Genus Aeromonas Research Articles

Fermented Wheat Bran Polysaccharides Improved Intestinal Health of Zebrafish in Terms of Intestinal Motility and Barrier Function

Intestinal barrier dysfunction and gut microbiota disorders have been associated with various intestinal and extraintestinal diseases. Fermented wheat bran polysaccharides (FWBP) are promising natural products for enhancing the growth performance and antioxidant function of zebrafish. The present study was conducted, in order to investigate the effects of FWBP on the intestinal motility and barrier function of zebrafish, which could provide evidence for the further potential of using FWBP as a functional food ingredient in the consideration of gut health. In Experiment 1, the normal or loperamide hydrochloride-induced constipation zebrafish larvae were treated with three concentrations of FWBP (10, 20, 40 μg/mL). In Experiment 2, 180 one month-old healthy zebrafish were randomly divided into three groups (six replicates/group and 10 zebrafish/tank) and fed with a basal diet, 0.05% FWBP, or 0.10% FWBP for eight weeks. The results showed that FWBP treatment for 6 h can reduce the fluorescence intensity and alleviate constipation, thereby promoting the gastrointestinal motility of zebrafish. When compared with control group, zebrafish fed diets containing FWBP showed an increased villus height (p < 0.05), an up-regulated mRNA expression of the tight junction protein 1α, muc2.1, muc5.1, matrix metalloproteinases 9 and defensin1 (p < 0.05), an increased abundance of the phylum Firmicutes (p < 0.05), and a decreased abundance of the phylum Proteobacteria, family Aeromonadaceae, and genus Aeromonas (p < 0.05). In addition, 0.05% FWBP supplementation up-regulated the intestinal mRNA expression of IL-10 and Occludin1 (p < 0.05), enhanced the Shannon and Chao1 indexes (p < 0.05), and increased the abundance of Bacteroidota and Actinobacteriota at the phylum level (p < 0.05). Additionally, 0.1% FWBP supplementation significantly improved the villus height to crypt depth ratio (p < 0.05) and increased the mRNA expression of IL-17 (p < 0.05). These findings reveal that FWBP can promote the intestinal motility and enhance the intestinal barrier function, thus improving the intestinal health of zebrafish.

Effects of Sulfamethoxazole and Florfenicol on Growth, Antioxidant Capacity, Immune Responses and Intestinal Microbiota in Pacific White Shrimp Litopenaeus vannamei at Low Salinity.

Antibiotic residue may pose a serious risk to aquaculture, and the culture of Litopenaeus vannamei in a low-salinity environment is a growing trend over the world. Here, we aimed to understand the combined effect of low salinity and sulfamethoxazole (SMZ) and florfenicol (FLO) antibiotics on L. vannamei. The growth performance, immune functions, antioxidant capacity and intestinal microbiota were investigated. Compared with the control group, the weight gain and survival rate significantly decreased (p < 0.05) in shrimp after they were exposed to low-salinity (salinity 3) water and the mixture of antibiotics and low-salt conditions for 28 days. The antioxidant activities of SOD and T-AOC, shown at low salinity and in the higher concentration of the SMZ treatment group (SMZH), were significantly decreased, while the GST activity was significantly increased in each treatment group in comparison with the control group. The expression of immune-related genes, including TOLL, LvIMD, PPO and HSP, in the low concentration of the SMZ treatment group (SMZL) was higher than that in the other groups. The diversity of intestine microbiota was disturbed with a lower Shannon index in the low-salinity and SMZH groups, and a higher Simpson index in the SMZH group. Proteobacteria, Actinobacteria and Bacteroidetes were the dominant phyla in the gut of L. vannamei. At the genus level, Microbacterium, Shewanella, Aeromonas, Acinetobacter, Gemmobacter, Paracoccus and Lysobacter were significantly decreased in the low-salinity group. However, the abundance of opportunistic pathogens belonging to the genus Aeromonas in the FLO group was increased. The predicted microbe-mediated functions showed that the pathway for "amino acid metabolism" and "replication and repair" was significantly inhibited in both the low-salinity and antibiotic-exposed groups. All the findings in this study indicate that the combined effect of antibiotics and low salinity on L. vannamei negatively impacted the physiological and intestinal microbiota functions.

Antibiotic Resistance, Virulence Gene Detection, and Biofilm Formation in Aeromonas spp. Isolated from Fish and Humans in Egypt.

The genus Aeromonas is widely distributed in aquatic environments and is recognized as a potential human pathogen. Some Aeromonas species are able to cause a wide spectrum of diseases, mainly gastroenteritis, skin and soft-tissue infections, bacteremia, and sepsis. The aim of the current study was to determine the prevalence of Aeromonas spp. in raw fish markets and humans in Zagazig, Egypt; identify the factors that contribute to virulence; determine the isolates' profile of antibiotic resistance; and to elucidate the ability of Aeromonas spp. to form biofilms. The examined samples included fish tissues and organs from tilapia (Oreochromis niloticus, n = 160) and mugil (Mugil cephalus, n = 105), and human skin swabs (n = 51) and fecal samples (n = 27). Based on biochemical and PCR assays, 11 isolates (3.2%) were confirmed as Aeromonas spp. and four isolates (1.2%) were confirmed as A. hydrophila. The virulence genes including haemolysin (hyl A) and aerolysin (aer) were detected using PCR in A. hydrophila in percentages of 25% and 50%, respectively. The antimicrobial resistance of Aeromonas spp. was assessed against 14 antibiotics comprising six classes. The resistance to cefixime (81.8%) and tobramycin (45.4%) was observed. The multiple antibiotic resistance (MAR) index ranged between 0.142-0.642 with 64.2% of the isolates having MAR values equal to 0.642. Biofilm formation capacity was assessed using a microtiter plate assay, and two isolates (18.1%) were classified as biofilm producers. This study establishes a baseline for monitoring and controlling the multidrug-resistant Aeromonas spp. and especially A. hydrophila in marine foods consumed in our country to protect humans and animals.

Cytotoxicity and Antimicrobial Resistance of Aeromonas Strains Isolated from Fresh Produce and Irrigation Water

The genus Aeromonas has received constant attention in different areas, from aquaculture and veterinary medicine to food safety, where more and more frequent isolates are occurring with increased resistance to antibiotics. The present paper studied the interaction of Aeromonas strains isolated from fresh produce and water with different eukaryotic cell types with the aim of better understanding the cytotoxic capacity of these strains. To study host-cell pathogen interactions in Aeromonas, we used HT-29, Vero, J774A.1, and primary mouse embryonic fibroblasts. These interactions were analyzed by confocal microscopy to determine the cytotoxicity of the strains. We also used Galleria mellonella larvae to test their pathogenicity in this experimental model. Our results demonstrated that two strains showed high cytotoxicity in epithelial cells, fibroblasts, and macrophages. Furthermore, these strains showed high virulence using the G. mellonella model. All strains used in this paper generally showed low levels of resistance to the different families of the antibiotics being tested. These results indicated that some strains of Aeromonas present in vegetables and water pose a potential health hazard, displaying very high in vitro and in vivo virulence. This pathogenic potential, and some recent concerning findings on antimicrobial resistance in Aeromonas, encourage further efforts in examining the precise significance of Aeromonas strains isolated from foods for human consumption.

Comparative Analysis of the Symbiotic Microbiota in the Chinese Mitten Crab (Eriocheir sinensis): Microbial Structure, Co-Occurrence Patterns, and Predictive Functions

Symbiotic microorganisms in the digestive and circulatory systems are found in various crustaceans, and their essential roles in crustacean health, nutrition, and disease have attracted considerable interest. Although the intestinal microbiota of the Chinese mitten crab (Eriocheir sinensis) has been extensively studied, information on the symbiotic microbiota at various sites of this aquatic economic species, particularly the hepatopancreas and hemolymph, is lacking. This study aimed to comprehensively characterize the hemolymph, hepatopancreas, and intestinal microbiota of Chinese mitten crab through the high-throughput sequencing of the 16S rRNA gene. Results showed no significant difference in microbial diversity between the hemolymph and hepatopancreas (Welch t-test; p > 0.05), but their microbial diversity was significantly higher than that in the intestine (p < 0.05). Distinct differences were found in the structure, composition, and predicted function of the symbiotic microbiota at these sites. At the phylum level, the hemolymph and hepatopancreas microbiota were dominated by Proteobacteria, Firmicutes, and Acidobacteriota, followed by Bacteroidota and Actinobacteriota, whereas the gut microbiota was mainly composed of Firmicutes, Proteobacteria, and Bacteroidota. At the genus level, Candidatus Hepatoplasma, Shewanella, and Aeromonas were dominant in the hepatopancreas; Candidatus Bacilloplasma, Roseimarinus, and Vibrio were dominant in the intestine; Enterobacter, norank_Vicinamibacterales, and Pseudomonas were relatively high-abundance genera in the hemolymph. The composition and abundance of symbiotic microbiota in the hemolymph and hepatopancreas were extremely similar (p > 0.05), and no significant difference in functional prediction was found (p > 0.05). Comparing the hemolymph in the intestine and hepatopancreas, the hemolymph had lower variation in bacterial composition among individuals, having a more uniform abundance of major bacterial taxa, a smaller coefficient of variation, and the highest proportion of shared genera. Network complexity varied greatly among the three sites. The hepatopancreas microbiota was the most complex, followed by the hemolymph microbiota, and the intestinal microbiota had the simplest network. This study revealed the taxonomic and functional characteristics of the hemolymph, hepatopancreas, and gut microbiota in Chinese mitten crab. The results expanded our understanding of the symbiotic microbiota in crustaceans, providing potential indicators for assessing the health status of Chinese mitten crab.

Aeromonas salmonicida isolates from Canada demonstrate wide distribution and clustering among mesophilic strains.

All the 36 known species to date of the genus Aeromonas are mesophilic except the species Aeromonas salmonicida, which includes both psychrophilic and mesophilic subspecies. For 20 years, more and more mesophilic A. salmonicida strains have been discovered. Only A. salmonicida subsp. pectinolytica has officially been classified as a mesophilic subspecies. Most mesophiles have been isolated in hot countries. We present, for the first time, the characterization of two new mesophilic isolates from Quebec (Canada). Phenotypic and genomic characterizations were carried out on these strains, isolated from dead fish from a fish farm. Isolates 19-K304 and 19-K308 are clearly mesophiles, virulent to the amoeba Dictyostelium discoideum, a surrogate host, and close to strain Y577, isolated in India. To our knowledge, this is the first time that mesophilic strains isolated from different countries are so similar. The major difference between the isolates is the presence of plasmid pY47-3, a cryptic plasmid that sometimes presents in mesophilic strains. More importantly, our extensive phylogenetic analysis reveals two well-defined clades of mesophilic strains with psychrophiles associated with one of these clades. This helps to have a better understanding of the evolution of this species and the apparition of psychrophilic subspecies.

A review on aeromoniasis in poultry: A bacterial disease of zoonotic nature

Aeromonas spp. are widely distributed in surface water, sewage, untreated and chlorinated, drinking water, as well as meats, fish, shellfish, poultry, and their products. A disease caused by Aeromonas spp. is designated as aeromoniasis. It can affect different aquatic animals, mammals, and birds in different geographic regions. Moreover, gastrointestinal and extra-intestinal disease conditions may be provoked in humans as a result of food poising with Aeromonas spp. Some Aeromonas spp. have been identified, however, Aeromonas hydrophila (A. hydrophila), A. caviae, and A. veronii bv sobria may be of public health significance. Aeromonas spp. are members of family Aeromonadaceae and genus Aeromonas. They are Gram-negative rod-shaped, facultative anaerobic, and oxidase and catalase-positive bacteria. The pathogenicity of Aeromonas in different hosts is mediated by several virulence factors such as endotoxins, cytotoxic enterotoxin, cytotoxins, hemolysins, adhesins, and extracellular enzymes such as proteases, amylases, lipases, ADP-ribosyltransferases, and DNases. Most avian species are susceptible to either natural or experimental infections with Aeromonas spp. Infection usually arises through feacal-oral route. Traveler's diarrhea as well as other systemic and local infections are the clinical picture of food poisoning associated with aeromoniasis in humans. Despite Aeromonas spp. being sensitive to various antimicrobials, multiple drug resistance has been commonly reported worldwide. Accordingly, this review highlights aeromoniasis in poultry regarding Aeromonas virulence factors epidemiology, pathogenicity, zoonosis, and antimicrobial resistance.

Antimicrobial Activity of Excoecaria Agallocha Mangrove Extract in Inhibiting the Growth of Aeromonas Hydrophila by in-Vitro

One of the bacterial diseases that often attack freshwater fish is the red-sore disease, also known as aeromoniasis or Motile Aeromonas Septicemia (MAS). It is caused by bacteria from Aeromonas genus, such as Aeromonas hydrophila. A safe alternative treatment for pathogenic bacteria is the use of antibacterial compounds made from natural ingredients. The Excoecaria agallocha mangrove extract is one of potential alternatives that is useful as natural antimicrobials due to its various contents of antibacterial bioactive compounds. The objective of this study was to determine the antimicrobial activity of E. agallocha mangrove extract in inhibiting the growth of A. hydrophila. The research method was experimental with complete randomized design and factorial pattern of 3 factors (type of plant organ, bacterial strain, and concentration of extract), in 48 treatments and 3 replications. The solvents used consisted of methanol (polar) and n-hexane (non-polar). The plant organs used included leaves and stems. The bacterial strains consisted of GPl-04, GB-01, GL-01, GL-02, GJ-01, and GK-01 with concentrations of leaves and stems extracts of 0, 10, 20, and 30%. The parameter measured was the diameter of inhibition zone, which was done by using disc paper (Kirby Bauer method). Non-parametric Kruskall-Wallis Test and qualitative-descriptive data analysis were applied. The methanol extract of E. agallocha leaves in GJ-01 strain at 10 and 20% concentrations had antimicrobial activity with inhibition zone in 2.55 mm and 2.46 mm, respectively. The n-hexane extract of E. agallocha stem at 20 and 30% concentrations had antimicrobial activity with inhibition zones of 1.88 mm in GPl-04 strain and 1.58 mm in GB-01 strain, respectively. Extract of E. agallocha mangrove exhibited potential as a natural antibacterial to prevent aeromoniasis in fish.

Isolation of vB_AsaM_LPM4 reveals the dynamics of Prophage 3 in Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida causes furunculosis, a major infection that affects fish farms worldwide. We isolated phage vB_AsaM_LPM4 (LPM4) from a diseased fish. Based on its DNA sequence, LPM4 is identical to the uncharacterized Prophage 3, a prophage present mostly in North American A. salmonicida subsp. salmonicida isolates that bear the genomic island AsaGEI2a. Prophage 3 and AsaGEI2a are inserted side by side in the bacterial chromosome. The LPM4/Prophage 3 sequence is similar to that of other prophages found in various members of the genus Aeromonas. LPM4 specifically infects A. salmonicida subsp. salmonicida strains that do not already bear Prophage 3. The presence of an A-layer on the surface of the bacteria is not necessary for the adsorption of phage LPM4 but seems to facilitate its infection process. We also successfully produced lysogenic strains that bear Prophage 3 using sensitive strains with different genetic backgrounds, suggesting that there is no interdependency between LPM4 and AsaGEIs. PCR analysis of the excision dynamics of Prophage 3 and AsaGEIs revealed that these genetic elements can spontaneously excise themselves from the bacterial chromosome independently of one another. Through the isolation and characterization of LPM4, this study reveals new facets of Prophage 3 and AsaGEIs.

Diversidad y perfiles de resistencia a los antibióticos en especies de Aeromonas aisladas de ecosistema acuático del ecuador

Introduction: In Ecuador, the diversity of Aeromonas species and their resistance profiles to antibiotics in most aquatic ecosystems are unknown. Objective: To know the diversity of species of the genus Aeromonas and their resistance profiles to antibiotics present in the mineralmedicinal water spa “Santagua de Chachimbiro” and in the water of the Cuicocha crater volcanic lake. Methodology: Water samples from the “Santagua de Chachimbiro” spa and from the volcanic crater lake Cuicocha, located in the province of Imbabura-Ecuador, were analyzed. The isolation of the Aeromonas species was carried out by the deep seeding technique and by membrane filtration using R2A agar and ampicillin starch agar as the culture medium. The bacteria were identified following the schemes proposed by Longa et al., (1997), complemented with tests of the Microgen galleries. The antibiotic resistance profile was determined by the plate diffusion method of Kirby and Bauer (1966) interpreted according to the CLSI (2019). Results: A total of 19 strains of the genus Aeromonas belonging to 6 species were identified. Antibiotic resistance profiles show different patterns for each of the species, although species multi-resistant to antibiotics amoxicillin, amoxicillin/clavulanic acid, aztreonam, erythromycin and tetracycline prevail. Conclusions: The presence of various species of Aeromonas in aquatic environments of Ecuador with varied profiles of resistance and multi-resistance to antibiotics is demonstrated. Keywords: aeromonas; aquatic ecosystem; mineralmedicinal water; crateric lake; resistance to antibiotics

Dietary betaine and/or TMAO affect hepatic lipid accumulation and glycometabolism of Megalobrama amblycephala exposed to a high-carbohydrate diet.

A 12-week experiment was conducted to explore the effects of betaine and/or TMAO on growth, hepatic health, gut microbiota, and serum metabolites in Megalobrama amblycephala fed with high-carbohydrate diets. The diets were as follows: CD group (control diet, 28.5% carbohydrate), HCD group (high-carbohydrate diet, 38.2% carbohydrate), HBD group (betaine-added diet, 38.3% carbohydrate + 1.2% betaine), HTD group (TMAO-added diet, 38.2% carbohydrate + 0.2% TMAO), and HBT group (diet added with both betaine and TMAO, 38.2% carbohydrate + 1.2% betaine + 0.2% TMAO). The results showed that the hepatosomatic index (HSI); whole-body crude fat; hepatic lipid accumulation; messenger RNA expression levels of gk, fpbase, g6pase, ahas, and bcat; serum branched-chain amino acids (BCAAs); ratio of Firmicutes-to-Bacteroidetes; and abundance of the genus Aeromonas were all significantly increased, while the abundance levels of the genus Lactobacillus and phyla Tenericutes and Bacteroidetes were drastically decreased in the HCD group. Compared with the HCD group, the HSI; whole-body crude fat; hepatic lipid accumulation; expression levels of fbpase, g6pase, pepck, ahas, and bcat; circulating BCAA; ratio of Firmicutes-to-Bacteroidetes; and abundance levels of the genus Aeromonas and phyla Tenericutes and Bacteroidetes were significantly downregulated in the HBD, HTD, and HBT groups. Meanwhile, the expression levels of pk were drastically upregulated in the HBD, HTD, and HBT groups as well as the abundance of Lactobacillus in the HBT group. These results indicated that the supplementation of betaine and/or TMAO in high-carbohydrate diets could affect the hepatic lipid accumulation and glycometabolism of M. amblycephala by promoting glycolysis, inhibiting gluconeogenesis and biosynthesis of BCAA, and mitigating the negative alteration of gut microbiota. Among them, the combination of betaine and TMAO had the best effect.

Removal of bacterial pathogens and antibiotic resistance bacteria by anaerobic sludge digestion with thermal hydrolysis pre-treatment and alkaline stabilization post-treatment

Primary sludge (PS) is associated with public health and environmental risks, so regulations focus on reducing the pathogenic and heavy metal contents of the treated material (biosolids), intended for soil amendments and land reclamation. The regulations set limits for Escherichia coli (or fecal coliforms), Salmonella spp., helminth eggs and enterovirus. However, the potential risk due to antibiotic resistant bacteria (ARB) and other human potential pathogenic bacteria (HPB) are not considered.In this work, three sludge treatment processes, having in common an anaerobic digestion step, were applied to assess the removal of regulated bacteria (fecal coliforms, Salmonella spp), ARB and HPB. The treatment arrangements, fed with PS from a full-scale wastewater treatment plant were: 1) Mesophilic anaerobic digestion followed by alkaline stabilization post-treatment (MAD-CaO); 2) Thermophilic anaerobic digestion (TAD) and, 3) Pre-treatment (mild thermo-hydrolysis) followed by TAD (PT-TAD). The results address the identification, quantification (colony forming units) and taxonomic characterization of ARB resistant to β-lactams and vancomycin, as well as the taxonomic characterization of HPB by sequencing with PacBio. In addition, quantification based on culture media of fecal coliforms and Salmonella spp. is presented.The capabilities and limitations of microbiological and metataxonomomic analyses based on PacBio sequencing are discussed, emphasizing that they complement each other. Genus Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Ochrobactrum, Pseudomonas and Raoultella, among others, were found in the PS, which are of clinical or environmental importance, being either HPB, HPB-ARB, or non-pathogenic ARB with the potentiality of horizontal gene transfer. Based on the analysis of fecal coliforms and Salmonella spp., the three processes produced class A (highest) biosolids, suitable for unrestricted agriculture applications. Mild thermo-hydrolisis was effective in decreasing ARB cultivability, but it reappeared after the following TAD. O. intermedium (HPB-ARB) was enriched in MAD and TAD while Laribacter hongkongensis (HPB) did persist after the applied treatments.

Potential Pathogenicity of Aeromonas spp. Recovered in River Water, Soil, and Vegetation from a Natural Recreational Area.

The genus Aeromonas is widely distributed in aquatic environments and is recognized as a potential human pathogen. Some Aeromonas species are able to cause a wide spectrum of diseases, mainly gastroenteritis, skin and soft-tissue infections, bacteremia, and sepsis. Currently, untreated river water is used for irrigation and recreational purposes. In this study, the Aeromonas spp. present in a river recreational environment was investigated by quantifying its presence in water, soil, and vegetation using three techniques: qPCR, plate counting in selective ADA medium, and Most Probable Number, in parallel. The presence of clones in the three types of samples was elucidated through genotyping with the ERIC-PCR technique, whereas the identification of the isolated Aeromonas was carried out by sequencing the rpoD gene. Finally, the pathogenic potential of some of the strains was explored by studying the presence and expression of virulence genes characteristic of the genus, their antimicrobial susceptibility profile, as well as the quantification of their cell damage and intracellular survival in an in vitro macrophages infection model. The results showed the presence of Aeromonas in all samples with the three quantification methods, with Aeromonas popoffii being the most prevalent species. The presence of strains with the same genotype (ERIC-PCR) was also confirmed in different samples. Some of the strains showed a high level of cell damage and intracellular bacterial survival, as well as the presence of various virulence factors. Furthermore, these strains showed resistance to some of the antibiotics tested and used therapeutically in both humans and animals. These results indicate that the presence of Aeromonas in this environment may represent a biosanitary risk that could be a public health problem.

Evaluation on the effects of phage cocktail in preventing Aeromonas veronii infection in Gibel carps (Carassius auratus gibelio)

Members in Aeromonas genus especially for Aeromonas veronii isolates are ubiquitous in aquaculture environments and have increasingly implicated in diseases of fish, and many of them have multidrug resistance. In this work, we used a phage cocktail as an alternative strategy to combat the infection of A. veronii 1810 in gibel carps. Three bacteriophages were isolated and designated as pAEv1810, pAEv1812 and pAEv1818. Electron microscopy observation revealed that all three phages belonged to the order of Caudovirales. Host spectrum assay showed that all three phages were lytic to A. veronii 1810 and pAEv1812 had a relatively broad host range. The cocktail combined with the three phages performed a significant inhibition (P < 0.05) of growth of A. veronii 1810 compared with the untreated cultures. During the 60 days of cultivation time in the in vivo trial, our results revealed that feeding the diet containing the phage cocktail cannot affect the growth performance and digestive enzyme activities of gibel carps. At the end of the 12-day observation in the challenge phase, the survival rate of gibel carps was calculated as 80% for the phage cocktail treatment, which was almost caught up with the rate of antibiotic treatment. For the digestive enzyme activities, our results indicated that the oral administrated phages helped the main enzymes including amylase, lipase and proteinase return to normal. Additionally, certain numbers of phages steadily existed in the cultured water, gills and intestines of gibel carps during the 60 days of phage feeding time. Subsequently, the intestinal microbiota of gibel carps in the group of CAbc (blank control), CABT (Bacterial challenge treatment) and CApt (phage cocktail treatment) were analyzed at the genus level. The Aeromonas abundance in the CApt group was significantly decreased compared to that of CABT (P < 0.05) and the intestinal microbiota in the CApt group presented high similarity compared to that of the CAbc group, suggesting the applied phages could effectively protect the structure and bacterial communities in the intestinal tracts of gibel carps. Together, the overall results of this experiment indicated that using a phage cocktail could be effective in combating A. veronii infections in gibel carps.

The variants of polymyxin susceptibility in different species of genus Aeromonas.

The aquatic environment is an important medium for the accumulation and dissemination of antibiotic-resistant bacteria as it is often closely related to human activities. Previous studies paid little attention to the prevalence and mechanism of polymyxin-resistant bacteria in the aquatic environment. As a Gram-negative opportunistic pathogen widely distributed in aquatic ecosystems, the antibiotic-resistant profile of Aeromonas spp. deserves much attention. In this study, we identified 61 Aeromonas spp. isolates from water samples in the section of the Yangtze River. The total polymyxin B (PMB) resistance rate of these strains was 49.18% (30/61), showing a high level of polymyxin resistance in Aeromonas spp. The MIC50 and MIC90 for PMB exhibited a significant discrepancy among different species (p < 0.001). The MIC50 and MIC90 for PMB in the Aeromonas hydrophila were 128 mg/L and above 128 mg/L while in Aeromonas caviae and Aeromonas veronii, the MIC50 and MIC90 value were both 2 mg/L. Only two A. veronii strains (MIC = 2 mg/L) and one A. caviae strain (MIC = 0.5 mg/L) were identified as carrying mobilized polymyxin resistant gene mcr-3.42, and mcr-3.16. All mcr genes were located in the chromosome. This is the first report that the downstream region of mcr-3.42 was the truncated mcr-3-like gene separated by the insertion sequences of ISAs20 (1,674 bp) and ISAs2 (1,084 bp). Analysis of epidemiology of mcr-positive Aeromonas genomes from GenBank database showed that the genus Aeromonas and the aquatic environment might be the potential container and reservoir of mcr-3. By the whole-genome sequencing and qRT-PCR, we inferred that the sequence differences in the AAA domain of MlaF protein and its expression level among these three species might be involved in the development of polymyxin resistance. Our study provided evidences of the possible mechanism for the variety of polymyxin susceptibility in different species of the genus Aeromonas and a theoretical basis for the surveillance of the aquatic environment.

Metabolite features of serum and intestinal microbiota response of largemouth bass (Micropterus salmoides) after Aeromonas hydrophila challenge

The enteric morphology, enteric microbiota structure and serum metabolomics of M. salmoides before and after infected by A. hydrophila were analysed to explore the pathogenic mechanism of A. hydrophila infection in M. salmoides. The results revealed that, after the infection of A. hydrophila, the villus boundary of largemouth bass became less obvious; the relative abundance of Proteobacteria and decreasing relative abundance of Tenericutes were increasing; genera relative abundance of putatively beneficial bacteria (Mycoplasma) were decreasing, whereas the genus Aeromonas increased after infection; serum metabolomic analysis showed that infection with A. hydrophila caused disorder to the metabolic processes of largemouth bass, particularly amino acid metabolism, and caused inflammation; several potential pathogen infection-related and significantly differential intestinal microbiota-related metabolite markers were identified, such as 6-hydroxy-5-methoxyindole glucuronide, zalcitabine, bilirubin, aciclovir. This study may provide new insights into the potential association between enteric microbiota and serum metabolism and the pathogenic mechanism of M. salmoides infected by A. hydrophila, providing a scientific basis for disease control in largemouth bass breeding.

Growth and Spoilage Potential of an Aeromonas salmonicida Strain in Refrigerated Atlantic Cod (Gadus morhua) Stored under Various Modified Atmospheres.

Aeromonas spp. are ubiquitous aquatic bacteria, frequently isolated from seafood. The growth and spoilage potential of an inoculated strain of Aeromonas salmonicida (SU2) in Atlantic cod (Gadus morhua) fillets were assessed as a function of various modified atmospheres (MA) (CO2 (67%) with O2 (33%) or N2 (33%), and vacuum packaging (VP)) under refrigerated storage (4 °C) for 13 days. Additionally, the general microbiological quality was assessed by quantifying total aerobic psychrotrophic count (PC), total aerobic plate count (APC), and H2S-producing bacteria. A selection of spoilage metabolites was quantified by nuclear magnetic resonance (NMR) spectroscopy. The MA containing CO2/O2 and CO2/N2 significantly inhibited the growth of the inoculated strain throughout storage (p < 0.001), whereas VP allowed for a 3-log increase of Aeromonas in 13 days. No significant effect of the inoculation with A. salmonicida on spoilage metabolite production was detected. Applying O2 instead of N2 in the CO2-enriched atmosphere resulted in a significantly lower PC for 10 days, and H2S-producing bacteria were inhibited in both MAs compared to vacuum. This study provides data that can be used as a basis to further elucidate the role of bacteria belonging to the genus Aeromonas as potential spoilage organisms in marine fish species.

Relative expression and validation of Aeromonas salmonicida subsp. salmonicida reference genes during ex vivo and in vivo fish infection.

The genus Aeromonas is found worldwide in freshwater and marine environments and has been implicated in the etiology of human and animal diseases. In fish, among Aeromonas species, A. salmonicida causes massive mortality and great economic losses in marine and continental aquaculture species. Currently, several aspects of the clinical signs and pathogenesis of this Gram-negative bacterium have been described; however, determination of an appropriate reference gene is essential to normalize cellular mRNA data remain unknown. Here we evaluate the stability of seven candidate reference genes to be used for data normalization during ex vivo and in vivo experiments conducted in Atlantic cod, Atlantic salmon, and lumpfish. To assess this, raw Ct values obtained were evaluated by using geNorm, NormFinder, BestKeeper, Delta Ct comparison, and the comprehensive ranking, through the bioinformatic open-access portal RefFinder. We determined that fabD and era were most suitable reference genes in Atlantic cod primary macrophages, hfq and era in Atlantic salmon primary macrophages, rpoB and fabD in lumpfish head kidney samples, and hfq and era in lumpfish spleen. Our study demonstrates that use of multiple reference genes and its validation before measurements helps to minimize variability arising in qPCR studies that evaluate A. salmonicida gene expression in fish tissues. Overall, this study provided with an expanded list of reliable reference genes for A. salmonicida gene expression using qPCR during fish infection studies.

Insights into gut microbiota communities of Poecilobdella manillensis, a prevalent Asian medicinal leech.

Medicinal leeches (Annelida: Hirudinea) are fresh water ectoparasitic species which have been applied as traditional therapy. However, gut microbiota could bring high risks of opportunistic infections after leeching and arouses great interests. Here, gut bacterial and fungal communities of an Asian prevalent leech Poecilobdella manillensis were characterized and analysed through culture-independent sequencing. With high coverage in 18 samples (>0.999), a more complicated community was apparent after comparing with previous leech studies. A total of 779/939 OTUs of bacteria and fungi were detected from leech guts. The bacterial community was dominated by the phylum Bacteroidetes and Synergistetes. Genera Mucinivorans and Fretibacterium accounted mostly at the genus level, and genus Aeromonas showed an extremely low abundance (2.02%) on average. The fungal community was dominated by the phylum Ascomycota and Basidiomycota. At the genus level, the dominant OTUs included Mortierella, Geminibasidium and Fusarium. The analysis of core taxa included those above dominant genera and some low-abundance genera (>1%). The functional annotation of the bacterial community showed a close correlation with metabolism (34.8±0.6%). Some fungal species were predicted as opportunistic human pathogens including Fusarium and Chaetomiaceae. The present study provides fundamental rationales for further studies of such issues as bacteria-fungi-host interactions, host fitness, potential pathogens, and infecting risks after leeching. It shall facilitate in-depth explorations on the safe utilization of leech therapy. Present paper is the first-ever exploration on microbiota of a prevalent Asian medicinal leech based on culture-independent technical. And it is also the first report of gut fungi community of medicinal leech. The diversity and composition of bacteria in P. manillensis was far different from that of the European leech. The main components and core OTUs indicate a particular gut environment of medicinal leech. Unknown bacterial and fungal species were also recovered from leech gut.

IDENTIFICATION OF BACTERIA FROM POST TIN MINING POND AND THEIR ABILITY TO FORM BIOFILMS AT DIFFERENT PH

A number of water quality indicators in the tin post-mined pond of a certain age indicate that the water condition is acidic, low dissolved oxygen content, low cation exchange capacity, and polluted by heavy metals. Restoration of the water quality of post-tin mining pond can use microorganisms as bioremediation agents. Microorganisms live by forming microbial community structures called biofilms. The aims of this study was to identify and find out the optimal pH of biofilm formation biofilm-forming bacteria from post-tin mining pond. The steps of research method was the isolation of bacteria by the spread plate technique, the biofilm formation test by the crystal violet technique, and the identification of bacteria macroscopically, microscopically, and physiologically. The isolation results showed that the highest bacterial density was at station 3 with a total of 8.1x103 cfu/ml. The results of the visualization of biofilm formation find out the A8 isolate at pH 5 with the most concentrated staining, while the highest Optical Density (OD) value for each pH was 0.11245 (pH 3) for A8 bacteria, 0.1901 (pH 5) for I1 bacteria and 0.1901 (pH 5) for A8 bacteria of 0.08945 (pH 7). There were 14 isolated bacterial belonging to the Genus Branhamella, Bacteroides, Aeromonas, Bacillus and Clostridium 08945 (pH 7). Based on identification results, biofilm-forming bacterial isolates from the tin-mining pond of Rebo Village there were 14 isolated bacterial belonging to the Genus Branhamella, Bacteroides, Aeromonas, Bacillus and Clostridium 08945 (pH 7). There were 14 isolated bacterial belonging to the Genus Branhamella, Bacteroides, Aeromonas, Bacillus and Clostridium.