Aquaculture represents an important sector in the Egyptian national income structure . However, this sector is challenged by a wide range of opportunistic pathogens that result in high mortalities and considerable economic losses. Bacterial pathogens are among these pathogens that are naturally present in the fish environment, and under some stressful conditions, they become the pathogens of the most important diseases in aquaculture . Among others, Psychrobacter spp. could be considered as a potential bacterial pathogen in fish that results in high mortalities and considerable economic losses [15, 16]. Clearly, providing updates and baseline information about the occurrence of Psychrobacter spp. infection among marine fishes, together with investigation of the pathogen characteristics, seems crucial for implementation of the appropriate measures to prevent and control the infection. Revising the available literature, no previous studies revealed the occurrence of the novel pathogenic strain of P. glacincola infection in fish either at a national or international level. However, several previous studies identified P. glacincola as a novel strain from sea ice cores in Antarctica , mud of the Shetland Islands , processed fresh edible sea urchin in Tokyo , and red tanner crab . In addition, other previous studies [23, 36] registered P. glacincola in NCBI from marine environment and sea urchin. Given the above information, the present work provides a novel contribution in relation to the occurrence of a novel pathogenic strain of P. glacincola in wild marine fishes in Egypt through the isolation, identification, and characterization of the bacterium by bacteriological, biochemical, and molecular methods.
In the present work, the bacteriological examination of the examined fish revealed that the overall prevalence of P. glacincola infection among examined fish was 6.7%, while the individual prevalence rates were 16.7%, 10%, and 13.3% among Lutjanus ehrenbergii, Rhabdosargus haffara, and Cheilinus lunulatus fish, respectively. On the other hand, P. glacincola could not be detected among Lethrinus borbonicus, Siganus rivulatus and Scarus ghobban. Taken into consideration, no previous studies reported P. glacincola infection among fishes all over the world, and the possible explanation for this prevalence may be attributed to the difference in susceptibility of fish species to the infection [37, 38]. In accordance with their clinical impact, the infected species showed several clinical signs and PM lesions as a result of P. glacincola infection. In this regard, Blackspot snapper (Lutjanus ehrenbergii), Haffara seabream (Rhabdosargus haffara) and Snubnose emperor (Lethrinus borbonicus) fishes of the Red Sea at Hurghada city showed lethargy and sluggish movement, haemorrhages and ulcers on the body and operculum, scale loss, fin congestion, and rot, especially tail fins, combined with congestion of the liver, spleen, and kidney.
It is noteworthy to state that there are no available literatures reported the clinical signs of P. glacincola infection, but our results were agreed to some extent with that of Hisar et al., (2002), who found skin darkness, gills paleness and abnormal swimming, internal organ congestion in rainbow trout infected with other Psychrobacter spp. The P. glacincola infected fishes showed frayed fins and fin rot that adversely affected the swimming activities and foraging behaviour of the diseased fish, leading to loss of condition and weakness [39–41]. The diffused haemorrhages on the fish body could be attributable to the secretion of some enzymes such as elastase enzyme and hemolysin that damage the blood vessels, leading to blood leakage . Also, the clinical signs and PM lesions of the diseased fishes may be attributed to the extracellular products of Psychrobacter spp. such as proteases and hyaluronidase, that are involved in the development of clinical pathology and lesions [43, 44].
Isolation and identification of the causative agents remain one of the main lines for infection control . Phenotyping is commonly used in combination with genotyping to identify and characterize bacterial pathogens [45–47]. Likewise, biochemical characterization has been proved to be a valuable method for the typing and differentiation of several bacterial fish pathogens [48–50]. In this study, the phenotyping of the recovered isolates showed that the morphological characteristics of the colonies were cream-colored, un-pigmented, smooth and opaque with a buttery consistency. In addition, colonies formed yellowish colonies on MacConkey agar with no hemolysis on blood agar. Biochemically, the isolates were homogeneous and positive for cytochrome oxidase, catalase, and citrate utilization, while negative for lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, indole production, H2S production, tryptophane deaminase, gelatin liquefaction test, and acid from all sugars. Our findings are in accordance with the results reported by Bowman et al. (1997)  and Garcia-Lopez et al. (2014) , who reported similar bio-chemical reactions with P. glacincola. Taken into account, the variation in any biochemical characteristic may be attributed to presence or absence of plasmid(s) or mobile genetic elements that controls its metabolic traits .
In accordance with the molecular methods, the phylogenetic analysis based on 16S rRNA gene sequence is an important tool, which confirmed the genetic relatedness and stands alongside the biochemical tests and bacteriological tests for accurate and quick identification of bacteria [53–55]. The 16S rRNA gene sequence alongside the biochemical tests provide accurate and rapid identification for the bacterial pathogen [4, 54, 56] and the phylogenetic analysis of 16S rRNA gene allows and confirms the identification of unknown bacterial isolates . In this study, the phylogenetic analysis identified the recovered strain as P. glacincola (MRB62) based on 16S rRNA gene sequence. Comparing the 16S rRNA gene sequence of the present strain (P. glacincola MR B62) with known 16S rRNA gene sequences of Psychrobacter spp. on GenBank databases revealed closed similarity of 100% with P. glacincola T (Accession No. AB334769.1) , and the draft genome sequence of this strain was deposited into NCBI and assigned accession number MZ413384.1.
Based on those results, the present study reports for the first time the occurrence of P. glacincola infection among Snubnose emperor (Lethrinus borbonicus), Haffara seabream (Rhabdosargus haffara) and Broomtail wrasse (Cheilinus lunulatus) marine fishes of the Red Sea at Hurghada city, Egypt. Furthermore, the recovered strain (P. glacincola MR B62) of the present study revealed an identity of 99.71%, 99.64%, 99.43%, and 99.07% with that of P. glacincola LMG 21274T (Accession No. AJ 430830.1) , P. glacincola DSM 12194T (Accession No. NR 042076.1) , P. adeliensis DSM 15333T (Accession No. 117634.1) , and P. immobilis NBRCT 15733 (Accession No. AJ NR113805.1) , respectively. The present findings confirm the hypothesis that bacteria with an identity of more than 98.7% in the 16S rRNA gene sequence are considered to be the same species .
In accordance with the results of the pathogenicity test, the pathogen was isolated and identified from the experimentally challenged Haffara seabream (Rhabdosargus haffara) fish to fulfil Koch's postulates. The present study proved that the present P. glacincola isolate was pathogenic to Haffara seabream. In this concern, the challenged fish showed 23.3% mortality rates and exhibited clinical signs similar to those of the naturally infected fishes that included skin hemorrhages, scale loss, tail fin rot and congestion, and liver congestion. The recorded clinical signs may be attributed to the extracellular products such as cytotoxins, hemolysin, protease, collagenase, and hyaluronidase that were released during the infection [61, 62]. The present study also showed that P. glacincola isolates were sensitive to Amikacin, Streptomycin Ciprofloxacin, Gentamycin, Chloramphenicol, Tobramycin and Ofloxacin and resist to Tetracycline, Cephalothin, Cefotaxime, Erythromycin, Oxolonic acid, Trimethoprim/Sulphamethoxazole, Clindamycin, Flucloxacillin and Amoxicillin/Clavulanic acid. Some of our results agreed with a previous study , where P. glacincola was sensitive to streptomycin and gentamycin and resist tetracycline and ampicillin. Generally, the high variations in the antibiotic sensitivity test results may be due to the dramatic anti-microbial resistance growth and the bacterial isolate variations.