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Biodiversity of chloramphenicol-resistant mesophilic heterotrophs from Southeast Asian aquaculture environments

Huys G, Bartie K, Cnockaert M, Hoang Oanh DT, Phuong NT, Somsiri T, et al.

Res. Microbiol., 2007 Apr;158(3):228-35.
PMID: 17350231

In the present study, samples of pond water, sediment and farmed species were collected at 12 fish and shrimp farms in Malaysia, Thailand and Vietnam to determine the biodiversity and environmental distribution of chloramphenicol-resistant (CmR) mesophilic heterotrophs in Southeast Asian aquaculture sites. Following isolation on Iso-Sensitest agar supplemented with 35mug ml(-1) Cm and dereplication using (GTG)(5)-PCR fingerprinting, 557 genotypically unique CmR strains were subjected to polyphasic identification. The 557 mesophilic heterotrophic CmR isolates represented 18 different genera largely dominated by the genera Escherichia (40.2%), Pseudomonas (11.7%), Acinetobacter (11.1%), Klebsiella (7.5%) and Bacillus (5.9%). A total of 439 CmR isolates were further assigned to 31 described species or species groups, mainly including organisms that have been associated with various human opportunistic infections such as Escherichia coli (n=219), Pseudomonas putida (n=47), Klebsiella pneumoniae (n=38) and Acinetobacter baumannii (n=23). Strains of Escherichia, and most notably, of E. coli, were the only common group of CmR heterotrophs irrespective of country, sample type or farm type. Together with other predominant but less widespread groups such as acinetobacters and pseudomonads, the results of this biodiversity study suggest that E. coli can be regarded as a potential indicator of Cm resistance in Southeast Asian aquaculture environments.

Matched MeSH terms: Fishes/microbiology
Prevalence and resistance to antibiotics for Aeromonas species from retail fish in Malaysia

Radu S, Ahmad N, Ling FH, Reezal A

Int J Food Microbiol, 2003 Mar 25;81(3):261-6.
PMID: 12485753

A total of 87 market fish samples representing five types of fish were evaluated for the presence of Aeromonas spp. Of the samples examined, 69%, 55%, 11.5% and 2.3% harbored Aeromonas spp., A. veronii biovar sobria, A. hydrophila and A. caviae, respectively. The 60 isolated Aeromonas spp. strains were further examined for hemolytic activity, resistance to antimicrobial agents and presence of plasmids. Hemolytic activity varied widely among the isolated strains. Though all the isolates demonstrated resistance to three or more of the antibiotics tested, all were susceptible to ceptazidime. Thirty-four (56.7%) of the sixty isolates harbored plasmids, with sizes ranging from 2.3 to 15.7 kb. These results indicate that hemolytic, multiple antibiotic resistant and genetically diverse aeromonads are easily recovered from fish in this region.

Matched MeSH terms: Fishes/microbiology*
Possible transmission routes of Vibrio spp. in tropical cage-cultured marine fishes

Nurliyana M, Amal MNA, Zamri-Saad M, Ina-Salwany MY

Lett Appl Microbiol, 2019 Jun;68(6):485-496.
PMID: 30834548 DOI: 10.1111/lam.13146

This study investigates the possible transmission routes of Vibrio spp. in a tropical cage-cultured marine fishes. Samplings of cultured Asian seabass, red snapper, hybrid grouper, wild fish, trash fish, fish fry, water and sediment samples were conducted from December 2016 to August 2017. All fish were dissected in situ and swabs were taken aseptically from the skin, eye, liver and kidney for bacterial isolation and identification. Bacterial isolation and identification from water, sediment and trash fish were also made. A total of 261 Vibrio spp. isolates recovered from the cultured, wild and fry fish, as well as from the sediment and water of the farm environment were analysed. Sequences of the pyrH gene were used to investigate the degree of relatedness and possible transmission routes existing between the isolated Vibrio spp. The population tree revealed the existence of selected Vibrio spp. that possibly transmitted between the newly introduced fish fry and wild fish into the cultured fish, while water also might possibly serves as natural transmission medium of certain Vibrio spp. in this fish farm. SIGNIFICANCE AND IMPACT OF THE STUDY: The source of transmission of Vibrio spp. into farmed marine fish remains unclear. This study highlights the possible transmission routes of Vibrio into cage-cultured marine fishes via newly introduced fish fry and wild fish. Understanding the routes of transmission of Vibrio spp. might help in controlling the disease in the near future.

Matched MeSH terms: Fishes/microbiology
Fulltext The microbial composition of dried fish prepared according to Greenlandic Inuit traditions and industrial counterparts

Hauptmann AL, Paulová P, Castro-Mejía JL, Hansen LH, Sicheritz-Pontén T, Mulvad G, et al.

Food Microbiol, 2020 Feb;85:103305.
PMID: 31500717 DOI: 10.1016/j.fm.2019.103305

The practices of preparing traditional foods in the Arctic are rapidly disappearing. Traditional foods of the Arctic represent a rarity among food studies in that they are meat-sourced and prepared in non-industrial settings. These foods, generally consumed without any heating step prior to consumption, harbor an insofar undescribed microbiome. The food-associated microbiomes have implications not only with respect to disease risk, but might also positively influence host health by transferring a yet unknown diversity of live microbes to the human gastrointestinal tract. Here we report the first study of the microbial composition of traditionally dried fish prepared according to Greenlandic traditions and their industrial counterparts. We show that dried capelin prepared according to traditional methods have microbiomes clearly different from industrially prepared capelin, which also have more homogenous microbiomes than traditionally prepared capelin. Interestingly, the locally preferred type of traditionally dried capelin, described to be tastier than other traditionally dried capelin, contains bacteria that potentially confer distinct taste. Finally, we show that dried cod have comparably more homogenous microbiomes when compared to capelin and that in general, the environment of drying is a major determinant of the microbial composition of these indigenous food products.

Matched MeSH terms: Fishes/microbiology*
Fulltext Ecological and Ontogenetic Components of Larval Lake Sturgeon Gut Microbiota Assembly, Successional Dynamics, and Ecological Evaluation of Neutral Community Processes

Abdul Razak S, Scribner KT

Appl Environ Microbiol, 2020 05 05;86(10).
PMID: 32169941 DOI: 10.1128/AEM.02662-19

Gastrointestinal (GI) or gut microbiotas play essential roles in host development and physiology. These roles are influenced partly by the microbial community composition. During early developmental stages, the ecological processes underlying the assembly and successional changes in host GI community composition are influenced by numerous factors, including dispersal from the surrounding environment, age-dependent changes in the gut environment, and changes in dietary regimes. However, the relative importance of these factors to the gut microbiota is not well understood. We examined the effects of environmental (diet and water sources) and host early ontogenetic development on the diversity of and the compositional changes in the gut microbiota of a primitive teleost fish, the lake sturgeon (Acipenser fulvescens), based on massively parallel sequencing of the 16S rRNA gene. Fish larvae were raised in environments that differed in water source (stream versus filtered groundwater) and diet (supplemented versus nonsupplemented Artemia fish). We quantified the gut microbial community structure at three stages (prefeeding and 1 and 2 weeks after exogenous feeding began). The diversity declined and the community composition differed significantly among stages; however, only modest differences associated with dietary or water source treatments were documented. Many taxa present in the gut were over- or underrepresented relative to neutral expectations in each sampling period. The findings indicate dynamic relationships between the gut microbiota composition and host gastrointestinal physiology, with comparatively smaller influences being associated with the rearing environments. Neutral models of community assembly could not be rejected, but selectivity associated with microbe-host GI tract interactions through early ontogenetic stages was evident. The results have implications for sturgeon conservation and aquaculture production specifically and applications of microbe-based management in teleost fish generally.IMPORTANCE We quantified the effects of environment (diet and water sources) and host early ontogenetic development on the diversity of and compositional changes in gut microbial communities based on massively parallel sequencing of the 16S rRNA genes from the GI tracts of larval lake sturgeon (Acipenser fulvescens). The gut microbial community diversity declined and the community composition differed significantly among ontogenetic stages; however, only modest differences associated with dietary or water source treatments were documented. Selectivity associated with microbe-host GI tract interactions through early ontogenetic stages was evident. The results have implications for lake sturgeon and early larval ecology and survival in their natural habitat and for conservation and aquaculture production specifically, as well as applications of microbe-based management in teleost fish generally.

Matched MeSH terms: Fishes/microbiology*
Fulltext Extraction of high-molecular-weight DNA from Streptococcus spp. for nanopore sequencing in resource-limited settings

Das S, Delamare-Deboutteville J, Barnes AC, Rudenko O

Microbiologyopen, 2024 Aug;13(4):e1432.
PMID: 39166362 DOI: 10.1002/mbo3.1432

The long-read sequencing platform MinION, developed by Oxford Nanopore Technologies, enables the sequencing of bacterial genomes in resource-limited settings, such as field conditions or low- and middle-income countries. For this purpose, protocols for extracting high-molecular-weight DNA using nonhazardous, inexpensive reagents and equipment are needed, and some methods have been developed for gram-negative bacteria. However, we found that without modification, these protocols are unsuitable for gram-positive Streptococcus spp., a major threat to fish farming and food security in low- and middle-income countries. Multiple approaches were evaluated, and the most effective was an extraction method using lysozyme, sodium dodecyl sulfate, and proteinase K for lysis of bacterial cells and magnetic beads for DNA recovery. We optimized the method to consistently achieve sufficient yields of pure high-molecular-weight DNA with minimal reagents and time and developed a version of the protocol which can be performed without a centrifuge or electrical power. The suitability of the method was verified by MinION sequencing and assembly of 12 genomes of epidemiologically diverse fish-pathogenic Streptococcus iniae and Streptococcus agalactiae isolates. The combination of effective high-molecular-weight DNA extraction and MinION sequencing enabled the discovery of a naturally occurring 15 kb low-copy number mobilizable plasmid in S. iniae, which we name pSI1. We expect that our resource-limited settings-adapted protocol for high-molecular-weight DNA extraction could be implemented successfully for similarly recalcitrant-to-lysis gram-positive bacteria, and it represents a method of choice for MinION-based disease diagnostics in low- and middle-income countries.

Matched MeSH terms: Fishes/microbiology
Gold-nanoparticle based electrochemical DNA sensor for the detection of fish pathogen Aphanomyces invadans

Kuan GC, Sheng LP, Rijiravanich P, Marimuthu K, Ravichandran M, Yin LS, et al.

Talanta, 2013 Dec 15;117:312-7.
PMID: 24209346 DOI: 10.1016/j.talanta.2013.09.016

Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry.

Matched MeSH terms: Fishes/microbiology
Fulltext Molecular characterization of Streptococcus agalactiae strains isolated from fishes in Malaysia

Amal MN, Zamri-Saad M, Siti-Zahrah A, Zulkafli AR, Nur-Nazifah M

J Appl Microbiol, 2013 Jul;115(1):20-9.
PMID: 23557382 DOI: 10.1111/jam.12210

AIMS: The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP-PCR) techniques.
METHODS AND RESULTS: A total of 181 strains of Strep. agalactiae isolated from red hybrid tilapia (Oreochromis sp.) and golden pompano (Trachinotus blochii) were characterized using RAPD and REP-PCR techniques. Both the fingerprinting techniques generated reproducible band patterns, differing in the number and molecular mass amplicons. The RAPD technique displayed greater discriminatory power by its production of more complex binding pattern and divided all the strains into 13 groups, compared to 9 by REP-PCR technique. Both techniques showed the availability to differentiate the genetic profiles of the strains according to their geographical location of origin. Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains isolated from red hybrid tilapia, while the strain of ATCC 27956 that recovered from bovine displayed a unique profile for both methods.
CONCLUSIONS: Both techniques possess excellent discriminative capabilities and can be used as a rapid means of comparing Strep. agalactiae strains for future epidemiological investigation.
SIGNIFICANCE AND IMPACT OF THE STUDY: Framework as the guideline in traceability of this disease and in the search for potential local vaccine candidates for streptococcosis in this country.

Matched MeSH terms: Fishes/microbiology*
Genome characterization of a novel vibriophage VpKK5 (Siphoviridae) specific to fish pathogenic strain of Vibrio parahaemolyticus

Lal TM, Sano M, Ransangan J

J Basic Microbiol, 2016 Aug;56(8):872-88.
PMID: 26960780 DOI: 10.1002/jobm.201500611

Vibrio parahaemolyticus has long been known pathogenic to shrimp but only recently it is also reported pathogenic to tropical cultured marine finfish. Traditionally, bacterial diseases in aquaculture are often treated using synthetic antibiotics but concern due to side effects of these chemicals is elevating hence, new control strategies which are both environmental and consumer friendly, are urgently needed. One promising control strategy is the bacteriophage therapy. In this study, we report the isolation and characterization of a novel vibriophage (VpKK5), belonging to the family Siphoviridae that was specific and capable of complete lysing the fish pathogenic strain of V. parahaemolyticus. The VpKK5 exhibited short eclipse and latent periods of 24 and 36 min, respectively, but with a large burst size of 180 pfu/cell. The genome analysis revealed that the VpKK5 is a novel bacteriophage with the estimated genome size of 56,637 bp and has 53.1% G + C content. The vibriophage has about 80 predicted open reading frames consisted of 37 complete coding sequences which did not match to any protein databases. The analysis also found no lysogeny and virulence genes in the genome of VpKK5. With such genome features, we suspected the vibriophage is novel and could be explored for phage therapy against fish pathogenic strains of V. parahaemolyticus in the near future.

Matched MeSH terms: Fishes/microbiology*
Diverse Profiles of Biofilm and Adhesion Genes in Staphylococcus Aureus Food Strains Isolated from Sushi and Sashimi

Puah SM, Tan JAMA, Chew CH, Chua KH

J Food Sci, 2018 Sep;83(9):2337-2342.
PMID: 30101982 DOI: 10.1111/1750-3841.14300

Staphylococcus aureus is able to form multilayer biofilms embedded within a glycocalyx or slime layer. Biofilm formation poses food contamination risks and can subsequently increase the risk of food poisoning. Identification of food-related S. aureus strains will provide additional data on staphylococcal food poisoning involved in biofilm formation. A total of 52 S. aureus strains isolated from sushi and sashimi was investigated to study their ability for biofilm formation using crystal violet staining. The presence of accessory gene regulator (agr) groups and 15 adhesion genes was screened and their associations in biofilm formation were studied. All 52 S. aureus strains showed biofilm production on the tested hydrophobic surface with 44% (23/52) strains classified as strong, 33% (17/52) as moderate, and 23% (12/52) as weak biofilm producers. The frequency of agr-positive strains was 71% (agr group 1 = 21 strains; agr group 2 = 2 strains; agr group 3 = 12 strains; agr group 4 = 2 strains) whereas agr-negative strains were 29% (15/52). Twelve adhesion genes were detected and 98% of the S. aureus strains carried at least one adhesion gene. The ebps was significantly (p < .05) associated with strong biofilm producing strains. In addition, eno, clfA, icaAD, sasG, fnbB, cna, and sasC were significantly higher in the agr-positive group compared to the agr-negative group. The results of this study suggest that the presence of ebps, eno, clfA, icaAD, sasG, fnbB, cna, and sasC may play an important role in enhancing the stage of biofilm-related infections and warrants further investigation.
PRACTICAL APPLICATION: This work contributes to the knowledge on the biofilm formation and the distribution of agr groups in S. aureus strains as well as microbial surface components in recognizing adherence matrix molecules of organisms isolated from ready-to-eat sushi and sashimi. The findings provide valuable information to further study the roles of specific genes in causing biofilm-related infections.

Matched MeSH terms: Fishes/microbiology
The effects of fish gender on susceptibility to acute Streptococcus agalactiae infection in Javanese medaka Oryzias javanicus

Amal MNA, Zarif ST, Suhaiba MS, Aidil MRM, Shaqinah NN, Zamri-Saad M, et al.

Microb Pathog, 2018 01;114:251-254.
PMID: 29217326 DOI: 10.1016/j.micpath.2017.11.069

This study describes the susceptibility of different fish gender following acute Streptococcus agalactiae infection by using Javanese medaka Oryzias javanicus as test fish. The fish were grouped into four groups, which were: (1) all-male; (2) all-female; (3) mixed-gender (1 male: 1 female ratio); and (4) control non-infected (1 male: 1 female ratio). The fish in group 1, 2 and 3 were intraperitoneally exposed to 5.4 × 108 CFU/mL of S. agalactiae, while for group 4, the fish were exposed using sterile broth. The main clinical signs and histopathological changes of infected Javanese medaka were commonly observed in S. agalactiae infected fishes. However, no difference on clinical signs and histopathological changes of fish in group 1, 2 and 3 were noticed. The Javanese medaka mortality in group 1, 2 and 3 were observed from 4 h post infection (hpi) to 6 hpi, with the cumulative mortality from 3% to 30%. Then, the mortality increased at 12 hpi, with the range from 53% to 80%. However, 100% of the infected fish dead at 24 hpi. No clinical sign, histopathological change and fish mortality recorded in group 4. Generally, the clinical signs, mortality patterns, cumulative mortality and histopathological changes of Javanese medaka infected by S. agalactiae did not show any difference between the all-male, all-female and mixed-gender groups. This indicates that the susceptibility of fish to S. agalactiae infection is not influenced by their gender.

Matched MeSH terms: Fishes/microbiology*
Sennetsu neorickettsiosis: a probable fish-borne cause of fever rediscovered in Laos

Newton PN, Rolain JM, Rasachak B, Mayxay M, Vathanatham K, Seng P, et al.

Am J Trop Med Hyg, 2009 Aug;81(2):190-4.
PMID: 19635868

Neorickettsia sennetsu has been described from Japan and Malaysia, causing a largely forgotten infectious mononucleosis-like disease. Because it is believed to be contracted from eating raw fish, frequently consumed in the Lao PDR, we looked for evidence of N. sennetsu among Lao patients and fish. A buffy coat from 1 of 91 patients with undifferentiated fever was positive by 16S rRNA amplification and sequencing and real-time polymerase chain reactions (PCR) targeting two N. sennetsu genes. Lao blood donors and patients with fever, hepatitis, or jaundice (N = 1,132) had a high prevalence (17%) of immunofluorescence assay IgG anti-N. sennetsu antibodies compared with 4% and 0% from febrile patients (N = 848) in Thailand and Malaysia, respectively. We found N. sennetsu DNA by PCR, for the first time, in a fish (Anabas testudineus). These data suggest that sennetsu may be an under-recognized cause of fever and are consistent with the hypothesis that it may be contracted from eating raw fish.

Matched MeSH terms: Fishes/microbiology*
Fulltext Virulence Factors and Antibiotic Susceptibility of Staphylococcus aureus Isolates in Ready-to-Eat Foods: Detection of S. aureus Contamination and a High Prevalence of Virulence Genes

Puah SM, Chua KH, Tan JA

Int J Environ Res Public Health, 2016 Feb;13(2):199.
PMID: 26861367 DOI: 10.3390/ijerph13020199

Staphylococcus aureus is one of the leading causes of food poisoning. Its pathogenicity results from the possession of virulence genes that produce different toxins which result in self-limiting to severe illness often requiring hospitalization. In this study of 200 sushi and sashimi samples, S. aureus contamination was confirmed in 26% of the food samples. The S. aureus isolates were further characterized for virulence genes and antibiotic susceptibility. A high incidence of virulence genes was identified in 96.2% of the isolates and 20 different virulence gene profiles were confirmed. DNA amplification showed that 30.8% (16/52) of the S. aureus carried at least one SE gene which causes staphylococcal food poisoning. The most common enterotoxin gene was seg (11.5%) and the egc cluster was detected in 5.8% of the isolates. A combination of hla and hld was the most prevalent coexistence virulence genes and accounted for 59.6% of all isolates. Antibiotic resistance studies showed tetracycline resistance to be the most common at 28.8% while multi-drug resistance was found to be low at 3.8%. In conclusion, the high rate of S. aureus in the sampled sushi and sashimi indicates the need for food safety guidelines.

Matched MeSH terms: Fishes/microbiology*
Phytochemical Analysis, Identification and Quantification of Antibacterial Active Compounds in Betel Leaves, Piper betle Methanolic Extract

Syahidah A, Saad CR, Hassan MD, Rukayadi Y, Norazian MH, Kamarudin MS

Pak J Biol Sci, 2017;20(2):70-81.
PMID: 29022997 DOI: 10.3923/pjbs.2017.70.81

BACKGROUND AND OBJECTIVE: The problems of bacterial diseases in aquaculture are primarily controlled by antibiotics. Medicinal plants and herbs which are seemed to be candidates of replacements for conventional antibiotics have therefore gained increasing interest. Current study was performed to investigate the presence of phytochemical constituents, antibacterial activities and composition of antibacterial active compounds in methanolic extract of local herb, Piper betle .
METHODOLOGY: Qualitative phytochemical analysis was firstly carried out to determine the possible active compounds in P. betle leaves methanolic extract. The antibacterial activities of major compounds from this extract against nine fish pathogenic bacteria were then assessed using TLC-bioautography agar overlay assay and their quantity were determined simultaneously by HPLC method.
RESULTS: The use of methanol has proved to be successful in extracting numerous bioactive compounds including antibacterial compounds. The TLC-bioautography assay revealed the inhibitory action of two compounds which were identified as hydroxychavicol and eugenol. The $-caryophyllene however was totally inactive against all the tested bacterial species. In this study, the concentration of hydroxychavicol in extract was found to be 374.72±2.79 mg g-1, while eugenol was 49.67±0.16 mg g-1.
CONCLUSION: Based on these findings, it could be concluded that hydroxychavicol and eugenol were the responsible compounds for the promising antibacterial activity of P. betle leaves methanolic extract. This inhibitory action has significantly correlated with the amount of the compounds in extract. Due to its potential, the extract of P. betle leaves or it compounds can be alternative source of potent natural antibacterial agents for aquaculture disease management.

Matched MeSH terms: Fishes/microbiology*

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