Janenuj Wongtavatchai

Comparative Sequence Analysis of a Multidrug-Resistant Plasmid from Aeromonas hydrophila

ABSTRACT Aeromonas hydrophila is a pathogenic bacterium that has been implicated in fish, animal, and human disease. Recently, a multidrug resistance (MDR) plasmid, pR148, was isolated from A. hydrophila obtained from a tilapia (Oreochromis niloticus) farm in Thailand. pR148 is a 165,906-bp circular plasmid containing 147 coding regions showing highest similarity to pNDM-1_Dok1, an MDR plasmid isolated from a human pathogen. pR148 was also very similar to other IncA/C plasmids isolated from humans, animals, food, and fish. pR148 contains a mercuric resistance operon and encodes the complete set of genes for the type 4 secretion system. pR148 encodes a Tn21 type transposon. This transposon contains the drug resistance genes qacH, blaOXA-10, aadA1, and sul1 in a class 1 integron; tetA and tetR in transposon Tn1721; and catA2 and a duplicate sul1 in a locus showing 100% similarity to IncU plasmids isolated from fish. The blaOXA-10 and aadA1 genes showed 100% similarity to those from the Acinetobacter baumannii AYE genome. The similarity of pR148 to a human pathogen-derived plasmid indicates that the plasmids were either transferred between different genera or that they are derived from a common origin. Previous studies have shown that IncA/C plasmids retain a conserved backbone, while the accessory region points to lateral gene transfer. These observations point out the dangers of indiscriminate use of antibiotics in humans and in animals and the necessity of understanding how drug resistance determinants are disseminated and transferred.

Characterization of antibiotic resistance in Vibrio spp. isolated from farmed marine shrimps (Penaeus monodon)

A total of 83 Vibrio isolates from farmed marine shrimps (Penaeus monodon) were tested for the presence of class 1, 2 and 3 integrons, SXT constin and tetracycline resistance-encoding genes. Mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were determined in fluoroquinolone-resistant Vibrio strains (n=17). Five isolates were found to carry class 1 integrons, of which only one contained the partial rumA gene in the variable region. All the Vibrio strains were devoid of class 2 and 3 integrons. Seven isolates harbored SXT constin. None of the Vibrio isolates were positive to the tet(K), tet(L), tet(M), tet(O) and tet(S) genes. Ten fluoroquinolone-resistant Vibrio strains carried a point mutation G-248-T in the gyrA QRDR, leading to a Ser-83-Ile substitution in GyrA, but none of these strains had mutations in the QRDR of the parC gene.

Molecular serotyping, virulence gene profiling and pathogenicity of Streptococcus agalactiae isolated from tilapia farms in Thailand by multiplex PCR

This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems.

White spot disease risk factors associated with shrimp farming practices and geographical location in Chanthaburi province, Thailand.

Over the past 2 decades, shrimp aquaculture in Thailand has been impacted by white spot disease (WSD) caused by white spot syndrome virus (WSSV). Described here are results of a survey of 157 intensive shrimp farms in Chanthaburi province, Thailand, to identify potential farm management and location risk factors associated with the occurrence of WSD outbreaks. Logistic regression analysis of the survey responses identified WSD risks to be associated with farms sharing inlet water and culturing shrimp year round and with a single owner operating more than 1 farm. The analysis also showed WSD risks to be reduced at farms that used probiotics and applied lime to pond bottoms when fallow to neutralize acidity and kill microorganisms. Regression modeling identified no association of geographical location with WSD. The data should assist shrimp farms in mitigating the effects of WSD in Thailand.

Francisella Infection in Cultured Tilapia in Thailand and the Inflammatory Cytokine Response

Francisella infections developed in freshwater Nile Tilapia Oreochromis niloticus and red tilapia Oreochromis spp. farms in Thailand during 2012-2014. The diseased fish were lethargic and pale in color and showed numerous white nodules in their enlarged spleens. Histopathological examination and electron microscopy suggested that the white nodules were multifocal granulomas consisting of coccobacilli within vacuolated cells. Isolation of Francisella-like bacteria was achieved from 42 of 100 samples, while polymerase chain reaction confirmed Francisella infections in all samples. Analysis of the 16S rRNA gene from samples obtained from three different geographical culture areas revealed more than 99% similarity with F. noatunensis subsp. orientalis. The influence of Francisella infection on inflammatory cytokines was determined on splenic cells of fish intraperitoneally injected with the bacteria (0.8 × 10(5) colony-forming units per fish). Infected tilapia showed significantly greater expression of the pro-inflammatory genes interleukin-1β (IL-1β) and tumor necrotic factor-α (TNF-α) within 24 h postinjection (hpi) and for up to 96 hpi. However, down-regulation of an anti-inflammatory gene, transforming growth factor-β (TGF-β) was observed as early as 24 hpi. This investigation demonstrates that an imbalance between pro- and anti-inflammatory cytokines in response to the infection may account for the substantial number of granulomas in fish hematopoietic tissues that was found in the later stage of the disease. Received September 9, 2015; accepted December 13, 2015.

Residual levels of 17α-Methyldihydrotestosterone in nile tilapia (Oreochromis niloticus) fry following feeding supplementation

Abstract Intensive tilapia culture requires male-monosex population for its better yield and allowing more effective management of a single crop. Androgenic hormones are usually applied during the fish farming process to produce male-monosex population. This study investigated the residue of a synthetic androgenic steroid, 17α-methyldihydrotestosterone (MDHT) after a course of feeding supplementation in Nile tilapia (Oreochromis niloticus) fry at a dose of 80 mg/kg feed for 15 and 23 consecutive days. An analytical method using liquid chromatography tandem mass spectrometry was developed to determine the residual MDHT in tilapia fry at 1, 2, 3, 5, 7, 14 and 21 days after the last dose. The levels of MDHT on day 1 after hormone withdrawal were 3.198 ng/g in the 15-day treatment, and 3.224 ng/g in the 23-day treatment. MDHT was not detectable in fry after hormonal withdrawal for 5 days in both treatments (limit of quantitation, 0.95 ng/g), which suggests that negligible levels of MDHT will be present in Nile tilapia after 6–8 months hormonal withdrawal during the grown out period.