in random pattern. The correlation of sul2 abundance was significantly positive in HP, while that of strA abundance was significantly negative in water (P < 0.05) during shrimp culture. Among all of the CB, 33.59% carried multiple ARGs. Temporal distance-decay analysis indicated that CB carrying ARGs in water were more resistant to the effects of human activity. CB carrying ARGs varied temporally in HP and pond water during shrimp culture. These results demonstrate that multiple ARGs are carried by CB, and these varied with the phase of aquatic culture.
Subject(s)
Crustacea/microbiology , Drug Resistance, Microbial/genetics , Environmental Monitoring/methods , Genes, Bacterial , Hepatopancreas/microbiology , Ponds/microbiology , Animals , Aquaculture , Bacteria/genetics , Bacteria/isolation & purification , China , Crustacea/growth & development , Humans , Seafood , Water MicrobiologyABSTRACT
Anti-lipopolysaccharide factors (ALFs) are a type of antimicrobial peptide (AMP) which show broad-spectrum antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, fungi and viruses. In this chapter, we review the discovery and classification of this kind of antimicrobial peptide in crustaceans. The structure and function, as well as the mechanism of antibacterial and antiviral activities of ALFs will be summarized and discussed. We will then describe the expression and regulation of various ALF genes in different crustacean species. Finally, the application prospects of ALFs in drug development and disease-resistant genetic breeding will be pointed out and discussed. The review will also discuss several key questions such as the systematic classification and expression regulation of the ALF genes, as well as the future application of ALFs and ALF-derived peptides.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Arthropod Proteins/pharmacology , Crustacea , Animals , Bacteria/drug effects , Crustacea/genetics , Crustacea/microbiology , Crustacea/virology , Drug Development , Fungi/drug effects , Lipopolysaccharides , Viruses/drug effectsABSTRACT
Antibiotic-resistant bacteria (ARB) are widespread in nature and represent a serious public and environmental problem. In the present study, we report for the first time the presence of bacterial ß-lactamases in two macroinvertebrate species with different feeding traits. The class A ß-lactamases, SHV-1 and TEM-1, were found in Citrobacter freundii isolated from Gammarus elvirae and Escherichia coli from water samples, respectively. The metallo-ß-lactamase CphA was found in Aeromonas veronii and Aeromonas hydrophila strains isolated from the predator Dina lineata. The presence of a large plasmid was ascertained only in E. coli strains isolated from water. In all strains studied, an integrase I typical of class I integrin was found. In contaminated freshwater habitats, ARB and antibiotic resistance genes could be disseminated through trophic links with important ecological implications. Transmission through the food chain may contribute to spreading and transferring antibiotic resistance not only in freshwater ecosystems but also outside the aquatic habitat.
Subject(s)
Aeromonas/isolation & purification , Anti-Bacterial Agents/pharmacology , Citrobacter freundii/isolation & purification , Drug Resistance, Microbial/physiology , Escherichia coli/isolation & purification , Invertebrates/microbiology , Aeromonas/drug effects , Aeromonas/genetics , Animals , Bacterial Proteins/isolation & purification , Citrobacter freundii/drug effects , Citrobacter freundii/genetics , Crustacea/microbiology , Drug Resistance, Microbial/drug effects , Drug Resistance, Microbial/genetics , Escherichia coli/drug effects , Escherichia coli/genetics , Integrons/genetics , Italy , Leeches/microbiology , Microbial Sensitivity Tests , Plasmids , Rivers , beta-Lactamases/isolation & purificationABSTRACT
Although increasing levels of attention have been targeted towards aquaculture-associated bacteria, the bacterial community of animal intestines and its relationship with the aquaculture environment need to be further investigated. In this study, we used high-throughput sequencing to analyze the bacterial community of pond water, sediment, and the intestines of diseased and healthy animals. Our data showed that Proteobacteria, Firmicutes, Cyanobacteria, and Bacteroidetes were the dominant taxa of bacteria across all samples and accounted for more than 90% of the total sequence. Difference analysis and Venn diagrams showed that most of the intestinal bacterial OTUs (operational taxonomic units) of diseased and healthy animals were the same as those of sediment and water, indicating that the aquaculture environment was the main source of intestinal bacteria. Compared with healthy animals, a considerable reduction of OTUs was evident in diseased animals. Welch's t test showed that the dominant bacterial taxa in sediment, water, and animal intestine were significantly different (p < 0.05) and each had its own unique dominant microorganisms. In addition, differences between the intestinal bacteria of healthy and diseased animals were represented by potential probiotics and pathogens, such as Bacillus, Vibrio, Oceanobacillus, and Lactococcus. Principal component analysis (PcoA) showed that a similar environment shaped a similar microbial structure. There was a large difference in the spectrum of intestinal bacteria in diseased animals; furthermore, the spectrum of intestinal bacteria in diseased animals was very different from the environment than in healthy animals. This study provides a theoretical basis for a relationship between the intestinal bacteria of healthy and diseased animals and the environment and provides guidance for environmental regulation and disease prevention in aquaculture areas.
Subject(s)
Bacteria/classification , Bacteria/genetics , Crustacea/microbiology , Gastrointestinal Microbiome , Geologic Sediments/microbiology , Water Microbiology , Animals , Aquaculture , High-Throughput Nucleotide Sequencing , MetagenomicsABSTRACT
Eatable crustaceans are susceptible to bacterial septicemia from injury or a compromised immune defense, which can possibly have detrimental effects in mammals that consume them. Since many crustaceans (i.e., crabs, lobsters and crayfish) are used for animal food and human consumption, it is of interest to understand the effects potential bacterial infections can have on their health as well as ours, including effects on cardiovascular and neuromuscular activities. The Red Swamp crayfish (Procambarus clarkii) was used as a model crustacean to investigate the effect of direct exposure to isolated endotoxin lipopolysaccharide (LPS) and the associated peptidoglycans from gram-negative bacteria (Serratia marcescens). S. marcescens is a common strain identified to cause septicemia in mammals and is prevalently found in nature. LPS injection into the hemolymph of crayfish revealed acute changes in heart rate and effects on survival. Direct LPS exposure on an in situ sensory-CNS-motor circuit produces a decrease in recruiting of the motor nerve at 500⯵g/ml but has no significant effect at 100⯵g/ml. At the isolated neuromuscular junction, the direct action of the LPS endotoxin (500⯵g/ml) enhances evoked synaptic transmission, while not altering facilitation. Also, the amplitude and the frequency of spontaneous vesicle fusion events was not altered by LPS exposure. However, the resting membrane potential of the muscle transiently hyperpolarizes. These direct actions on tissues appear to be independent of innate immune responses and suggest the LPS targets on these tissues have a role in excitability of cellular function. {242 words}.
Subject(s)
Crustacea/drug effects , Endotoxins/pharmacology , Heart Rate/drug effects , Lipopolysaccharides/pharmacology , Animals , Astacoidea/drug effects , Astacoidea/physiology , Central Nervous System/drug effects , Crustacea/microbiology , Crustacea/physiology , Endotoxins/toxicity , Heart/drug effects , Hemolymph/drug effects , Lipopolysaccharides/toxicity , Mammals/microbiology , Mammals/physiology , Motor Neurons/drug effects , Neuromuscular Junction , Polysaccharides/pharmacology , Polysaccharides/toxicity , Sensory Receptor Cells/drug effects , Serratia marcescens/pathogenicity , Synaptic Transmission/drug effectsABSTRACT
A Gram-stain-positive, aerobic, spore-forming, moderately halophilic bacterium, SSKP1-9T, was isolated from traditional salted shrimp paste (Ka-pi) produced in Samut Sakhon Province, Thailand. This strain grew optimally at 37-40 °C, pH 7.0 and in the presence of 8-16â% (w/v) NaCl. The 16S rRNA gene sequence similarity values between strain SSKP1-9T and Lentibacillus juripiscarius TISTR 1535T and Lentibacillus halophilus TISTR 1549T were 98.7 and 97.2â%, respectively. Based on 16S rRNA gene sequence similarity, strain SSKP1-9T represents a distinct novel species, as shown by phenotypic traits, DNA-DNA hybridization and average nucleotide identity values. In addition, the whole-cell protein profile confirmed the novelty of the taxon. The genomic DNA G+C content was 44.6 mol%. The major isoprenoid quinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid. Polar lipid analysis revealed the presence of phosphatidylglycerol, diphosphatidylglycerol, four unidentified lipids, an unidentified phospholipid and an unidentified glycolipid. The major cellular fatty acids were anteiso-C15â:â0, anteiso-C17â:â0 and iso-C16â:â0. The results of phenotypic and chemotaxonomic characteristics and whole-genome analysis support that strain SSKP1-9T represents a novel species of Lentibacillus, for which the name Lentibacilluslipolyticus sp. nov. is proposed. The type strain is SSKP1-9T (=JCM 32625T=TISTR 2597T).
Subject(s)
Bacillaceae/classification , Food Microbiology , Phylogeny , Seafood/microbiology , Animals , Bacillaceae/isolation & purification , Bacterial Typing Techniques , Base Composition , Crustacea/microbiology , DNA, Bacterial/genetics , Diaminopimelic Acid/chemistry , Fatty Acids/chemistry , Nucleic Acid Hybridization , Peptidoglycan/chemistry , Phospholipids/chemistry , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Thailand , Vitamin K 2/analogs & derivatives , Vitamin K 2/chemistryABSTRACT
Microbial food spoilage is responsible for significant economic losses. Brochothrix thermosphacta is one of the major bacteria involved in the spoilage of meat and seafood. Its growth and metabolic activities during food storage result in the production of metabolites associated with off-odors. In this study, we evaluated the genotypic and phenotypic diversity of this species. A collection of 161 B. thermosphacta strains isolated from different foods, spoiled or not, and from a slaughterhouse environment was constituted from various laboratory collections and completed with new isolates. A PCR test based on the rpoB gene was developed for a fast screening of B. thermosphacta isolates. Strains were typed by MALDI-TOF MS, rep-PCR, and PFGE. Each typing method separated strains into distinct groups, revealing significant intra-species diversity. These classifications did not correlate with the ecological origin of strains. The ability to produce acetoin and diacetyl, two molecules associated with B. thermosphacta spoilage, was evaluated in meat and shrimp juices. The production level was variable between strains and the spoilage ability on meat or shrimp juice did not correlate with the substrate origin of strains. Although the B. thermosphacta species encompasses ubiquitous strains, spoiling ability is both strain- and environment-dependent.