Biochemical and Molecular Identification of the Most Common Streptococci Affecting Common pandora ( Pagellus erythrinus Linnaeus, 1758 ) From the Mediterranean Coast of Tripoli

Streptococcosis is one of the septicaemic bacterial diseases of public health concern due to its zoonotic potential. Its luxurious existence in wild marine fish from an open water body strongly suggests presence of massive sewage


INTRODUCTION
The direct dumping of sewage into the seashores of several Mediterranean countries has a tremendous negative impact on the aquatic fauna and flora of The Mediterranean Sea (Abdallah and Abdallah, 2008).The direct discharge of chemical wastes; oil refineries, agrochemicals, car industries, shipping run-offs, and ballistic water; has been accused of being the major immunosuppressive factor in aquatic vertebrates and invertebrates (Claudet and Fraschetti, 2010).These adverse effects on the integrity of the immune barriers in aquatic animals have facilitated the direct biological invasion with an enormous number of bacterial, viral, mycotic, and parasitic invaders (Shahidul Islam and Tanaka, 2004).
Streptococcal infections in fish can result in significant mortality rates (more than 50%) within 3 to 7 days (Yanong and Francis-Floyd, 2002).On the other hand, Some outbreaks are more chronic, with mortalities lasting several weeks and only a few fish dying each day (Austin and Austin, 2016).A typical history indicating that Streptococcus is the cause of the disease in a group of fish, may include reports of abnormal swimming behavior, commonly described as spinning (Yanong and Francis-Floyd, 2002).
Streptococcus iniae is a threatening fish pathogen that induced invasive diseases and massive outbreaks in fish farms (Weinstein et al., 1997).Streptococcal infections have been linked to a considerable morbidity and mortality in marine mammals.Several streptococcal species have been isolated, including Streptococcus iniae from captive Amazon river dolphins (Inia geoffrensis) (Bonar et al., 2003), Streptococcus equi subsp.zooepidemicus from harbor seals (Phoca vitulina), Grey seals (Haliochoerus grypus) (Akineden et al., 2007).
The haem-agglutination activity of S. dysgalactiae may be attributed to its fimbria-like structures, but in the case of L. garvieae, it's unclear which function these structures serve (Abdelsalam et al., 2009).S. dysgalactiae showed high adherence to the EPCs, while L. garvieae strains didn't satisfactorily adhere to the EPCs.streptolysin S (SLS); a poreforming secreted cytotoxin; that has been shown to be a virulence factor in the human pathogen S. pyogenes, has a homolog in S. iniae (Locke et al., 2007).
Several candidates, such as surface proteins, capsular polysaccharides, and extracellular secreted products, have been identified by recent molecular studies into factors that contribute to the virulence of S. iniae.Furthermore, the recently completed sequencing of S. iniae's genome will expedite the identification of new virulence factors and serve as a guide for the development of effective vaccine targets for farmed fish, thereby mitigating the risk of zoonotic infection (Baiano and Barnes, 2009).
The bacterial resistance from different ecosystems is rising at an alarming rate and is becoming a major global concern.The prevalence of resistant bacteria in the environment is determined by the bacteria's exposure to various antibiotics, which results in the development of certain strains of resistant bacteria to specific antibiotics (Al-Bahry et al., 2009).The discovery of resistant Enterococci in The Mediterranean aquaculture site shows the existence of a marine reservoir of antibiotic resistances that could be transmitted to virulent strains affected by mariculture in an antibiotic-independent way (Di Cesare et al., 2012).Several reports of antibioticresistant Streptococci have been reported among marine fishes exposed to long-acting sewage pollution (Rani et al., 2013;Sherif et al., 2020).
One of the biggest issues facing aquaculture is the emergence of antimicrobial resistance (AMR) in farmed fish.Due to the high frequency of bacterial infections in fish, antibiotics are often used, which prolongs their survival in the aquatic environment and promotes the growth of the antibiotics' resistant bacteria (Preena et al., 2020) The rise in antibiotic resistance among bacterial pathogens is a significant challenge in human medicine.Researchers studying antibiotic resistance in aquaculture have produced findings that could shed light on how pathogens found in hospitals and other clinical settings pick up and modify genetic structures necessary for their survival when subjected to antibiotic therapy (Sørum, 2005).Resistance genes are most likely regularly transported into hospitals and other comparable settings by human clinical pathogens.The 51-kb R plasmid pTP10 from the multiresistant clinical isolate Corynebacterium striatum M82B could serve as an example to illustrate such scenario.DNA fragments originally discovered in soil bacteria as well as in pathogens of plants, animals, fish (P.damselae subsp.piscicida), and humans make up this R plasmid (Kim et al., 2006).In bacteria from terrestrial, animal, and human bacterial floras and environments, R plasmids of fish pathogens and aquatic bacteria, like the IncU plasmid, have been found (Sørum, 2005;Kim et al., 2006).
The current study was performed to identify and characterize most common streptococci affecting common pandora (Pagellus erythrinus) inhabiting the Mediterranean coast of Tripoli.

Fish sampling
A total of 270 live common pandora (Pagellus erythrinus) were captured by fishermen through autumn, summer and spring seasons from the coastal areas between Tajoura (east to Tripoli) to Janzour (west to Tripoli).Live fish were anaesthetized in an anaesthetic bath containing a sedative dose of MS-222 (AL-Taee et al., 2021) (Sigma Aldrich Co., St. Louis, MO, USA) before sample collection and clinical examination to minimize the possible handling stress Samples were divided equally over three seasons (summer, autumn, and spring) (90 fish / season).The captured fish' average weights and lengths were 250±15 g and 20±5 cm respectively.
It is worthy to mention that winter season was void of samples due to bad climatic conditions including wind-driven sea waves and storms through the western Libyan Mediterranean coast.Euthanized fish were kept on crushed ice in an insulated icebox and sent to the Poultry and Fish Diseases laboratory, Faculty of Veterinary Medicine, University of Tripoli 2-4 hours after collection at most.

Clinical examination
To remove external contaminants, the fish were swapped with 70% ethanol.Using the three lines technique, fish was cut open from the left side to expose the liver and other internal organs (Eissa, 2016).Before sampling, fish were checked externally and internally for any apparent gross findings.Any observed clinical findings were directly photographed with a digital camera.

Bacteriological examination
Following clinical examination, loopfuls from kidneys and brains were streaked onto blood agar (BA) (CM0055, Oxoid, UK) supplemented with 2 % NaCl, and KF Streptococcus agar base (SAB) plates ( BO0914T,Fisher-Scientific, Carlsbad , USA) with triphenyl tetrazolium chloride (TTC) supplement.The inoculated plates were then incubated at 25-37°C for 24-72 hrs.The bacteriological isolation protocol was adopted from Eissa et al. (2021).The colonial characteristics were compared to the standard criteria published by Austin and Austin (2016).

Biochemical identification
The recovered isolates were biochemically identified following identification scheme descried by Austin and Austin (2016) morpho-chemical criteria for Streptococcus / Enterococcus species.The retrieved isolates were presumptively characterized using conventional biochemical tests (Gram reaction; oxidase; catalase; hemolysis pattern on blood agar; salt tolerance (2%, 3%, and 6.5%); culture at 25°C, and 37°C; growth on bile esculin agar; carpenicillin sensitivity), which were further confirmed using the commercial miniaturized API 20 Strep system (Biomérieux, France) according to the manufacturer's instructions.

Antibiogram
The antibiotic susceptibility of the retrieved bacterial isolates was evaluated using the Kirby Bauer disc diffusion method described by Bauer (1966), and NCCLS (1999).Mueller-Hinton agar (MHA) (Oxoid, Hampshire, UK) supplemented with 2% (w/v) sodium chloride was used for in vitro antimicrobial susceptibility testing , which was incubated at 25°C and 37°C for 24-48 hrs.Antibiotic inhibition zones were measured in mm using a measuring caliber at the end of the incubation time.

Sequencing of 16S rRNA
The extraction of bacterial DNA was performed using the PrepMan® Ultra Sample Preparation Reagent protocol (Applied Biosystems, USA).The PCR amplification of the 16S rRNA gene was performed using the universal pair primers (F-5′-AGAGTTTGATCCTGGCTCAG-3′) and (R-5′-AAGGAGGTGATCCAGCC-3′); described by Gross et al. (2012).PCR reactions was done according to the protocol described by Eissa et al., (2020).PCR products were purified using GeneJET™ Gel extraction kit (Thermo Scientific, Waltham, USA).The sequencing process of the 16S rRNA genes of four bacterial strains was performed in two directions using ABI 3730XL DNA sequencer at Macrogen sequencing company (Macrogen, Seol, South Korea).The raw sequences were edited and assembled using Bio Edit version 7.0 (Hall, 2004).The identity of bacterial isolates was confirmed by aligning the assembled sequences with other interrelated isolates deposited in the database of GenBank.The neighbor-joining phylogenetic tree was created using MEGA version X, and the evolutionary distance calibrated by the Kimura2-parameter method with 1000 replicates of bootstrap value was applied (Kumar et al., 2016).

Prevalence of infection among sampled common pandora
The prevalence of infection among different P. erythrinus collected through different sampling intervals were estimated by calculating the number of infected fishes divided by the total number of examined fish multiplied by 100.

Ethical declaration
The current study was ethically carried in compliance with University of Tripoli Animal Care Committee and following regulations for the care of animals in research.All the examined fishes were anaesthetized in an anaesthetic bath containing a sedative dose of MS-222 (Sigma Aldrich Co., St. Louis, MO, USA) before the clinical examination and the sample collection to minimize the possible handling stress (AL-Taee et al., 2021).Fish euthanasia for sample collection was done according to the most recent guidelines of AVMA (2020).

RESULTS 1. Clinical examination
There were no apparent pathognomonic signs, and most of the examined fish were apparent healthy and close to normal.However, some fish showed fin congestion and erosions (Fig. 1).Internally, the clinical examination showed moderate hemorrhage in the kidney, stomach, and swim bladder, as well as congestion with moderate hemorrhage in brains of the examined fish (Fig. 2).

Biochemical identification
The results of biochemical examination using the conventional biochemical test as well as the miniaturized API 20 Strep test strips have revealed that the retrieved Streptococci were categorized into 4 primary isolates S. iniae, S. dysgalactiae, S. phocae, and Enterococcus faecalis (Table 2).
Fig. 3: The neighbor-joining phylogenetic tree showed the comparative analysis of the 16S rRNA gene sequence of S. dysgalactiae, S. iniae, S. phocae, E. faecalis, and other interrelated bacterial strains on GenBank.

DISCUSSION
Streptococcus. iniae has not yet been placed to a Lancefield group and is β-hemolytic on blood agar, with some clinical strains reported from Asia being more mucoid than others (Lau et al., 2006).Furthermore, due to their abundance in faeces and lengthy survival in the environment, Enterococcus and Streptococcus species have been considered as biomarkers of faecal pollution in water.Despite the fact that the ratio of faecal coliforms to faecal Streptococci has been ruled out as an indicator (Rivera et al., 2010), species identification associated with a specific environment or host may provide additional information about the origin of isolates and the source of faecal contamination (Gerba, 2009).
This study proposes a method for quickly identifying clinical and environmental Enterococcus species.That was determined by 12 biochemical tests.With some exceptions, the identification limit was more than 99 % (87, 91.5, and 97 % for E. avium, E. hirae, and E. durans, respectively).The use of sucrose fermentation to discriminate E. avium from E. raffinosus reduced the identification threshold in the key.2016).Interestingly, the prevalence of E. faecalis in the sampled P. erythrinus during spring (6.66 %), and summer (7.77%) was less than that achieved in autumn (8.88 %).These results apparently contradicted the achieved results in several epidemiological studies where summer and spring were commonly the highest seasons in E. faecalis prevalence (Blanch et al., 2003;Austin and Austin, 2016).
In the current study, the achieved morphochemical criteria for S. iniae suspect isolates were consistently accordant with the standard criteria of ATCC S. iniae isolates and many other S. iniae confirmed isolates described in several fish microbiological literatures edited by Buller (2014).The prevalence of the retrieved S. iniae isolates was the highest in summer (13.33 %), followed by spring (10%), and autumn of the same year, which represented the lowest prevalence (7.7%).Summer season allows an enriched media for the establishment, growth, and spread of parasites which are famous vectors for transmitting bacterial fish pathogens, including S. iniae.Moreover, summer is the peak of the breeding cycle of the P. erythrinus, where they exist in larger numbers in the coastal areas of Tripoli with a consequent increase in the number of exposed fish to biological pollutants sheded from parasitic vectors heavily attached to them, or from the continuously dumped sewage.However, the higher temperature is a detrimental factor in the dynamics and water chemistry of the sea, whereas levels of oxygen are highly declined in hot water together with a gradual increase of ammonia.Together with the external parasites' breakdown, these deteriorated water chemistries would have triggered remarkable immunosuppression in the cohabitating fishes with an ultimate heavy invasion with S. iniae and other seasonal bacteria such as Photobacteriaum damsalae subspecies damselae, which have been concurrently isolated from the sampled P. erythrinus during summer, 2019.These assumptions coincided with a similar epidemiological survey of the most prevalent pathogens in gilthead seabream and sea bass from The Mediterranean coasts of Egypt (Moustafa et al., 2014;Moustafa et al., 2015).
ß-haemolytic S. phocae was isolated in seals and has been linked to Atlantic salmon disease in Chile since 1999; Chilean isolates are genetically, serologically, and phenotypically homogeneous (Valdés et al., 2009).Our retrieved isolates were Gram-positive cocci arranged in chains and hemolytic on blood agar supplemented with 5% sheep RBCs in the ongoing study.The isolates were biochemically capable of generating acid from D-Fructose, Maltose, N-Acetylglucosamine, and Ribose.Isolates were positive for alkaline phosphatase production.No growth was obtained on 40 % bile, 10°C, and 45°C.The isolates were susceptible to Bacitracin.The above-mentioned results coincided with the standard morpho-chemical criteria of S. phocae (Romalde et al., 2008).The highest prevalence of S. phocae was reported in autumn (11 %), while that in summer, and spring were equal (5.5 %).This result is not epidemiologically understandable except if the harbor host (seals, otters, dolphins) are gathered after their migration during the autumn season at closer distances to areas where P. erythrinus was collected than in summer or spring.Also, in autumn, P. erythrinus are scanty in number in the deep zones of The Mediterranean Sea, where they travel to warmer coastal zones for food and stabilization.This would have triggered seals to travel to more coastal zones where their prey fish are available.Consequently, the exposure time between S. phocae harbor hosts (seals) would have increased with the ultimate increase in prey fish infection or at least carrying the pathogen on their mucous or skin.This assumption is relatively consistent with the epidemiological patterns of S. phocae in seals reported by Skaar et al. (1994).
Streptococcus dysgalactiae is the only pyogenic Streptococcus that is not β-hemolytic (Igwe et al., 2003).The causative agent was identified as the α-hemolytic Lancefield group C Streptococcus dysgalactiae subsp.dysgalactiae [GCSD].The syndrome caused by GCSD is characterized by a systemic multifocal inflammatory reaction, microabscessation, acute septicemia, and significant mortality rates in fishes with pathognomonic necrotic ulcers at the caudal peduncle area (Abdelsalam et al., 2010b;Sherif et al., 2020).GCSD has been identified as the primary cause of various mammalian diseases, including streptococcal mastitis/endometritis in domestic mammals, as well as skin lesions, meningitis, and bacteremia in humans (Koh et al., 2009).In the current study, our isolates were α hemolytic on blood agar supplemented with 5 % sheep RBCs which have turned into β-hemolytic after a long time.Also, there were no growths at 10, 45⁰C or pH 9.6.All suspect isolates were able to grow in the presence of 40 % bile salts.The isolates were negative for V.P., bile esculin, sodium hippurate hydrolysis, pyridinyl amidase, and α or βgalactosidase (Yang and Li, 2009).They were cable of producing acid from Trehalose and amygdalin.
The above-mentioned results were accurately consistent with the standard morpho-chemical criteria of S. dysgalactiae reported by Yang and Li (2009).The achieved prevalence of the retrieved S. dysgalactiae was the highest in summer (8.88 %), followed by spring (7.77 %), then autumn (6.6 %).These prevalence are consistent with similar results obtained by Abdelsalam et al., (2010a), and Buller, (2014).Those studies have confirmed that the highest prevalence of S. dysgalactiae is remarkably high during the summer season and, to a small extent, the late spring.Further, the deteriorated water quality during the summer season, as previously described, would have resulted in favoring the growth of S. dysgalactiae, and E. faecalis more than S. iniae, and S. phocae.Since the beginning of this century, Enterococci, formerly known as faecal Streptococci, have been recognized as being of faecal origin.
Enterococcus species typically live in the intestines of humans and other animals.Enterococci.On the other hand, they are ubiquitous and can be found free-living in soil, on plants, or in dairy products (Fortina et al., 2004;Naser et al., 2005;Gerba, 2009).Enterococci are part of the normal intestinal microbiota of humans and animals, and they are regarded as indicators of fecal contamination of recreational water.However, they can also be isolated from natural ecosystems that haven't been contaminated by faecal material (Wright et al., 2009).Depending on the disease type, infections are treated with a course of antimicrobial drugs such as penicillin, ampicillin, amoxicillin, cloxacillin, cefazolin, and/or gentamicin, doxycycline, and trimethoprim/sulfamethoxazole for 1 to several weeks (Koh et al., 2004;Koh et al., 2009).This could explain the type of antibiotic resistances recorded in this study.

CONCLUSION
The uprising dangers of sewage pollution through the southern Mediterranean coasts were confirmed to have very deleterious effects on living aquatic animals including fish, shellfish and marine mammals occupying these coasts.The luxurious isolation of pathogenic of pathogenic Streptococci originating from marine mammal (S. phocae, S.iniae), human / animal (S. dysgalactiae), and sewage (E.feacalis) explains the magnitude of sewage pollution through the Southern Mediterranean coasts.Moreover, the reported bacterial resistance against wide-spectrum of commercial antibiotics is an alarming indicator about the potentials/magnitudes of Multidrug resistances expected to occur among consumers of marines fishes caught from these polluted Mediterranean coasts.Ultimately, the current study highlighted the urgent need for large scale regional study to determine the actual potentials of Multidrug resistant bacteria and their public health impacts on inhabitants of The North African Mediterranean coasts.
Enterococcus faecalis isolates were morphochemical confirmed based upon consistency with standard criteria published by Blanch et al. (2003), and Austin and Austin, (

Table 2 :
Phenotypic and biochemical characteristics of the retrieved Streptococci.