B.C.J. De Silva

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

&NA; 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 &mgr;g/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 &mgr;g/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‐1&bgr;, tnf‐&agr;, il‐6, il‐10, cxcl‐18b, ccl34a.4, cxcl‐8a, lyz‐c, def&bgr;l‐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 &mgr;g/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 &mgr;g/mL could enhance the immune responses and develop the disease resistance against A. hydrophila, which could be attributed to its strong immune modulatory properties. Graphical abstract Figure. No caption available. HighlightsSynthesized CNPs at 5 &mgr;g/L exposure was non‐toxic to zebrafish at 96 hpf stage.CNPs showed immune modulatory and disease resistant against A. hydrophila in 5 dpf stage zebrafish after non‐toxic exposure.Biodegradable CNPs could be a potential immunostimulant for larval aquaculture.

Incidence of antibiogram, antibiotic resistance genes and class 1 and 2 integrons in tribe Proteeae with IMP27 gene for the first time in Providencia spp. isolated from pet turtles

Proteeae is a tribe which consists of three genera: Proteus, Providencia and Morganella. The objective of this study was to determine antimicrobial resistance profile, virulence genotype and class 1 and 2 integrons in Proteeae isolated from pet turtles and to determine the impact of antibiotic resistance on virulence and antimicrobial resistance genes. Integron‐positive isolates were used to detect their gene cassette array. Sixty four Proteeae were isolated and all were resistant to macrolides (100%). Among 64 isolates 56, 52, 36 and 25 were resistant to nitrofurans, β‐lactams, tetracycline and aminoglycoside respectively. Sixteen (25%) isolates were positive for intI1 while 14 (21·87%) were positive for integrase 2 (intI2). Eleven (17·18%) isolates were positive for class 1 variable region while 7 (10·93%) were positive for class 2 variable region. IMP27, a novel metallo β‐lactamase gene was found in Providencia isolates. Proteus sp. were positive for every tested virulence genes and UreC gene was detected in 48·44% followed by zapA (17·19%), mrpA (17·19%) and hlyA (14·06%) genes. In this study, integron associated‐antibiotic resistance genes have been identified in Proteeae isolates in a considerable range representing clear threats to public health.

Characterization of virulence properties and multi-drug resistance profiles in motile Aeromonas spp. isolated from zebrafish (Danio rerio)

Aeromonas spp. are opportunistic pathogenic bacteria associated with a multitude of diseases in ornamental fish. In this study, virulence properties and antibiotic resistance patterns of 43 Aeromonas strains isolated from 46 zebrafish were investigated. The isolates were identified as Aeromonas veronii biovar veronii (n = 26), A. veronii biovar sobria (n = 3), Aeromonas hydrophila (n = 8), A. caviae (n = 3), Aeromonas enteropelogenes (n = 2) and Aeromonas dhakensis (n = 1) by gyrB gene sequencing. The sequence divergence within and between the species ranged from 0–5·80% and 4·90–8·00%. Each species formed a distinct group in a neighbour‐joining phylogenetic tree. The lipase production, biofilm formation, DNase activity, gelatinase production, caseinase production and β‐hemolysis were phenotypically observed in 34 (79·07%), 33 (74·74%), 30 (69·77%), 25 (58·14%), 22 (51·18%) and 21 (48·84%) isolates. The virulence genes were detected by polymerase chain reaction (PCR) in following frequencies‐ aer (86·05%), hlyA (83·72%), gcaT (83·72%), lip (72·09%), act (67·44%), fla (65·12%), ascV (58·14%), ast (55·81%), ser (41·86%), ahyB (39·53%) and alt (25·58%). Every isolate was resistant to at least four antibiotics in disk diffusion test. The multiple antibiotic resistance (MAR) index values ranged from 0·22–0·50 among the isolates. Our study suggests that zebrafish can be a potential reservoir of virulent and multi‐drug resistant Aeromonas spp.

Antimicrobial property of lemongrass (Cymbopogon citratus) oil against pathogenic bacteria isolated from pet turtles

The usage of essential oils as antimicrobial agents is gaining attention. Besides, pet turtles were known to harbor a range of pathogenic bacteria while the turtle keeping is a growing trend worldwide.The current study examined the antimicrobial activity of lemon grass oil (LGO) against seven species of Gram negative bacteria namely; Aeromonas hydrophila, A. caviae, Citrobacter freundii, Salmonella enterica, Edwardsiella tarda, Pseudomonas aeruginosa, and Proteus mirabilis isolated from three popular species of pet turtles. Along with the results of disc diffusion, minimum inhibitory and minimum bactericidal concentration (MIC and MBC) tests, LGO was detected as effective against 6 species of bacteria excluding P. aeruginosa. MIC of LGO for the strains except P. aeruginosa ranged from 0.016 to 0.5% (V/V). The lowest MIC recorded in the E. tarda strain followed by A. hydrophilla, C. freundii, P. mirabilis, and S. enterica. Interestingly, all the bacterial species except E. tarda were showing high multiple antimicrobial resistance (MAR) index values ranging from 0.36 to 0.91 upon the 11 antibiotics tested although they were sensitive to LGO.

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.