I declare that I am the sole author of the thesis entitled Antimicrobial activity of some marine microalgae against common bacterial diseases in aquaculture in Chattogram, Bangladesh. Suchandan Sikder, Associate Professor, Department of Medicine and Surgery, CVASU for his valuable supervision and guidance.
Significance of the study
Since this bioactive compound is present in the microalgae, it inhibits the pathogenic bacteria from infecting the host and then eliminates it. Microalgae can easily be grown in high density and used commercially in aquaculture or fish farming.
Marine microalgae available in Bangladesh
45 taxa under 17 genera of green algae have been reported from the coasts of Bangladesh (Islam et al., 2000). Among the several green species, Chlorella miminutissima, Tetrselmis chuii, Nannochloropsis sp., Arthrospira platensis, Isochrysissp., Chondruscrispus, Mastocarpus stellallatus, Ascophyllum nodosum, Alaria esculentus, Sprirulina platenesculentussis, Na platennoclentussis, Na platennochloropula, Chlorulina platennochloropula, Chlorina platennochloropula, found in St.
Characteristics of common marine microalgae species
- Nannochloropsis sp
- Tetraselmis sp
- Chlorella sp
It has since been shown that the causative agent is Rhabdovirus carpio (spring viremia in carp) (Fijan et al., 1988). Aeromonas hydrophila is generally regarded as an opportunistic pathogen or secondary invader (Austin et al., 2012). Lysinibacillus sphaericus has been shown to be sensitive to tetracycline, a common broad-spectrum antibiotic (Priest et al., 1988).
Exophthalmos, congestion and common symptoms of staphylococcal infections in fish and tail ulcers are observed (Kusuda et al., 1998). Animals and the rise of antibiotic resistance in humans and livestock pathogens (Teuber et al., 1999). Other antimicrobial phenolic compounds isolated from the marine environment include anthraquinones coumarins and flavonoids (Amaro et al., 2011).
This compound was effective against Gram-positive and Gram-negative bacteria (Vello et al., 2014). The comparison of the antimicrobial activity of the extract was a relative percentage inhibition and the following formula was used for monitoring (Kumar et al., 2016). They are used in food, pharmaceutical and food applications (Guerin et al., 2003). The antibiotic content of algal species can vary depending on many ecological and biogeographical factors.
A previous study showed that Nannochloropsis oculata had moderate antibacterial activity against Bacillus subtilis (Qusem et al., 2016).
Antimicrobial activity of some marine microalgae
Marine microalgae as potential alternative to antibiotics in aquaculture
- Bacterial diseases of aquaculture in Bangladesh
- Common antimicrobials used in aquaculture industry
- Antimicrobial resistance (AMR) as a major obstacle in aquaculture sector…
Aeromonas hydrophila, Aeromonas sorbia, Aeromonas caviae, Aeromonas schuberti and Aeromonas veronii include (Azad et al., 2001). Another parasite that affects tilapia is Streptococcus agalactiae, which is associated with intensive breeding of brood stock (Hernandez et al., 2009). The bacterium is commonly distributed in fresh water and organic matter containing bottom sediments, as well as in the intestinal tract of fish (Dumontel et al., 1996).
Lysinibacillus sphaericus was thought to cause a type of human pathogenicity associated with tropical ulcer forms and dermal and/or respiratory infections in the 20th century (Wenzler et al., 2015). Staphylococcal infections in fish were also observed by Couch et al., (1992) under specific and severe stress imposed on the fish by the environment. The challenges are how to use antimicrobials wisely and minimize the risk of resistance (Kemper et al., 2008).
The use of 11 banned antimicrobial drugs has been identified in aquaculture in China and Vietnam, including chloramphenicol, ciprofloxacin, florfenicol, nitrofurans and enrofloxacin (Lulijwa et al., 2019). Although global economic losses from diseases in aquaculture have not been compiled, reports from many regions of the world have increased with advances in the trade of live aquatic animals (Tomova et al., 2015).
Spread of AMR and risks to public health
These environments and aquaculture food production are potentially important in terms of the occurrence, persistence and transmission of AMR (Taylor et al., 2011). Many aquatic bacteria harbor a wide variety of mobile genetic elements, such as plasmids, integrons and transposons, that can easily move, recombine and mobilize, promoting the emergence of new mobile combinations of antimicrobial resistance genes (ARGs), giving bacteria the ability to proliferate can be adjusted quickly. to new environments in which antimicrobials are present (Cabello et al., 2013). Common fish pathogens that infect fish handlers include Aeromonas hydrophila, Mycobacterium marinum, Streptococcus iniae, Vibrio vulificus and Photobacterium damselae (Haenen et al., 2013).
Described aquatic environments can shape the evolution of potential resistance profiles as genetic reactors or hotspots for AMR genes where substantial genetic exchange and recombination can occur (Watts et al., 2017). Antibiotic tolerance can be conferred by chromosomal or mobile genetic elements and can be achieved using four major techniques, including the reduction of antibiotic membrane permeability, drug inactivation and accelerated antibiotic efflux and cellular target mutation (Baker et al., 2006). These food products are a major source of human infections, leading to an increase in antibiotic use, which facilitates the development of antibacterial resistance (Aminov et al., 2009).
Pathogenic bacteria prevalent in fish include Aeromonas species, which are abundant in freshwater ecosystems (Ibrahim et al., 2014). Due to the possibility of transmission of resistant pathogens and commensal bacteria to the human population, the presence of antibiotic resistance in animal bacteria has caused serious concern (Cabello et al., 2006).
Alternatives to antibiotics
Marine microalgae as potential alternative to antibiotics
The essential properties of these polysaccharides: anticoagulant, anti-thrombotic, antiviral, antitumor, anti-inflammatory, antioxidant and immunomodulatory (Li et al., 2013). Antioxidants are very effective devices to combat oxidative stress and therefore increase the welfare status of the population (Rani et al., 2013). The characteristics of the profile of saturated and unsaturated fatty acids in algae with a predominance of myristic, palmitic, oleic and eicosapentaenoic acids (EPA) is a specific feature related to the antimicrobial potential of algal species (El Shoubaky et al., 2014) .
Culture of microalgae
- Conway medium preparation
- Mass culture of microalgae
- Preparation of microalgae crude extracts
The culture was gradually increased from an initial stock culture volume of 20 ml to 20 L. Initially, 20 ml of microalgae stock culture was mixed with 30 ml of medium in each flask (total culture volume 50 ml), with batch cultures in growth. volume (250 ml, 500 ml, 1 L, 10 L) as inocula for the next step after which they were transferred to a larger container with 20 L culture medium. The microalgae were collected by centrifugation at 5000 rpm for 5 min to get rid of the water content.
The dried biomass was taken in clean 70 ml volume screw-top bottles and immersed in methanol solvents for 48 h at room temperature (Senhorinho et al., 2012).
Isolation and identification of bacteria from fish
- Samples collection from fish
- Preparation of bacterial culture media
The medium was heated with frequent stirring and boiled for 5 minutes to ensure complete dissolution of the medium. To prepare TSB, a total of 30 g of the solid ingredients was dissolved in 1 liter of distilled water and mixed thoroughly (Table 3.4). Nutrient broth was prepared by mixing 13 g of solid ingredients, including gelatin peptone, beef extract, yeast extract, sodium chloride, in 1 liter of distilled water (Table 3.5).
To prepare MSA, a total of 111.025 g of the solid ingredients including proteose peptone, beef extract, sodium chloride, D-mannitol, phenol red and agar were suspended in 1 L of distilled water (Table 3.6). The medium was heated with frequent stirring and boiling for 5 minutes to ensure complete dissolution of the medium. Blood agar was prepared by mixing a total of 40 g of the solid ingredients including agar, beef extract, peptone and sodium chloride in 1 L of distilled water (Table 3.7).
The medium was heated with frequent stirring and boiled for 5 min to ensure complete dissolution of the medium. The medium was gently poured into a petri dish at 60 °C in laminar flow and stored at 4 °C.
Isolation and identification of bacteria
- Aeromonas hydrophila
- Lysinibacillus sphaericius
- Staphylococcus
Gram staining of bacterial samples
- Preparation of Gram’s stain
- Gram staining procedure
After that, the secondary stain safranin was added to the slide for 1 min and washed with a gentle stream of water.
Biochemical tests
- Principles of VITEK 2 biochemical identification method
- Materials and reagents required
- Preparation of test suspension
- Results and interpretation
A sufficient number of colonies were transferred from 24h culture on the prescribed culture medium to the salt tube with a sterile stick or swab to obtain a density equivalent to McFarland 0.50 to 0.63 with the VITEK 2 DENSICHEK. The culture was tested by the VITEK 2 GP card system within 30 min of the suspended culture preparation. The culture tube VITEK 2 GP card is placed in the VITEK 2 cassette and refer to the user manual (supplied with the instrument) for guidance on the use of the instrument.
A slash line or low discrimination identification were appropriate results for the VITEK 2 GP procedure as shown in the VITEK 2 GP product details provided to end users, requiring additional testing to better identify the organism. VITEK 2 interprets the operation if a certain identification pattern is recognized; reported findings indicate a high probability of suitability for an individual animal. If the potential sample is not known, the device will recommend additional tests to distinguish between 2 or 3 very similar species, or report the result as a non-specific organism (>3 species may show a detected sample, or the biological sample is quite typical and not shown in the database).
Antimicrobial activity test
Minimal Inhibitory Concentration (MIC)
Statistical analysis
Isolation and identification of bacteria
- Aeromonas hydrophila
- Lysinibacillus sphaericus
- Staphylococcus sp
On trypticase soy agar, Aeromonas was observed as circular, creamy white colonies marked by arrows. To identify Lysinibacillus, bacterial culture from BPW was inoculated onto blood agar and characteristic purple color large colonies were observed (Figure 4.3). On mannitol salt agar, Staphylococcus was observed as colorless or yellow, shiny, large colonies charged by arrows.
Antimicrobial activity test
- Antimicrobial activity of microalgae against Aeromonas hydrophila…
The agar plate shows a clear zone of bacterial growth surrounding the antibiotic (A), Tetraselmis (T) and Chlorella (C) with a smaller zone of inhibition and no zone around the control disc (N). b) Statistical analysis of microalgae with control showed no significant difference in antibacterial activity. The antimicrobial activity of the microalgae extracts was calculated using the formula mentioned earlier in the Materials and Methods section. The MIC of Chlorella and Tetraselmis extracts grown under different conditions was determined against Aeromonas hydrophila and Staphylococcus.
The MIC value was defined as the minimum concentration of microalgae capable of preventing bacterial growth. Various species of microalgae are known to produce intracellular and extracellular metabolites with various biological activities such as antialgal, antibacterial, antifungal and antiviral activity. The effects of the microalgae extracts used were sensitive to Gram-positive and Gram-negative bacteria.
This is due to the difference in cell wall structures of the Gram-positive and Gram-negative bacteria. The penetration, binding and antimicrobial activity of the compound was determined by the difference cell wall structures of bacteria. Further studies are directed to identify the specific components of microalgae responsible for the sensitivity to bacterial infections.
Isolation and identification of microalgae directly from marine sources was not possible in the current study due to time constraints.
