Antibacterial activity of silver nanoparticles on Flavobacterium johnsoniae infection in common carp (Cyprinus carpio)
Mohamed Shaalan*1, Boglárka Sellyei2, Mansour El-Matbouli3, Csaba Székely2
1Department of Veterinary Pathology, Faculty of Veterinary Medicine, Cairo University, Egypt
2Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary
3Clinical Division of Fish Medicine, University of Veterinary Medicine, Vienna, Austria
Flavobacterial infections cause of significant losses in fish farms with the emergence of antibiotic-resistant isolates. Silver nanoparticles (Ag-NPs) are known for their potent antimicrobial activity against different types of bacteria. In this study, we evaluated the antibacterial properties of silver nanoparticles (diameter = 23 nm) against Flavobacterium johnsoniae infection in common carp (Cyprinus carpio). The fish experiments included the artificial infection with F. johnsoniae which was followed with Ag NPs treatment (immersion or intraperitoneal injection). Assessment of antibacterial activity and the evaluation of their effect to the fish tissue were conducted. In this experiment, mortality rates reduced from 45% in infected non-treated group to 30% and 15% in intraperitoneal injection and immersion-treated groups, respectively. Both treated groups did not show any clinical signs or histopathological lesions two weeks after infection. The single dose treatment with Ag-NPs during early infection with F. johnsoniae aided in minimizing fish losses.
Fish infections caused by Flavobacterium species represent major threats to commercial aquaculture worldwide (Crump et al. 2005, Flemming et al. 2007). However, the genus Flavobacterium encompasses species essentially confined to soil and water environments, some of them have been associated with clinical disease in fishes.
Flavobacterium johnsoniae affiliated with diseased fish was first mentioned by Christensen (1977), followed by the report of Carson et al. (1993) of F. johnsoniae causing superficial erosion on the skin of juvenile cultured barramundi (Lates calcarifer) in Australia. Then, it has become a significant pathogen in the aquaculture industry worldwide (Loch & Faisal 2015). Most recently, F. johnsoniae and F. johnsoniae-like isolates were associated with false columnaris disease in aquacultured longfin eels (Anguilla mossambica), rainbow trout (Oncorhynchus mykiss), and koi in South Africa (Flemming et al. 2007), in Russian sturgeon (Acipenser gueldenstaedtii) in Turkey (Karatas et al. 2010) and in farmed rainbow trout in Korea (Suebsing & Kim 2012). F. johnsoniae antibiotic resistant isolates were also reported in common carp (Cyprinus carpio) in Hungary (Varga et al. 2016).
Prevention of flavobacteria epizootics remains difficult due to their ubiquitous presence in soils and waters. Outbreaks in fish farms occur under intensive farming conditions link to increases in suspended solids in the water and sudden falling in water temperature.
Besides optimizing and adjusting management practices, antibiotics have been the preferred treatment method for bacterial infections because of their cost-effectiveness and powerful outcomes. But, the rampant use of antibiotics has led to the emergence with high frequency of multidrug resistant (MDR) strains indicating that the control of disease outbreaks caused by Flavobacterium spp. will remain a significant challenge (Aly et al. 2013, Declercq et al. 2013, Miranda et al. 2016, Varga et al. 2016). Moreover, it is a major concern about the transfer of antibiotic resistance genes from aquatic bacteria to human bacteria (Cabello et al. 2016).
Nanoparticles (NPs) are increasingly used to target bacteria as a novel alternative to antimicrobial agents (Wang et al. 2017). Silver nanoparticles (Ag-NPs) disrupted the bacterial cell membrane and directed complete cell lysis and leakage of intracellular content (Shaalan et al. 2017).
Recently, in vitro antimicrobial activity of silver nanoparticles (Ag-NPs) on pathogenic bacteria in aquatic environments has been studied (Soltani et al. 2009, Swain et al. 2014, Dananjaya et al. 2016, Shaalan et al. 2017).
The aim of our study was to evaluate the efficacy of silver nanoparticles as a novel approach to treat antibiotic-resistant flavobacterial infections. In addition to the assessment of the safety of short-term exposure of silver nanoparticles on fish tissues.
Material and Methodes
Synthesis and characterization of silver nanoparticles
The synthesis of silver nanoparticles was conducted by chemical reduction method, sodium borohydride and trisodium citrate were the reducing agents while poly vinyl pyrrolidone (PVP) acted as the capping agent (El-Mahdy et al. 2015, Shaalan et al., 2017). The newly formed nanoparticles were imaged by transmission electron microscopy (TEM) to reveal their morphology while their concentration was measured with inductively-coupled plasma mass spectrometry (ICP-MS).
Bacterial strains, media, and growth condition
F. johnsoniae type strain from the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH (DSM No.:2064) were used for the bacterial infection. Before use, the dry stocks were desolved in physiological saline solution and streak on Hsu-Shott’s-agar. The plates were incubated aerobically at room temperature (RT, approx. 22-24ºC) for 72 h in humidity chamber. Then single colonies were inoculated into 10 ml nutrient broth and incubated for 48h at RT with shaking 180 rpm on an orbital shaker for the MIC assay and for challenge trials.
Fish management conditions
Specific pathogen-free and clinically healthy juvenile common carp (Cyprinus carpio) (n=70) (length: 6 ± 1.2 cm, weight: 4.5 ± 1.6 g) were acclimatized for 2 weeks in 100 L water tanks. In all treatments, tanks were aerated continuously with natural photoperiod cycle. The water temperature was kept on 15 ± 2.0 ºC and pH 7.4. Fish were fed on the basis of 1%/body weight daily with commercial pellet.
The type strain of F. johnsoniae (DSM No.:2064) as an infective agent was used in the challenge studies. The experiment was conducted according to the guidelines of experiments on fish with the approval number (PEI/001/1002-13/2015). Fish in the experimental groups (n=60) were anaesthetized with 20 ppm clove oil (Javahery et al. 2012) and then injected intraperitoneally with 0.1 ml of a suspension containing 107 bacterial cells then transferred to six tanks with ten fish in each. Ten fish were stocked in a separated tank and injected with PBS only served as a negative control group. On the third day post-challenge, the silver nanoparticles treatments were executed. Ten fish were treated by injection of 100 µl of silver nanoparticles solution (34 µg ml-1) in the peritoneal cavity, and ten by immersion bath for 3 hours in a water bath containing 100 µg l-1 of silver nanoparticles, in duplicate. In parallel, fish (n=20) in the positive control groups were administrated with 0.1 ml of mock PBS. No feed was supplied during the experimental period. The fish were observed daily several times for any abnormal clinical appearances. Freshly dead fish were collected immediately and collected tissue from the gill and the head kidney (abdominal fluid in occasionally) for bacterial detection by PCR.
From all experimental groups, tissues samples (gill, liver, spleen, kidney, muscles) were collected at the 15th and 30th days of post infection for histopathological analysis.
The selected colonies of bacteria grew on Hsu-Shott’s-agar recovered from gills and kidney of dissected fish were used as a template for a species-specific PCR designed for the 16S rRNA gene by Bader Shoemaker & Klesius (2003). The amplified products were separated by electrophoresis in 1% agarose gel stained with ethidium bromide and visualised by UV transillumination. The length of PCR fragments was verified by GeneRuler 100bp Plus DNA ladder (Thermo Fisher Scientific Inc., Waltham, MA, USA).
For histopathologic examination, control and diseased fish were euthanised with an overdose of clove oil before sampling. Tissue samples were excised from the gills, liver, spleen, head kidney, fixed in neutral buffered formalin 10% for 24h thereafter processed, embedded and sectioned at 4-5 µm thickness and stained H&E (Suvarna et al. 2018).
Results and Conclusions
Characterization of shape, size and concentration of silver nanoparticles
TEM microphotographs showed spherical shape of silver nanoparticles (electron dense particles). The mean of the nanoparticles’ diameter was calculated to be 23 nm and the silver concentration in the synthesized Ag NPs was 34 µg ml-1 as measured by ICP-MS.
The efficacy of silver nanoparticles treatment against F. johnsoniae infection was examined in the in vivo study. The artificially-infected juvenile carps by bacterial injection to the peritoneal cavity (I/P) were treated by immersion bath for 3 hours in a water bath containing 100 µg l-1 of silver nanoparticles or by I/P injection of 100 µl of silver nanoparticles solution (34 µg ml-1).
The bacterial infection was performed through I/P injection, which was effective to produce the disease as water bath infection trial had not worked. Moyer & Hunnicutt (2007) reported that only injection with F. johnsoniae was effective to infect zebrafish. Then, after the first deaths, we applied two methods of silver nanoparticles administration, the immersion and I/P injection, to have a conclusion about the best method for application.
In the immersion method, we applied silver nanoparticles in concentration of 100 ng ml-1. Lee et al. (2012) reported that common carp did not show severe adverse health effects or deaths after immersion in 200 ng ml-1 of silver nanoparticles. In other study, water bath containing 100 ng ml-1 of silver nanoparticles was not lethal to rainbow trout (Shaalan et al. 2018). For the dose of I/P injection we applied the same concentration as the MIC in our in vitro study.
The manifestation of disease and the mortalities were detected mainly during the first 15 days of the observation period. Affected fish showed erratic swimming with curved body, darkened colour and abrasion of the body surface, lost or protrusion of scales, petechial haemorrhage on the skin in addition to fluid accumulation in the abdominal cavity (dropsy).
In our study, a remarkable reduction of the mortality rates was recorded in both treated groups, especially in the immersion-treated one. The reduction of mortalities in fish after treatment with silver nanoparticles matched with previous experiment performed on rainbow trout infected with A. salmonicida (Shaalan et al. 2018).
In the gills and kidney samples of all moribund and freshly-dead fish, the presence of F. johnsoniae was confirmed by bacteriological isolation and the PCR reaction.
The histopathological lesions were mainly confined to the positive control group at 15 days post infection (DPI). In the gills, severe and massive gill necrosis was observed in the positive control fish group, while the treated groups showed a mild lamellar fusion. Regarding renal lesion, positive control group fish showed diffuse severe depletion of hematopoietic tissue, this lesion was reported in another study after experimental infection with F. psychrophilum (Marancik, et al. 2014) while there were no tubular lesions in fish of treated groups. The liver showed diffuse severe vacuolar degeneration of the hepatocytes with some necrotic and fragmented cells only in the positive control group.
The clinical signs and histopathologic lesions in gills, kidney and liver were noted in the positive control groups only, confirming the therapeutic effects of silver nanoparticles when applied during the early stage of F. johnsoniae infection.
In conclusion, silver nanoparticles are promising as alternative to antibiotics, especially at the onset of bacterial infection including infections with antibiotic-resistant bacteria. Single-time dosage offers convenience of treatment during bacterial outbreaks. As a therapeutic application to the fish farms, it is worth mentioning that the silver nanoparticles treatment should be conducted in the night. The presence of solar light enhanced the toxicity of silver nanoparticles to fish cell lines and embryos of zebrafish (George et al. 2014).
Keywords: Flavobacterium johnsoniae, silver nanoparticles, common carp, antibacterial, fish diseases
The authors thank G. Pataki and A. Doszpoly for the preparation of histopathological slides and fish cell lines, respectively. We acknowledge the TEMPUS scholarship (Bilateral state scholarship) awarded to Mohamed Shaalan (AK-00362-002/2018). The research costs were covered by the European Regional and Development Fund and the Government of Hungary within the project GINOP-2.3.2-15-2016-00025 (GOODFISH).
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