Aerobic bacteria and fungi from skin lesions of fish in Khartoum state

Objective: This cross-sectional study was conducted from April to July 2014 in Khartoum state, the Sudan, to investigate aerobic bacteria and fungi of skin lesions of fish in 3 different areas in Khartoum. Material and methods: A total of 50 samples were collected from the skin lesions of different types of fish including Synodontis species (n=17), Tilapia niloticus (n=15), Labeo niloticus (n=10), Hydrocynus species (n=4), and Clarias species (n=4). Liquid, semi-solid, and solid culture media like nutrient broth, blood agar, MacConkey agar, sabouraud dextrose agar (SDA), and Simmon's citrate medium were used for the isolation and identification of bacteria and fungi. Besides, Gram staining and biochemical characterization were also conducted. Results: Culturing of the collected samples revealed growth of bacteria from all (100%), and growth of fungi could be found from 32% samples. A number of 188 bacteria were isolated, mainly Staphylococcus species, Bacillus species, Aeromonas species, Pseudomonas species, and Vibrio species. Besides, 16 fungi could be identified containing Aspergillus niger, A. flavus, A. fumigatus, and Phycomycete. Conclusion: Fishes with skin lesions are harboring many pathogenic bacteria and fungi and may act as a source of zoonotic infections and can transmit several pathogens to workers in fish industry and consumers. Therefore, thorough and strict routine inspection of fish is recommended to ensure safety and that there are no serious risks to consumers.


INTRODUCTION
Due to the big amount of surface and underground water resources in the Sudan, there exist a hug wealth of fish too (MARF, 2001a, b;Saaed, 2004;De Young, 2006).In the last few years, Sudanese peoples' interest in fish consumption is noticeably rising as in other parts of the world.This is because of the unstable and escalating prices of red meat and because of the general believe that regular fish consumption is a possible practice for health improvement (Goja, 2013;Deliens et al., 2014).Consequently, there is an increase in fish trade and investments.However, if the fish habitats are contaminated with pathogens, consumption of these fishes may impose risk to humans (Goja, 2013).
Fishes are susceptible to a wide variety of pathogens especially when they are physiologically unbalanced or nutritionally deficient and subjected to stresses, i.e. poor water quality, and over stocking (Plumb, 1997;Goja, 2013;Rowe et al., 2014).Infected fishes show many disease symptoms but in case of skin problems the symptoms are mainly ulcerative and hemorrhagic skin patches (Bruno and Wood, 1994;Adeyemo, 2003; Haj-Ali, 2010; Hassan et al., 2010).
Edwardsiella tarda, Aeromonas species, Pseudomonas species and many other pathogenic bacteria have been isolated from diseased fishes and often these bacteria are fatal if not treated early enough (Yagoub, 2009;Goja, 2013;Kar, 2015;Sebastião et al., 2015).Besides to that, fungal infections are common among fish populations and can lead to heavy economic losses as well (Scarfe et al., 2005;Ramaiah, 2006).Most fish fungal infections are caused by the fungi of the family Saprolegniaceae, Phycomycetes, Aspergillus and Penicillium (Yagoub, 2004;Gozlan et al., 2014).This study was aiming at identifying the bacterial and fungal causative agents of fish skin infections in Khartoum state.

Study area and samples collection:
A cross-sectional study was carried out from April to July 2014 in Khartoum state.Three areas were investigated, namely; El-Shagarah Center for Fish Research, Almawrda Fish Market, and El-Haj Yousif Fish Market.
A total number of 50 samples were collected from the skin lesions of different types of fish including Synodontis species (n=17), Tilapia niloticus (n=15), Labeo niloticus (n=10), Hydrocynus species (n=4), and Clarias species (n=4).Samples were collected as described by Buller (2004).For investigation of bacteria, a sterile cotton swab was rubbed all over the lesion, while for fungi a piece of tissue from the lesion was taken using a sterile scalpel blade.All samples were placed in an ice container and transported to the laboratory of the Department of Microbiology, Faculty of Veterinary Medicine, University of Khartoum, and were cultured within 2 h of collection.
Culture media and reagents: Different types of culture media including liquid, semi-solid, and solid media, and chemicals, and reagents were used for isolation and identification of bacteria and fungi and all were either bought from companies ready-to-use or prepared according to Barrow and Feltham (2003) and Ochei and Kolhatkar (2000).The culture media were peptone water, nutrient broth, glucose phosphate peptone broth, nutrient agar, blood agar, MacConkey agar, sabouraud dextrose agar (SDA), starch agar, nutrient gelatin media, urea agar, Simmon's citrate medium, Hugh and Leifson's (O-F) medium, motility medium, and Arginine media.Chemicals and reagents included Gram solution, crystal violet, Lugol's iodine, decolorizing reagent, counter stain, Tetra methyl-p-phenylenediamine dihydrochloride, and Alpha-naphthol solution, as well as Voges-Proskauer (VP) test, Methyl red, Kovac's reagent and Nessler's, Andrade's indicator, Neutral red, Phenol red, Bromothymol blue, and Lead acetate paper.
Culturing: Collected samples were first inoculated into liquid media and incubated at 37°C for 24 h, after that all were streaked onto blood and MacConkey agars and incubated aerobically at 37°C for 24 h also.Further incubation was continued for another 24 h and if no growth was evident, then the plates were discarded and the sample was considered negative.All cultures were examined by naked eye for growth, colony morphology, and any changes in the medium.Purification of isolates was done by sub-culturing of a single well separated colony onto blood agar or nutrient agar and finally pure cultures were stored at 4°C.

Identification of isolates:
Gram staining was carried out according to Barrow and Feltham (2003)

Fungal investigations:
According to Barrow and Feltham (2003), samples were also cultured onto sabouraud dextrose agar media and incubated at 22°C for 1-2 weeks, during which period the plates were examined daily.Identification of isolated fungi was done by examining the mold in wet mount by transporting a portion of a colony to a drop of Lacto phenol cotton blue (LPCB) stain on sterile slide.A cover slip was applied on the preparation and examined microscopically.

RESULTS
Culture of the 50 specimens of the investigated fish lesions revealed growth of bacteria from all (100.0%) and growth of fungi from some of (32.0%).Isolates of bacteria were 188 and of fungi were 16.
Gram-positive bacteria: Gram stained smears from the cultures and biochemical tests (Table 1 and 2) showed that 78.7% (n=148) of the detected bacteria were Grampositive cocci and rods.Staphylococcus species and Bacillus species were detected with Staphylococcus auricularis (n=17) and Bacillus badius (n=11) being the most isolated micro-organisms.Micrococcus lylae (n=7) was the most frequent among the detected 6 species of Micrococcus.

DISCUSSION
Aquatic creatures, like fish, could potentially be harboring many infectious zoonotic micro-organisms that are able to cause health problems in humans.Hunt  or ii) by ingestion of raw or undercooked aquatic products, and lately, their incidence is escalating.Moreover, infectious diseases are very important in fish industry, because of their consequential heavy economic losses, especially in aquacultures (Haenen et al., 2013;Lafferty et al., 2015).These diseases are commonly caused by pathogens that are either indigenous; originate from the aquatic environment itself or exogenous; occur due to contamination (Haenen et al., 2013).Skin infections in fish are mainly caused by bacteria, parasites and Saprolegnia species, but also by oomycetes and fungi (Cutuli et al., 2015).
Many aerobic Gram positive and gram negative bacteria have been isolated from skin lesions of different types of fish in this study.These findings were similar to the findings of Buller (2004) 2014) who were able to isolate Staphylococcus species from healthy and diseased fish.The ability of the members of the genus Staphylococcus to establish themselves, colonize their hosts and survive could be explained by their ability to express and/or possess one or more potential virulence factors.These factors are adherence factors or exotoxins and include i) surface proteins that promote colonization of host tissues, ii) invasins that promote bacterial spread in tissues like leukocidin, kinases, and hyaluronidase, iii) inhibition of phagocytic engulfment by surface factors such as capsule and protein A, iv) biochemical properties that enhance survival in phagocytes like carotenoids and catalase production, v) immunological disguises, vi) hemolysins, leukotoxin, leukocidin toxins for membrane-damaging        (2004).Iqbal and Saleemi (2013) indicated that fungal infection in fish might occur because of the use of contaminated feed or alternatively decomposed feed in the aquatic environment of the fish.Certainly, infection of fish by pathogenic fungi diminishes the market value of the fish and the nutritional value of its flesh.

Hemolysis
The detected bacteria and fungi in the present study could have been recovered from lesions of primary or secondary infections.Aly (1996) indicated that in primary infections, the causative agent usually affects the normal skin and these infections are clinically characteristic and have specific disease course and often are caused by a single pathogen.In primary infections of many fungi, they invade the keratinized tissue of the skin, because of their strong affinity to keratin.However, in secondary infections, the skin that is already diseased and because of the underlying disease, the clinical picture and course of these infections vary.

CONCLUSION
This study pointed out to that fish with skin lesions in the investigated area could perhaps be a source of zoonotic infections and transmit the detected pathogens to workers in fish industry and consumers.Hence, it is recommended to raise the awareness of workers in fish industry and consumers about the possible biological hazards that could be contracted from fish with topical lesions.Additionally, thorough and strict inspection of fish should be a routine to ensure safety and that there are no risks to consumers.
et al. (2008), Igbinosa et al. (2012), Waltzek et al. (2012), Haenen et al. (2013), and Harper and Erickson (2016) indicated that consumers, fishermen, aquarium workers, and salesmen normally glean these zoonoses either by-i) direct contact, , Yiagnisis and Athanassopoulou (2011), Haenen et al. (2013), Tanekhy (2013), and Saad and Atallah (2014) who were able to isolate aerobic bacteria from different types of fish from different places around the world.Other workers from the Sudan, Egypt, and Nigeria like Selma (2006), Hassan et al. (2010), Ajayi (2012), and Goja (2013) have indicated that indicated that many bacteria can colonize intact healthy or injured and diseased skin of fish and internal organs.Their ability to do so, is underlined by many factors like ability to express virulence factors to invade the host and produce pathological effects.Other important factors would be environmental hygiene and immunity and resistance of the fish.Staphylococcus species was the most frequent species among all isolated bacteria in the present study.This was different from the observation of Süheyla and Osman (2004) who mentioned that staphylococci are the second most frequently isolated bacteria from clinical specimens after Enterobacteriaceae.Nonetheless, it was in agreement with the findings of Majumder et al. (2001), Yiagnisis and Athanassopoulou (2011), Carbajal-González et al. (2011), and Ali ( ND ND ND ND ND ND ND α β β α α α ND ND ND ND ND ND ND Starch hydrolysis ND ND ND ND ND ND ND d d + d --ND ND ND ND ND ND ND Glucose ND ND ND ND Aerobic growth ND ND ND ND ND ND ND ND ND ND ND ND ND and biochemical test, 1=Staphylococcus

Table 1 :
Characters and biochemical reactions of the isolated Staphylococcus species, Streptococcus species, Gemella haemolysans, Leuconostoc species, Aerococcus species, Micrococcus species, and Stomatococcus species from skin lesions of fish in Khartoum state, the Sudan (April to July 2014).

Table 2 :
Characters and biochemical reactions of the isolated Bacillus species, Corynebacterium species, Listeria species and Kurthia species from skin lesions of fish in Khartoum state, the Sudan (April to July 2014).

Table 4 :
Number and percentage of Gram-positive cocci and rods bacteria isolated from skin lesions of fish in Khartoum state, the Sudan (April to July 2014).

Table 5 :
Number and percentage of Gram-negative bacteria isolated from skin lesions of fish in Khartoum state, the Sudan (April to July 2014).

Table 6 :
Cutuli et al. (2015)(1983)he isolatedAspergillus species and Phycomycete species isolated from skin lesions of fish in Khartoum state, the Sudan (April to July 2014).Four fungi have been found in this study, namely A. niger, A. flavus, A. fumigates and Phycomycetes.This was corresponding to the observations ofOlufemi (1985)who indicated that a number of Aspergillus species were responsible of aflatoxicosis in fish.It was also in agreement withYagoub (2004)andSharma et al. (2013)who reported Aspergillus species in fish in the Sudan and from skin ulcers of fish in India.Moreover,Olufemi and Roberts (1983)found that Aspergillus species play a significant role as pathogens in farmed fishes.Cutuli et al. (2015)reported skin infections of tilapia by Fusarium oxysporum species complex.The detection of Phycomycetes species was in agreement with the findings of Yagoub Gram-negative bacteria including Aeromonas species, Pseudomonas species, Klebsiella species, Proteus species, Serratia marcescens, Providencia rettgeri and Yersinia pseudotuberculosis were detected in the present study.These