Detection of virulence, antimicrobial resistance, and heavy metal resistance properties in Vibrio anguillarum isolated from mullet (Mugil cephalus) cultured in Korea.

In the present study, we identified and characterized 22 strains of V. anguillarum from 145 samples of mullets (Mugill cephallus) cultured in several fish farms in South Korea. They were subjected to pathogenicity tests, antimicrobial susceptibility test, and broth dilution test to detect virulence markers, antimicrobial resistance, and heavy metal resistance properties. All the isolates showed amylase and caseinase activity, followed by gelatinase (90.9%), DNase (45.5%), and hemolysis activities (α = 81.1% and ß = 18.2%). The PCR assay revealed that isolates were positive for VAC, ctxAB, AtoxR, tdh, tlh, trh, Vfh, hupO, VPI, and FtoxR virulence genes at different percentages. All the isolates showed multi-drug resistance properties (MAR index ≥ 0.2), while 100% of the isolates were resistant to oxacillin, ticarcillin, streptomycin, and ciprofloxacin. Antimicrobial resistance genes, qnrS (95.5%), qnrB (86.4%), and StrAB (27.3%), were reported. In addition, 40.9% of the isolates were cadmium-tolerant, with the presence of CzcA (86.4%) heavy metal resistance gene. The results revealed potential pathogenicity associated with V. anguillarum in aquaculture and potential health risk associated with consumer health.

Occurrence of potential virulence determinants in Aeromonas spp. isolated from different aquatic environments.

Aeromonas species are bacterial pathogens that cause significant economic losses in the aquaculture industry worldwide. They are widely distributed in aquatic environments and cause several diseases in both humans and aquatic animals. The presence of various virulent Aeromonas spp. in aquatic environments predisposes infections in aquatic animals and humans. Concern about the transfer of pathogens from fish to humans also increased with the substantial increase in seafood consumption. Aeromonas spp. are also primary human pathogens that cause local and systemic infections, both in immunologically compromised and immunologically competent hosts. The most common Aeromonas spp. causing infections in aquatic animals and humans are A. hydrophila, A. salmonicida, A. caviae, and A. veronii biotype sobria. The ability of Aeromonas spp. to produce a variety of virulence factors enhances their pathogenic ability. Literature has supplied evidence for the presence of various virulence factors, including proteases, enterotoxins, hemolysin, and toxin genes of Aeromonas spp. in aquatic environments. The high prevalence of Aeromonas spp. in the aquatic environment is also a threat to public health. Because the Aeromonas spp. infections in humans are generally the result of ingestion or exposure to contaminated food and water. This review summarizes the recently published information on various virulence factors and virulence genes of Aeromonas spp. isolated from various aquatic environments, including seawater, freshwater, wastewater, and drinking water. It is also intended to highlight the risks associated with Aeromonas species' virulence properties for both aquaculture and public health.

Biofilm formation of pathogenic bacteria isolated from aquatic animals.

Bacterial biofilm formation is one of the dynamic processes, which facilitates bacteria cells to attach to a surface and accumulate as a colony. With the help of biofilm formation, pathogenic bacteria can survive by adapting to their external environment. These bacterial colonies have several resistance properties with a higher survival rate in the environment. Especially, pathogenic bacteria can grow as biofilms and can be protected from antimicrobial compounds and other substances. In aquaculture, biofilm formation by pathogenic bacteria has emerged with an increased infection rate in aquatic animals. Studies show that Vibrio anguillarum, V. parahaemolyticus, V. alginolyticus, V. harveyi, V. campbellii, V. fischeri, Aeromonas hydrophila, A. salmonicida, Yersinia ruckeri, Flavobacterium columnare, F. psychrophilum, Piscirickettsia salmonis, Edwardsiella tarda, E. ictaluri, E. piscicida, Streptococcus parauberis, and S. iniae can survive in the environment by transforming their planktonic form to biofilm form. Therefore, the present review was intended to highlight the principles behind biofilm formation, major biofilm-forming pathogenic bacteria found in aquaculture systems, gene expression of those bacterial biofilms and possible controlling methods. In addition, the possibility of these pathogenic bacteria can be a serious threat to aquaculture systems.

Aquatic environments: A potential source of antimicrobial-resistant Vibrio spp.

Vibrio spp. are associated with water and seafood-related outbreaks worldwide. They are naturally present in aquatic environments such as seawater, brackish water and freshwater environments. These aquatic environments serve as the main reservoirs of antimicrobial-resistant genes and promote the transfer of antimicrobial-resistant bacterial species to aquatic animals and humans through the aquatic food chain. Vibrio spp. are known as etiological agents of cholera and non-cholera Vibrio infections in humans and animals. Antimicrobial-resistant Vibrio species have become a huge threat in regard to treating Vibrio infections in aquaculture and public health. Most of the Vibrio spp. possess resistance towards the commonly used antimicrobials, including ß-lactams, aminoglycosides, tetracyclines, sulphonamides, quinolones and macrolides. The aim of this review is to summarize the antimicrobial resistance properties of Vibrio spp. isolated from aquatic environments to provide awareness about potential health risks related to Vibrio infections in aquaculture and public health.

Occurrence of Virulence and Antimicrobial Resistance Determinants in Vibrio harveyi Isolated from Marine Food Fish Cultured in Korea.

Vibrio harveyi is a significant cause of infection in both marine animals and humans. It has been reported frequently in seafood-borne infections worldwide. This study was conducted to determine the potential health impact of the V. harveyi isolated from marine food fish cultured in Korea concerning their virulence and antimicrobial resistance. A total of 49 V. harveyi samples were isolated by biochemical tests and multiplex PCR. Phenotypic detection of virulence factors resulted DNase activity (81.63%), hemolysis (α = 75.51% and ß = 12.25), gelatinase activity (71.43%), protease production (71.43%), phospholipase activity (65.31%), and lipase production (34.69%). Virulence genes, including VPI, tlh, tdh, toxR, VAC, and ctxAB, were detected in 57.14%, 44.90%, 36.73%, 22.45%, 12.24%, and 8.16% of the isolates, respectively. Resistance to ampicillin (77.55%), oxacillin (69.39%), nalidixic acid (53.06%), amoxicillin (46.94%), oxytetracycline (46.94%), colistin sulfate (34.69%), fosfomycin (34.69%), chloramphenicol (32.65%), streptomycin (32.65%), cephalothin (28.57%), oxytetracycline (26.53%), ceftriaxone (20.41%), erythromycin (14.29%), and cefoxitin (12.24%) was detected in disc diffusion assay. Most of the isolates were classified as multidrug resistant as they scored multiple antimicrobial resistance index ≥0.2. Furthermore, antimicrobial resistance genes tetB, qnrA, intI1 (Class 1 integron integrase), aac(6')-Ib, blaSHV, blaCTX-M, strA-strB, tetA, aphAI-IAB, qnrC, qnrS, and blaTEM were found in 81.63%, 67.35%, 61.22%, 46.94%, 44.90%, 44.90%, 36.73%, 18.37%, 10.20%, 10.20%, 8.16% and 6.12% of the isolates, respectively. In conclusion, the development of antimicrobial resistance among V. harveyi will ultimately reduce the efficacy of antimicrobials used for treating and can favor the development of more virulent V. harveyi strains.

Detection of Antimicrobial and Heavy-Metal Resistance Genes in Aeromonas spp. Isolated from Hard-Shelled Mussel (Mytilus Coruscus).

Hard-shelled mussel (Mytilus coruscus) is a popular seafood in South Korea because of its delicacy and high nutritional value. Our study aimed to identify antimicrobial and heavy-metal resistance determinants in Aeromonas isolates from marketed hard-shelled mussel in South Korea. A total of 33 Aeromonas species were isolated, and antimicrobial disk diffusion test was done to observe antimicrobial resistance patterns. In addition, broth microdilution test was performed to determine resistance to heavy-metals. PCR amplification was done to detect resistance genes. High resistance to amoxicillin (100.0%), ampicillin (93.9%), rifampicin (78.8%), and cephalothin (48.5%) was observed where least resistance to other antimicrobials was also detected. In addition, the isolates showed high resistance to cadmium (Cd) (57.6%), and 42.4% and 27.3% were resistant to chromium (Cr) and copper (Cu). The occurrence of antimicrobial resistance genes, such as blaTEM, blaSHV, blaCTX-M, tetB, tetE, and intI1 genes, was observed in 9 (27.3%), 8 (24.2%), 8 (24.2%), 6 (18.2%), 5 (15.2%), and 9 (27.3%) isolates, respectively. Also, heavy-metal resistance genes, czcA, copA, and merA were detected in 17 (51.5%), 11 (33.3%), and 7 (21.2%) of the isolates, respectively. The results suggest that mussels are a reservoir of multidrug and heavy-metal-resistant Aeromonas spp.

Pet-turtles: a potential source of human pathogenic bacteria.

Human infections caused by the bacterial pathogens transmitted from pet-turtles are becoming very common and getting more importance as the turtles are considered unsafe pet animals, mainly for children and immunocompromised people. Pet-turtles are known as the reservoir of different bacterial species as their intestinal microflora. Extrinsic stressors, such as crowding, unhygienic handling, poor water quality, polluted feeding and inadequate nutrition, can predispose pet-turtles to bacterial infections. The presence of different virulence genes leads to the virulent potential of bacteria. The virulent bacteria can cause infections in turtles and humans, if the turtle owners or shopkeepers don't practice proper sanitation while handling turtles. The aim of this review paper was to provide an overview of different bacterial species isolated from pet-turtles for awareness-raising about potential health risks related to raise pet-turtles.

Biological Activity of Porcine Gastric Mucin on Stress Resistance and Immunomodulation.

Purified porcine gastric mucin (PGM) is an alternative biomaterial to native mucin which displays multifunctional properties for exploring a wide range of biomedical applications. The present study evaluated the in vitro (RAW 264.7 macrophage cells) and in vivo (zebrafish embryos and larvae) bioactivities of PGM. The median lethal concentration (LC50) of PGM was 197.9 µg/mL for embryos, while it was non-toxic to RAW 264.7 cells, even at 500 µg/mL. Following PGM exposure (100 µg/mL), a higher embryo hatching rate (59.9%) was observed at 48 h post fertilization, compared to the control (30.6%). Protective effects of PGM from pathogenic Aeromonas hydrophila were demonstrated by high larvae survival rates of 85.0% and 94.0% at 50 and 100 µg/mL of PGM exposure, respectively. Heat tolerance effect of PGM (50 and 100 µg/mL) on larvae (40 °C for 48 h) was confirmed by 75% and 100% of survival rates, respectively. Additionally, PGM reduced the A. hydrophila-induced reactive oxygen species (ROS) generation in larvae. The qRT-PCR results in PGM exposed larvae exhibited induction of immune-related genes (tlr5a and tlr5b, myd88, c-rel, il1ß, tnf-α, il6, il10, cxcl18b, ccl34a.4, defbl1, hamp, ctsd, muc2.1, muc5.1, muc5.2, and muc5.3), stress response (hsp70, hsp90aa1.1, and hsp90ab1), and antioxidant genes (cat and sod1). Moreover, our results revealed that PGM involved in the regulation of transcriptional gene induction increases Hsp90 protein in the zebrafish larvae. Furthermore, upregulation of Il6, Il10, Tnfα, Ccl3, Defa-rs2, Defa21 and Camp and antioxidant genes (Sod2 and Cat) were observed in PGM-exposed RAW 264.7 cells. Overall findings confirmed the activation of immune responses, disease resistance against pathogenic bacteria, heat tolerance, and ROS-scavenging properties by PGM, which may provide insights into new applications for PGM as a multifunctional immunomodulator.

Aeromonas spp. Isolated from Pacific Abalone (Haliotis discus hannai) Marketed in Korea: Antimicrobial and Heavy-Metal Resistance Properties.

Antimicrobial and heavy-metal resistance of 29 Aeromonas spp. (Aeromonas hydrophila n = 9, Aeromonas enteropelogenes n = 14, Aeromonas veronii n = 3, Aeromonas salmonicida n = 2, and Aeromonas sobria n = 1) isolated from Pacific abalone marketed in Korea were analyzed. All isolates were found to be resistant against ampicillin. High level of resistant to cephalothin (86%), rifampicin (73%), imipenem (42%), and oxytetracycline (35%) were also detected. Thirteen (45%) of the isolates showed multiple antimicrobial resistance (MAR) index ≥ 0.2. The PCR assays implied the presence of qnrS, qnrB, qnrA, tetB, tetA, aac (3')- IIa, aac(6')-Ib, aphAI-IAB, blaCTX, blaTEM, and intI1 genes among 76%, 28%, 14%, 17%, 3%, 3%, 41%, 10%, 41%, 28%, and 66% of the isolates, respectively. Class 1 integron gene cassette profiles aadA1(3%) and aadA2 (3%) were also identified. Lead (Pb) resistance was the highest (69%) among 5 heavy metals tested, whereas 38%, 27%, and 20% of the isolates were resistant to Cadmium (Cd), Chromium (Cr), and Copper (Cu), respectively. Heavy-metal resistance genes, CopA, CzcA, and merA were positive in 83%, 75%, and 41% of the isolates, respectively. In conclusion, observed genotypic and phenotypic resistance profiles of Aeromonas spp. against antimicrobials and heavy metals reveal the ability of serving as a source of antimicrobials and heavy-metal-resistant traits.

Occurrence of Virulence and Extended-Spectrum ß-Lactamase Determinants in Vibrio spp. Isolated from Marketed Hard-Shelled Mussel (Mytilus coruscus).

This study investigated the occurrence of virulence and antimicrobial resistance properties in Vibrio spp. isolated from hard-shelled mussel (Mytilus coruscus) marketed in Korea. A total of 32 Vibrio spp. isolates including Vibrio diabolicus (n = 16), Vibrio alginolyticus (n = 13), Vibrio parahaemolyticus (n = 2), and Vibrio harveyi (n = 1) were identified by gyrB gene sequencing. Every isolate grouped with their respective reference sequence excluding V. diabolicus and V. alginolyticus that were intermixed in a neighbor-joining phylogenetic tree. Every Vibrio spp. showed DNase and gelatinase activities. Phospholipase, slime, caseinase, lipase, ß-hemolysis, and α-hemolysis were positive in 31 (96.8%), 31 (96.8%), 29 (93.5%), 28 (87.5%), 17 (53.1%), and 9 (28.1%) isolates, respectively. The PCR amplification of virulence genes displayed that V. alginolyticus-specific (toxR, tlh, VAC, and VPI) genes were detected in 23 (71.9%), 18 (56.3%), 21 (65.6%), and 26 (81.3%) isolates, respectively. V. parahaemolyticus-specific (toxR and tlh) genes were harbored by 2 (6.3%) and 2 (6.3%) isolates, respectively. The other virulence genes including ctxAB and hupO genes were observed in 23 (71.9%) and 16 (50.0%) isolates, respectively. The antimicrobial resistance was prevalent for amoxicillin (100.0%), ampicillin (100.0%), and streptomycin (37.5%) in disk diffusion test. Multiple antimicrobial resistance index ranged from 0.09 to 0.22. The extended-spectrum ß-lactamase (ESBL) genes, blaCTX, blaTEM, blaSHV, and blaOXA were detected in 28 (87.5%), 13 (40.6%), 7 (21.8%), and 1 (3.1%) isolates, respectively. Non-ESBLs such as streptomycin resistance (strA.B), kanamycin resistance (aphA-IAB), and tetracycline resistance (tetE) genes were found in 5 (15.6%), 3 (9.4%), and 1 (3.1%) isolates. Class 1 integron-related Integrase 1 (intI1) gene was found in 8 (25.0%) isolates. Our results reveal that the consumption of raw mussels may cause a potential public health risks owing to the virulent and antimicrobial-resistant Vibrio spp.

Antimicrobial resistance of pathogenic Aeromonas spp. isolated from marketed Pacific abalone (Haliotis discus hannai) in Korea.

AIMS: The object of this study was to identify potential health concerns of the Aeromons spp. isolated from marketed Pacific abalone (Haliotis discus hannai) with respect to their virulence and antimicrobial resistance patterns. METHODS AND RESULTS: We identified 29 strains of aeromonads consisting of five species; Aeromonas hydrophila (n = 9), Aeromonas enteropelogenes (n = 14), Aeromonas veronii (n = 3), Aeromonas salmonicida (n = 2) and Aeromonas sobria (n = 1), by employing series of biochemical tests and gene sequencing. In the phenotypic virulence assays, all isolates showed gelatinase and caseinase activities, while lipase formation (69%), phospholipase production (90%), DNase formation (82%), slime production (49%) and haemolysis activity (α = 18% and ß = 82%) were also detected among isolates. Prevalence of virulence genes; aerA (100%), fla (66%), ahyB (73%), act (52%), alt (42%), ast (35%), ser (52%), gcat (69%), ascV (43%), hlyA (83%), lip (52%) and exu (59%) were detected by PCR assays. In disc diffusion test, 100% resistance was detected against ampicillin while cephalothin, rifampicin, oxytetracycline, colistine sulphate, nalidixic acid and piperaciliin were resisted by 86, 73, 42, 35, 28, 20 and 20% of the isolates respectively. Thirteen (45%) of the isolates showed multiple antimicrobial resistance (MAR) indices ≥ 0·2. CONCLUSIONS: Our findings suggest that the potential health risk posed by the abalone-borne Aeromonas spp. should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time to evaluate possible public health risks upon consumption of abalone harbored Aeromonas spp. and also to isolate potential pathogenic and multidrug-resistant Aeromonas spp. from Pacific abalone in Korea.

Phylogenetic characteristics, virulence properties and antibiogram profile of motile Aeromonas spp. isolated from ornamental guppy (Poecilia reticulata).

Aeromonas spp. are opportunistic pathogenic bacteria related to an assembly of infectious diseases in ornamental fish. In the present study, virulence properties and antibiotic susceptibility of 52 guppy-borne Aeromonas spp. were investigated. The isolates were identified as A. veronii (n = 34), A. dhakensis (n = 10), A. hydrophila (n = 3), A. caviae (n = 3) and A. enteropelogenes (n = 2) by gyrB gene sequencing. The gyrB sequence deviation within and among the species ranged from 0 to 2.6% and 2.7-9.2%. Each species formed a distinct group in the unrooted neighbor-joining phylogenetic tree. The phenotypic virulence factors such as ß-hemolysis, slime, caseinase, DNase, gelatinase and lipase production were observed in 28 (53.9%), 33 (63.5%), 28 (53.9%), 42 (80.8%), 37 (71.2%) and 42 (80.8%) isolates, respectively. The virulence genes were detected by PCR assay in the following proportions- act (84.6%), hly (80.8%), aer (73.1%), lip (73.1%), gcaT (73.1%), ascV (53.8%), ahyB (53.8%) fla (51.9%), alt (48.1%), ast (36.5%) and ser (34.6%), respectively. The amoxicillin, ampicillin, imipenem, nalidixic acid, oxytetracycline and rifampicin were resistant to more than 70.0% of the isolates in antibiotic susceptibility test. Our study suggests that the ornamental guppy can be a potential reservoir of virulent and multi-drug resistant Aeromonas spp.

Isolation and characterization of phage AHP-1 and its combined effect with chloramphenicol to control Aeromonas hydrophila.

To develop an alternative bio-control measure for multi-drug resistant pathogenic Aeromonas hydrophila, which causes motile Aeromonas septicemia in fish, novel virulent phage (AHP-1) was isolated from carp tissues. Morphological analysis by transmission electron microscopy revealed that AHP-1 belongs to Myoviridae family. AHP-1 displayed 81% of moderate adsorption by 25 min, and latent period of 40 min with burst size of 97 PFU mL-1 at an optimal multiplicity of infection (MOI) 0.1. AHP-1 was stable over a broad range of pH (4-11), temperature (4-50 °C), and salinity (0.1-3.5%). Both time and MOI dependent in vitro A. hydrophila growth inhibition was observed with AHP-1. AHP-1 (10 MOI) showed higher growth inhibition against A. hydrophila than chloramphenicol (5 µg mL-1), and combined treatment was more promising than individuals. Immune gene expression analysis of zebrafish upon continuous bath exposure to AHP-1 resulted significantly higher (il-6 and sod-1) or slight induction (tnf-α, il1-ß, il-10, and cxcl-8a) than controls at beginning of the phage exposure, but those lowered to basal level by day 12 post-phage exposure. It suggests no adverse immune responses have occurred for the AHP-1 dose that used, and have potential for the phage therapy. Further detailed in vivo studies are needed to confirm the protective efficacy of newly isolated AHP-1 against A. hydrophila infection.

Characterization of Quinolone-Resistant Determinants in Tribe Proteeae Isolated from Pet Turtles with High Prevalence of qnrD and Novel gyrB Mutations.

Development of antibiotic resistance in bacteria has challenged significantly in both veterinary and human medicine. In this study, we analyzed the potential risk of pet turtles harboring tribe Proteeae as a source of quinolone-resistant determinants, including plasmid-mediated quinolone resistance (PMQR) genes and target gene alterations in the quinolone resistance-determining region (QRDR). Antimicrobial susceptibility of 54 Proteeae isolates against ciprofloxacin, ofloxacin, levofloxacin, and nalidixic acid was examined. The PMQR genes and QRDR alterations were identified using conventional PCR assays and sequencing. Four isolates were resistant to all quinolones tested in this study. Nine isolates showed resistance to nalidixic acid and showed either intermediate resistance or susceptibility to other tested quinolones. All isolates resistant to one or more tested quinolones harbored mutations in gyrB and some also had gyrA and parC mutations. Of 54, 12 Proteeae isolates displayed the novel E466D, N440T, Q411S, and F417L mutations in gyrB. Among the PMQR genes, 41 (76%) isolates harbored the qnrD gene with the highest prevalence, whereas aac(6')Ib-cr, qnrS, qnrA, and qnrB genes were detected in 28 (52%), 9 (17.0%), 7 (13.0%), and 1 (1.9%) study isolates, respectively. The QRDR analysis of selected mutants revealed that increasing quinolone selective pressure led to a predominance of gyrA mutants. All results indicate that a healthy pet turtle can play as a potential reservoir for quinolone-resistant Proteeae, which it might cause public health risk on pet owners.

Outcome of co-infection with opportunistic and multidrug resistant Aeromonas hydrophila and A. veronii in zebrafish: Identification, characterization, pathogenicity and immune responses.

Fish can be potentially co-infected by two or more bacterial strains, which can make synergistic influence on the virulence of infection. In this study, two opportunistic and multidrug resistant Aeromonas strains were isolated from wounds of morbid zebrafish with typical deep skin lesions similar to Motile Aeromonas Septicemia. Isolates were genetically identified as A. hydrophila and A. veronii by 16 S rRNA sequencing and phylogenetic analysis. Both isolates were positive for virulent genes (aerA, lip, ser, exu gcaT) and selected phenotypic tests (DNase, protease, gelatinase, lipase, biofilm production and ß-haemolysis). A. hydrophila and A. veronii had strong antibiotic resistance against ampicillin, tetracycline, nalidixic acid, kanamycin, erythromycin, clindamycin and trimethoprim-sulfamethoxazole. Histopathological studies revealed that co-infection causes severe necrosis and hypertrophy in the muscles, kidney and liver of zebrafish. Naturally co-infected zebrafish showed highly induced tnf-α, il-1ß, il-6, il-12, ifn, ifn-γ, cxcl18 b and ccl34a.4 at transcription level compared to healthy fish, suggesting virulence factors may activate immune and inflammatory responses of zebrafish. Experimentally infected zebrafish showed significantly higher mortality under co-infection with A. hydrohila and A. veronii (87%), followed by individual challenge of A. hydrophila (72%) or A. veronii (67%) suggesting that virulence of A. hydrophila have greater pathogenicity than A. veronii during co-infection.

Frozen White-Leg Shrimp (Litopenaeus vannamei) in Korean Markets as a Source of Aeromonas spp. Harboring Antibiotic and Heavy Metal Resistance Genes.

As the most consumed shrimp variety, white-leg shrimp (Litopenaeus vannamei) owns a high market demand in Korea. This study sought to screen the frozen white-leg shrimp for Aeromonas spp. harboring antimicrobial and heavy metal resistance characteristics. A total of 44 Aeromonas spp. strains were isolated and tested for antibiotic susceptibility and heavy metal tolerance followed by PCR-based detection of resistance genes and integrons. It was observed that resistance to ampicillin and oxacillin was 100% among isolates. Besides, 95%, 89%, 86%, 80%, 66%, and 43% of the isolates were resistant to nalidixic acid, tetracycline, cephalothin, streptomycin, trimethoprim-sulfamethoxazole, and imipenem, respectively, and less resistance to other antibiotics was also observed. Cr resistance was the highest (91%) among five heavy metals tested, whereas 57%, 32%, 20%, and 9% of the isolates were tolerant to Cu, Pb, Cd, and Hg, respectively. The PCR assays implied the presence of qnrB, qnrS, tetA, tetE, aac(6')-Ib, and aphAI-IAB, and intI1 genes among 80%, 77%, 18%, 30%, 9%, 0.25%, and 82% of the isolates, respectively. There were 35 (80%) integron 1-positive isolates harboring qacE2, dfrA1, orfC, orfD, aadB, catB3, oxa-10, and aadA1 genes in varying combinations. In addition, heavy metal resistance genes, CopA, merA, and CzcA were positive in 9%, 7%, and 27% of the isolates, respectively. According to these outcomes, the frozen white-leg shrimp in Korean markets can be suggested as a source of multidrug and heavy metal-resistant Aeromonas spp. that carries genetic determinants.

Chitosan nanoparticles: A positive immune response modulator as display in zebrafish larvae against Aeromonas hydrophila infection.

Chitosan nanoparticles (CNPs) were synthesized by ionic gelation method and its immunomodulatory properties were investigated in zebrafish larvae. Average particle size and zeta potential were 181.2 nm and +37.2 mv, respectively. Initially, toxicity profile was tested in zebrafish embryo at 96 h post fertilization (hpf) stage using medium molecular weight chitosan (MMW-C) and CNPs. At 5 µg/mL, the hatching rate was almost similar in both treatments, however, the survival rate was lower in MMW-C compared to CNPs exposure, suggesting that toxicity effect of CNPs in hatched larvae was minimal at 5 µg/mL compared to MMW-C. Quantitative real time PCR results showed that in CNPs exposed larvae at 5 days post fertilization (5 dpf) stage, immune related (il-1ß, tnf-α, il-6, il-10, cxcl-18b, ccl34a.4, cxcl-8a, lyz-c, defßl-1, irf-1a, irf-3, MxA) and stress response (hsp-70) genes were induced. In contrast, basal or down regulated expression of antioxidant genes (gstp-1, cat, sod-1, prdx-4, txndr-1) were observed. Moreover, zebrafish larvae (at 5 dpf stage) exposed to CNPs (5 µg/mL) showed higher survival rate at 72 h post infection stage against pathogenic Aeromonas hydrophila challenge compared to controls. These results suggest that although CNPs can have toxic effects to the larvae at higher doses, CNPs exposure at 5 µg/mL could enhance the immune responses and develop the disease resistance against A. hydrophila, which could be attributed to its strong immune modulatory properties.

Distribution of Antimicrobial Resistance Genes and Class 1 Integron Gene Cassette Arrays in Motile Aeromonas spp. Isolated from Goldfish (Carassius auratus).

Aeromonas spp. are opportunistic pathogens related to multiple infectious diseases in ornamental fishes. In the present study, the antimicrobial susceptibility, resistance genes, and integrons of 65 goldfish-borne Aeromonas spp. were evaluated. The isolates were identified as A. hydrophila (n = 30), A. veronii (n = 32), and A. punctata (n = 3) by gyrB sequencing. The antimicrobial susceptibility testing of the isolates designated that most of the isolates were resistant to amoxicillin (100.00%), nalidixic acid (100.00%), ampicillin (98.46%), tetracycline (92.31%), rifampicin (86.15%), and cephalothin (61.54%) and each of the isolates showed multiple antimicrobial resistance phenotype (resistant to ≥3 classes of antimicrobials). PCR amplification of antimicrobial resistance genes revealed that the plasmid-mediated quinolone resistance gene, qnrS, was the most prevalent (73.85%) among the isolates. The other antimicrobial resistance genes were detected in the following proportions: qnrB (26.15%), aac(6')-Ib-cr (4.60%), tetA (16.92%), tetE (21.54%), aac(6')-Ib (29.23%), and aphAI-IAB (7.69%). The IntI gene was found in 64.62% isolates, and four class 1 integron gene cassette profiles (incomplete dfrA1, catB3-aadA1, dfrA1-orfC, and qacE2-orfD) were identified. These data suggest that goldfish-borne Aeromonas spp. serve as a reservoir of antimicrobial resistance genes and class 1 integrons.

Antibacterial activity of essential oil from lavender (Lavandula angustifolia) against pet turtle-borne pathogenic bacteria.

Pet turtles are well-known to harbor an array of bacterial pathogens which can cause zoonotic infections in humans as well as opportunistic infections in the turtles itself. Essential oils are the natural plant extracts which have been traditionally used for disease treatment. In the present study, the essential oil of lavender (EOL) was examined for its antibacterial activity against thirty-eight strains of turtle-borne pathogenic bacteria belonging to seven species; Aeromonas hydrophila, A. caviae, A. dhakensis, Citrobacter freundii, Proteus mirabilis, Salmonella enterica and Pseudomonas aeruginosa. Antibacterial activity of EOL was tested by means of disk diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) tests. In addition, the antimicrobial susceptibility pattern of 11 commonly used antimicrobials was examined and the multiple antibiotic resistance (MAR) index was calculated. The results revealed that EOL was active against all tested turtle-borne pathogenic bacteria except P. aeruginosa. The range of MIC and MBC values of EOL against isolates except P. aeruginosa were recorded as 0.5-1% (V/V) and 0.5-2% (V/V), respectively. The MBC/MIC ratio was detected as <4, revealing that the tested EOL was bactericidal. Besides, most of the isolates were resistant to different antimicrobials in antimicrobial disk diffusion test. MAR index values of the tested strains were ranging from 0.27 to 0.91. The outcomes indicate that EOL has a potential to be used as an antibacterial agent against pathogenic bacteria isolated from pet turtles.

Quinolone susceptibility and genetic characterization of Salmonella enterica subsp. enterica isolated from pet turtles.

Turtle-borne Salmonella enterica owns significance as a leading cause in human salmonellosis. The current study aimed to determine the quinolone susceptibility and the genetic characteristics of 21 strains of S. enterica subsp. enterica isolated from pet turtles. Susceptibility of four antimicrobials including nalidixic acid, ciprofloxacin, ofloxacin, and levofloxacin was examined in disk diffusion and MIC tests where the majority of the isolates were susceptible to all tested quinolones. In genetic characterization, none of the isolates were positive for qnr or aac(6')-Ib genes and no any target site mutations could be detected in gyrA, gyrB, and parC quinolone resistance determining regions (QRDR). In addition, neighbor-joining phylogenetic tree derived using gyrA gene sequences exhibited two distinct clads comprising; first, current study isolates, and second, quinolone-resistant isolates of human and animal origin. All results suggest that studied strains of S. enterica subsp. enterica isolated from pet turtles are susceptible to quinolones and genetically more conserved with regards to gyrA gene region.