Characterization and susceptibility of streptococci and enterococci isolated from Nile tilapia (Oreochromis niloticus) showing septicaemia in aquaculture and wild sites in Egypt

Background The present investigation was an endeavor into the elucidation of the disease-causing pathogen of streptococcosis in Nile tilapia (Oreochromis niloticus) in Egypt affecting adult fish cultured and wild fish in the Nile river. Fish were obtained from commercial fishermen, collected as part of their routine fishing activities. The researchers observed the routine fishing process and selected fish for use in the study, at the point of purchase from the fisherman. Results Diseased fish showed exophthalmia with accumulation of purulent and haemorrhagic fluid around eyes, and ventral petechial haemorrhages. The Post mortem examination revealed, abdominal fat haemorrhage, pericarditis and enlargement of the liver, spleen and kidney. Gram-stained smears revealed the presence of Gram-positive cocci, β-hemolytic, oxidase and catalase negative. Analysis of the 16S rRNA gene confirmed that the 17 tilapia isolates studied were 6/17 Enterococcus faecalis, 2/17 Enterococcus gallinarum, 3/17 Streptococcus pluranimalium, 2/17 Aerococcus viridans, 1/17 isolate of each Streptococcus dysgalactiae, Streptococcus anginosus, Lactococcus garvieae and Granulicetella elegans/Leuconostoc mesenteroides cremoris. It should be noted that there was no mixed infection. Multiple resistance was observed and the most frequent antibiotic combination was penicillin, ampicillin, vancomycin, chloramphenicol, rifampicin, ofloxacin, clindamycin, erythromycin and tetracycline representing eight classes. Conclusions Consequently, we concluded that Streptococcus species are an emerging pathogen for Nile tilapia aquaculture in Egypt and to be considered as a new candidate in the warm water fish diseases in Egypt with special reference to L. garvieae, S. dysgalactiae in addition to L. mesenteroides cremoris which was not reported before from tilapia and taking into consideration their zoonotic implications for public health.


Background
The common name Nile tilapia (Oreochromis niloticus) is a species of Tilapia (Oreochromis sp.) which in turn is the regular name for about a hundred species of cichlid fish from the tilapiine cichlid lineage [1]. They are freshwater fish species, local to Africa and Middle East areas [2], and the third biggest group of bony fish. Delineations from Egyptian tombs underscored that Nile tilapia have been developed for more than 4000 years [3]. By the second half from the twentieth century, tilapia were brought into a myriad of tropical, sub-tropical and mild locales of the globe [4] becoming a standout asset amongst the most refined fish species adding to the global nourishment in the fish industry [5] where the sustenance of more than half a billion people in the Third World are supported by fisheries and aquaculture [6]. The market price of farmed tilapia boosted up from 154 million USD in 1984 to 4000 million USD in 2010 [7]. China, with a production capacity of 806,000 MT represents almost 50% of the total global production positioned itself as the largest Nile tilapia producer, with Egypt to follow with 200,000 MT, the Philippines with 111,000 MT, Thailand with 97,000 MT and Indonesia with 72,000 MT. Five other countries come next in their capacity for Nile tilapia production, The Lao People's Democratic Republic, Costa Rica, Ecuador, Colombia and Honduras [8]. Also, although significant quantities of tilapia are also produced annually by Cuba, Israel, Malaysia, the USA, Viet Nam and Zimbabwe, yet it should be noted that, their production is reported to FAO as 'tilapias nei' (which may include other tilapia species) and 'freshwater fishes nei' [8].
Streptococcosis is a worldwide threat and epidemiological studies revealed 500 streptococcal isolates in more than 50 sites in 13 countries (http://www.thefishsite.com/articles/190/streptococcus-in-tilapia/#sthash.G4T 0XzsI.dpuf ). Streptococcosis, also known as 'pop-eye' , is contagious with high mortality and has assumed its importance due to being the most crushing threat as it can bring about huge number of deaths of large size fish causing heavy commercial losses in Australia, Israel, Italy, Japan, Korea, South Africa, Colombia, Indonesia and USA [9,10]. The global commercial losses estimated to reach 250 million USD in 2008 [11]. Within 3 to 7 days, acute Streptococcus infections in fish induce >50% mortality rates [12] while chronic infections the mortalities could extend to several weeks, with a daily death of one or two of the fish. In most cases, the clinical symptoms of Streptococcus infection, with no species differences (http://www.thefishsite.com/articles/190/streptococcus-in-tilapia/#sthash.G4T0XzsI.dpuf), is usually in the form of lethargic, erratic swimming (spiraling or spinning swimming), dark skin pigmentation, exophthalmia with opacity and haemorrhage in the eye, abdominal distension, diffused haemorrhage in the operculum, around the mouth, anus and base of the fins and enlarged blackened spleen [8,10,[13][14][15].
Zoonotically, streptococcicosis is of great significance to delineate the infectious etiology of streptococcosis in Nile tilapia as a potential cause of disease in humans [16,17] which caused a significant increase in the use of antibiotics in aquaculture. The present investigation was an endeavor into the elucidation of the disease-causing pathogen of streptococcosis in Nile tilapia (O. niloticus) in Egypt affecting adult fish cultured and wild fish in the Nile river by studying the phenotypic, antibiotic resistance and molecular characterization of the streptococcal strains isolated from Nile tilapia demonstrating septicemia.

Fish sampling
Fish were obtained from commercial fishermen, collected as part of their routine fishing activities. The researchers observed the routine fishing process and selected fish for use in the study, at the point of purchase from the fisherman the fish were deceased and as such no ethical approval was required for this study. A total number of 80 Nile tilapia were collected as freshly dead or moribund fish ( Fig. 1a-d) presenting at least one or more of the clinical signs of septicaemia (eye lesions: in the form of unilateral or bilateral eye redness/opacity, skin lesions: detached scales, extensive skin congestion, ulcers, hemorrhage, or dark discoloration in the form of strips, fins: congestion at the base of fins, or even hemorrhages, abdomen: slightly distended in some cases, anal opening: congested protruded anal opening) were collected from different locations in Egypt (River Nile in Giza, Kafr Elzayat, Bani-sweif and a breeding farm at El-Tal El-Kebir) during the period 2013-2014. Selection of locations and farms from which the diseased fish were collected depended on their availability at time sampling, accessibility and proximity. After collection, the sampled fish were placed in expanded polystyrene boxes, covered with a plastic film, transported refrigerated to the laboratory and processed for microbiological analysis within 3 h in order to detect Streptococcus spp.. Septicaemic fish samples were submitted to the Fish Disease Department, Animal Health Research Institute, Dokki, Egypt.

Isolation and phenotypic characterization of bacteria
The swabs from the fish matrix that were collected (liver, kidney, brain, spleen and ascitic fluid) were immediately streaked onto Columbia agar supplemented with 5% defibrinated sheep blood (CBA) (Oxoid Ltd., Basingstone, England) plates and incubated at 30°C for 24 h in 5% CO2 atmosphere. Suspected colonies were picked, purified and subjected to phenotypic differential analysis by conventional procedures by their morphological, physiological and biochemical plate and tube tests and in addition to the Gram-positive identification (GPI) cards of the Vitek 2 system (bioMérieux, France) which was Fig. 1 a Body shape of Tilapia fish laterally compressed to oval. Lateral line interrupted with cycloid scales. Caudal fin has distinct vertical stripes and strong body vertical bands. Healthy transparently clear and spherical eye lenses. Normal glistening skin with no pathological lesions observed. b Tilapia fish showing externally eye redness, hemorrhage and ulcers on different parts of the body, hemorrhagic dorsal and tail fins, extensively detached scales and distended abdomen with signs of bleeding beneath the skin appearing as small dots the size of a pinprick or as a patch. c Shows normal appearance of Tilapia fish with normal liver, kidney, gall bladder and intestines with bright red gills. d Tilapia fish postmortum internal findings revealed tipical signs of septicemia in which severely congested gill, kidney, spleen, intestine and heart, dark gall bladder, in addition to hemorrhagic ascites, friable pale liver, muscle redness, and parts of the intestine were devoid of food used following the manufacturer's instructions. The ability of the strains to grow at 10 and 45°C was tested in Brain-heart infusion medium over a period of 10 days.

Preparation of genomic DNA and genetic characterization
The suspected Streptococcus isolates were cultured in Todd Hewitt Broth at 25 C for 24 h, and genomic DNA was extracted using the Qiagen Genomic-tip 500/G kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA concentration was estimated spectrophotometrically before use. The extracted DNA was used in the process of amplification of the 16S rRNA gene of the Streptococcus species -specific isolates with a pair of specific primers for each species using polymerase chain reaction (PCR) as outlined in Table 1. Seven μl of the amplified products was electrophoresed using 1.0% (w/v) agarose gel in 1× TBE electrophoresis buffer (0.1 mM Tris/HCI, 0.1 mM boric acid, 0.002 mM EDTA, pH 8.3). The gel was stained with GelRed Nucleic Acid Gel Stain. A negative control (no template DNA) and positive controls consisting of E. gallinarum ATCC 49673, E. faecalis ATCC 19433, L. garvieae ATCC 43921, S. pluranimalium ATCC 700864 T , A. viridans ATCC 11536 T (accession no. M58797), S. anginosus ATCC33397 were included in each run.

Serological assays
The Lancefield streptococcal group of the isolated strains was identified using the Oxoid Streptococcal Grouping Kit DR0585 (Oxoid Ltd., Basingstone, England) following the manufacturer's instructions.

Antimicrobial susceptibility testing
The resistance of the isolated strains to antibiotics was assayed by the disc diffusion method [18,[29][30][31]. E. faecalis ATCC 29212, Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658, Pseudomonas aeruginosa ATCC 27853 were used as Quality control strains for disk diffusion susceptibility testing of aquatic bacterial isolates [32]. The upcoming antibiotic discs (Oxoid, Ltd., Basingstoke, England) selected for testing are the most frequently used and prescribed in Egypt which are inhibitors of protein synthesis: phenicols (chloramphenicol), tetracyclines (tetracycline) and macrolides (erythromycin) for both Enterococcus and Streptococcus species, while aminoglycosides (gentamicin, streptomycin and amikacin) only for Enterococcus species; cell wall synthesis: penicillins (penicillin, ampicillin, amoxicillin-clavulonic acid and pipercillin-tazobactum) and glycopeptides (vancomycin) for both Enterococcus and Streptococcus species and polymyxins (colistin) for Enterococcus species; nucleic acid synthesis: ansamycins (rifampicin) and nitrofurantoins (nitrofurantoin) were used for both Enterococcus and Streptococcus species, fluoroquinolones (ciprofloxacin for Enterococcus species and ofloxacin for Streptococcus species), quinolones (nalidixic acid for Enterococcus species) and lincosamides (clindamycin) for Streptococcus species.

Statistical analysis
The experimental data obtained was subjected to multiple linear regressions using Microsoft Excel 2007 to evaluate the statistical analysis between the percentage of antimicrobial resistance (y-range) and actual concentration (x-axis) for all antibiotics to enable us to plot percent inhibition versus drug concentration for each strain.

Biochemical and molecular characterization
The microbiological cultures on CBA from the swabs taken from the internal organs (kidney, spleen and brain) and ascitic fluid revealed the presence of pinpoint white colonies (0.5 mm Ø) in pure culture or as major colony type. The Gram-stained smears that were taken from the presumptive Streptococcus colonies revealed Grampositive chain forming cocci (0.6-0.9 μm Ø) small or medium sized in diploids, short chain, chain in the form of Y / V-shape, or in long chain, β-haemolytic, catalase and aesculin hydrolysis test negative, ability to grow at 25 and 37°C and at pH 9.6 in the absence of 6.5% NaCl or 40% bile salts. Table 2 illustrates the effect of incubation temperature and NaCl 6.5% and bile salts on the growth of different isolates their role in identification of different types of isolates. The seventeen isolates were identified using Vitek 2 compact through the biochemical characterization of different isolates. None of our 16 isolates could be serologically typed to any Lancefield group and in the Vitek 2 inconclusive results were obtained. The only result obtained was with the S. dysgalactiae subspecies equisimilis (one isolate) which was grouped in the C group.

Prevalence and identification of Enterococcus, Lactococcus and Streptococcus species isolated from diseased fish
Overall, 17 Streptococcus species were collected and the Streptococcus spp. occurred in 8/80 (10%) liver samples, 4/80 (5%) spleen samples, in 3/80 (3.8%) kidney samples and 1/80 (1.3%) brain and ascitic fluid samples each. The logistic regression showed that the recovery of Streptococcus spp. was significantly affected by fish matrix (P < 0.001). The test of independence showed a significant association between the recovery of Streptococcus spp. from a given matrix and the location ( Table 3).
The rate of bacteria isolated from the diseased tilapia fish, were isolated in a rate of 21.25%. E. faecalis showed the highest number of isolates (6 isolates) in a rate of 7.5%, followed by S. pluranimalium (3.75) ( Table 3). E. faecalis was the most predominant isolate (6/80; 1.9%) and the liver was the most prominent site for the infection with E. faecalis (3.75%). Also, E. faecalis was the sole isolate from the ascitic fluid (1.25%). S. pluranimalium was the second predominant isolate (0.93%) being also predominantly localized in the liver (2.5%) and spleen (1.3%). Similarly, E. gallinarum was isolated at a rate of 1.3% from the liver and kidney. S. dysgalactiae subspecies equisimilis was only isolated from the brain (1.25%).

Antimicrobial susceptibility
The susceptibility of 17 Streptococcus strains was assessed against 18 different antibiotics (Fig. 2). Highest resistant to Tetracycline 94.1% (16/17) the lowest resistance was observed for Nitrofurantoin, Streptomycin, Gentamicin and Trimethoprim/Sulphamethaxozol 5.9% (1/17 each). The 17 isolates showed total susceptibility to Amoxicillin/Clavulanic acid, Piperacillin/Tazobactam, Nalidixic acid, Colistin and Amikacin. There was no significant relationship between the percentage of antimicrobial resistance (y-range) and actual concentration (x-axis) for all combined antibiotics and consequently no regression line could be drawn (Table 4).

Discussion
In this work, a collection of 17 strains of Streptococcus species isolated from diseased tilapia were biochemically, serologically and genetically characterized and data on their prevalence of the disease in Egypt. In contrast to other results [18], 16 of our isolates could not be serologically typed to any Lancefield group and in the Vitek 2 inconclusive results were obtained, a phenomenon which was also previously observed by Figueiredo et al. [33]. On the other hand, our sole isolate of S.
dysgalactiae subspecies equisimilis which was grouped in the C group was also identified in Japan by Nomoto et al. [34,35] and Abdelsalam et al. [36], in the amberjack (Seriola dumerili) and yellowtail (Seriola quinqueradiata) and in Amur sturgeon (Acipenser schrenckii) in China [37] and in Nile tilapia (Oreochromis niloticus (L.) in Brazil [20]. Our detection of Streptococcus species in the three organ tissues (liver, spleen and kidney) implicates them as the target organs of the organism [38][39][40]. Fish pathogenic strains have been described, as either an αor ßhaemolytic or as non-haemolytic [41]. Superficially, this data could surmise heterogeneity amid the pathogens,  inspite of the fact that some confirmed ranks such as S. agalactiae, incorporates both αand ß-haemolytic strains. It should be noted that, many features are common within the predominance of fish pathogens [9]. Yet, this does not exclude some discrepancies reported in isolates recovered from the Transvaal in South Africa [42] and other isolates from Japan [43][44][45]. Such discrepancies could be due to the dissimilarity and absence of a standardized test protocols and/or point to heterogeneity in the species composition of the organisms. The development and application of molecular methodology has been of great benefit and advantage through facilitating identification precision, classification of streptococci [17] and time-saving. Thus, our use of the PCR in the species -specific analysis of the 16S rRNA gene was of great assistance in the present investigation and unequivocally demonstrated that all of them belonged to the Streptococcus species. To a certain degree, geographical discrepancies have been reported from South Africa [42], Japan [46] and Italy [47]. It is of interest to reveal that according to the available literature, L. garvieae although being rarely isolated from tilapia with streptococcosis, it was identified in two countries only, Brazil [19] and Indonesia [10]. This makes our detection of L. garvieae in our isolates, the third report. Interestingly, the isolation of Leuconostoc mesenteroides cremoris in our investigation was not reported before from tilapia.
Aquaculture is a developing field where antibiotics are intensely used, either specifically to the water or in fish sustenance, as prophylactics to control infections, which has brought about the blooming of AR genes in the aquatic environment [48]. The measure of AMR in aquaculture is under dispute as there is an absence of consent on a standard indicator organism. Ratably, the utilization of antimicrobials in aquaculture seems, by all accounts, to be of substantially more prominent greatness when compared with terrestrial animals. A shortage of solid ratable and subjective information on antimicrobial use (AMU) in different animal production systems over the area is a noteworthy hole in the exploration. Subjective and ratable accessible information (for the most part AMU recurrence of particular antimicrobials) from particular examinations recommends a high assorted variety of antimicrobials utilized both as development promoters (AGPs), and in addition for prophylactic and treatment purposes, despite the fact that outcomes are hard to look at crosswise over investigations. As a result of the extensive contrasts in underway frameworks, sampling and assay techniques, phenotypic evaluations of AMR pervasiveness in this audit should be deciphered with extraordinary care. Generally, the collection of samples are liable to have an expansive testing mistake. The antibiotics used in aquaculture represent different classes of antibiotics, some of which are classified as "Highest Priority Critically Important Antimicrobials" in medical treatment in the WHO list of  Critically important antimicrobials for human medicine (CIA list) such as fluoroquinolones, 3rd and 4th Generation cephalosporins, Macrolides and ketolides and Glycopeptides [49]. Fortunately, the isolated strains in the present investigation were not resistant to any of the CIA list with the exception of erythromycin. Globally, the use of antimicrobial agents is regulated differently from country to country, being either very strict or under-regulated. Possible hazards associated with drug abuse in fish farming are the presence of residues in food and the development of antibiotic resistance in the bacterial population.
In agreement, to our surprise numerous multi-ABR-bacteria (49%) were observed among our isolates, irrespective of their traditional resistance to beta lactam antibiotics [50,51]. It was theorized that, resistance to these antibiotics could be a result of their overuse or/and misuse in the aquaculture industry, could arise from gene mutations or by acquisition of transferable genetic elements such as integrons [50,52]. The property of horizontal spread of resistance genes was incriminated to be responsible for the recorded levels of multi-resistance. Notwithstanding AMU in aquaculture, the basic routine with regards to releasing fertilizer from earthbound creatures into water frameworks leaves the aquatic environments especially powerless against the improvement of AMR.

Conclusion
Worldwide death of fish caused by streptococcal infection remains an extortionate issue for growers of Nile tilapia and other warmwater fish, yet can be handled effectively with a cohesive, supportable scheme incriminating vaccines and new-generation antibiotics developed specifically for aquaculture. Strains of Streptococcus associated with infection in humans can cause people handling affected whole raw fish, primarily tilapia, to become at risk of developing neonatal meningitis and sepsis (early-onset and late-onset disease), cellulitis, subdural empyema, endocarditis or arthritis following a puncture wound [53][54][55]. Substantially, our results highlight the necessity to encourage conscientious fish producers, efficient husbandry performances and judicious practise of antibiotics in aquaculture. It should be emphasized that, S. dysgalactiae has emerged as a causative agent in fish disease, with a highly medical significance in mammalian and human health as alpha-hemolytic Lancefield group C S. dysgalactiae of fish origin, was implicated in ascending upper limb cellulitis in humans [56].