Introduction

An important source of income in many developing countries is marine fish, besides being one of the major investment choices for national fishermen [1]. Egypt is bordered by the Red Sea on the east, which is 2250 km in length with an average depth of 490 m. Importantly, the Red Sea has a unique composition of fish species, which consists of 1166 species. Some of the most economically important Red Sea marine fish species in Egypt include marbled spinefoot (Siganus rivulatus), blackspot snapper (Lutjanus ehrenbergii), snubnose emperor (Lethrinus borbonicus), blue-barred parrotfish (Scarus ghobban), Haffara seabream (Rhabdosargus haffara), and broomtail wrasse (Cheilinus lunulatus). However, this important sector is challenged by a wide range of serious pathogenic organisms, affecting diverse marine fishes and shellfish [2]. Among others, bacteria are the most prevalent cause of morbidity and mortality among wild populations of fish, resulting in major economic losses in this sector [3]. Generally, several bacterial strains are normally present in aquatic environments and their simple presence in marine environments is insufficient to cause a disease outbreak; however, they might become highly pathogenic under stressful conditions [4].

Among others, Psychrobacter species have been isolated from various marine environments and seawater, which is considered a good habitat for these species [5]. The presence of Psychrobacter spp. was also significantly associated with other environmental factors, that is, temperature, neutral pH, high salinity, higher concentrations of potassium and magnesium [6], and hydrocarbon-contaminated aquatic environments [7, 8]. Furthermore, members of the genus Psychrobacter have been isolated from the gastrointestinal tract, skin, and gills of apparently healthy Atlantic salmon [9], Atlantic cod [10], juvenile grouper [11], and Atlantic mackerel [12]. To enhance the growth rate and immune system efficiency in fish, Psychrobacter spp. have been used as probiotics [13] since they induce growth inhibition of some pathogenic bacteria and enhance the growth of many beneficial or neutral bacteria in the gut of fish [14]. For some fish species, some Psychrobacter strains were recorded as opportunistic pathogenic microorganisms. Some previous studies [15, 16] reported P. immobilis infection in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon. Psychrobacter isolates such as P. immobilis and P. phenylpyruvicus have been isolated in clinical samples from brain tissue, urine, ears, wounds, cerebrospinal fluid, and blood and have been reported to be opportunistic pathogens in humans [17,18,19,20,21]. Another species named P. glacincola was isolated as a novel strain from sea ice cores in Antarctica [22], mud of the Shetland Islands [23], processed fresh edible sea urchin in Tokyo [24], red tanner crab [16], water of aquaculture and agriculture run off of Ria de Aveiro [25], and sediment samples of King George Island, Antarctica [26].

Revising the available literature, the prevalence of bacterial diseases was documented in several cultured and wild freshwater fish species from Egypt. However, only a few bacteriological surveys were performed on marine fish species to track disease outbreaks. More importantly, no available data was reported regarding the P. glacincola infection among fishes worldwide. Given the above information, the present study investigated the occurrence of P. glacincola among some fish species inhibited by the Red Sea in Hurghada, Egypt, through clinical examination, bacteriological isolation, phenotyping, and biochemical and molecular identification of the isolated strains. Combined with antimicrobial susceptibility testing of the recovered P. glacincola isolates, the study also included a pathogenicity test for pathogenicity verification of the isolated strains.

Material and methods

Study area and sampling

From October 2019 to March 2020, a total of 180 fish were obtained from Hurghada, Red Sea governorate, Egypt. The examined fish species were as follows: snubnose emperor (Lethrinus borbonicus), blackspot snapper (Lutjanus ehrenbergii), marbled spinefoot (Siganus rivulatus), Haffara seabream (Rhabdosargus haffara), blue-barred parrotfish (Scarus ghobban), and broomtail wrasse (Cheilinus lunulatus). The rate of sampling was 30 fish from each species. Fish were collected and transported immediately to the indoor aquarium at the National Institute of Oceanography and Fisheries in Hurghada, Egypt, for clinical and bacteriological examinations. We confirm that all experimental methods were performed in accordance with the relevant guidelines and regulations of the National Institute of Oceanography and Fisheries Committee for ethical care of marine organisms and experimental animals (NIOF-AICUC).

Clinical and postmortem examination

Clinical and postmortem examinations of fish were conducted to detect external and internal clinical abnormalities according to the method described by Schäperclaus [27]. Fishes were anesthetized with tricaine methanesulfonate (MS222-Sigma-Aldrich) prior to examination.

Bacterial isolation

Under completely aseptic conditions, bacteriological samples from the liver, kidney, and spleen were collected by sterile loop. The collected samples were then inoculated into brain heart infusion Broth supplemented with 1.5% NaCl (Oxoid, England) and incubated aerobically at 22 °C for up to 48 h, then streaked onto tryptone soy agar (Oxoid, England) supplemented with 1.5% NaCl and incubated at 22 °C for up to 48 h [28]. The recovered isolates were then preserved at − 80 °C in tryptone soy broth supplemented with 25% glycerol for further identification.

Phenotyping and biochemical identification of the isolated bacteria

The suspected isolates were identified through their morphological characteristics, Gram staining, motility test, oxidase test, and API 20E system (BioMerieux, France) according to the manufacturer’s instructions.

Molecular identification of the recovered isolates and sequence analysis

Using the GeneJET genomic DNA purification kit (Thermo Fisher Scientific, EU), the bacterial DNA was extracted from the recovered isolates according to the manufacturer’s instructions. The extracted DNA was then stored at − 20 °C until use. Later, to amplify the hypervariable segment of 16S rRNA using a set of universal primers, polymerase chain reaction (PCR) was conducted [29], which is shown in Table 1. PCR was conducted in 50-μL mixtures, according to the Master Mix manufacturer’s instructions, which contained 25-μL of Master Mix, 2 μL of each primer, 4 μL of the extracted DNA, and nuclease-free water up to 50 μL. As shown in Table 1, PCR was conducted in a thermocycler (Applied Biosystems, USA) under the conditions previously described [30]. In brief, the initial denaturation was performed at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1.5 min, followed by a final extension step at 72 °C for 10 min. The amplicons (1500 base pairs) were purified and sequenced by the 3500 Genetic Analyzer (Applied Biosystems, USA). The draft genome sequence of strain MR-B62 was sequenced at SolGent Co., Ltd. Bio-Industry development site (South Korea) using Sanger dideoxy sequencing technology. The sequences of the recovered isolates were analyzed using MEGA 7.0 software and compared to those available in the GenBank database. Using the maximum composite likelihood method, evolutionary distances were computed. A phylogenetic tree based on the 16S rRNA gene sequences was reconstructed by the neighbor-joining method [31].

Table 1 Primer sets used in molecular characterization of P. glacincola isolates
Full size table

Pathogenicity testing

A total of 40 acclimated healthy Haffara seabream (Rhabdosargus haffara) fish with an average body weight 50 ± 5 g were obtained from the National Institute of Oceanography and Fisheries, Hurghada, Egypt. These fish were experimentally infected with the bacterial suspension (P. glacincola) recovered from naturally infected marine fish. Fish were divided into four equal groups; three groups were intraperitoneally (IP) injected with P. glacincola suspension at a dose of 0.1 mL (3 × 107 CFU) [32], and the fish of the 4th group were IP injected with 0.1 mL of sterile saline and used as a control. Fish were closely observed daily for 2 weeks, and the clinical signs and mortalities were recorded. Freshly dead fish were subjected to postmortem examination, bacteriological isolation, and identification of P. glacincola from the liver, spleen, and kidney.

Antimicrobial susceptibility test of the recovered P. glacincola isolates

Using the Kirby Bauer disc diffusion method, the antimicrobial susceptibility of the recovered isolates was determined. The following antibiotic discs were used: tetracycline (30 µg), ciprofloxacin (5 µg), ofloxacin (5 µg), oxolinic acid (2 µg), erythromycin (15 µg), chloramphenicol (30 µg), amoxicillin/clavulanic acid (30 µg), cephalothin (30 µg), amikacin (30 µg), streptomycin (10 µg), cefotaxime (30 µg), trimethoprim/sulfamethoxazole (25 µg), gentamycin (10 µg), clindamycin (2 µg), flucloxacillin (5 µg), and tobramycin (5 µg). The recovered isolates were streaked into Mueller–Hinton agar (Oxoid, England), the antibiotic discs were placed, and the inoculated plate was incubated at 25 °C for 48 h. Diameters of the inhibition zones were measured and interpreted according to the Clinical and Laboratory Standards Institute (2012) (33).

Statistical analysis

The Kaplan–Meier curve was used to calculate the infected and control trials' survival rates over time. The number of fish that survived is divided by the number of dead samples.

Results

Clinical signs of P. glacincola infection

The infected marine fishes showed a series of clinical signs that included lethargy and sluggish movement, hemorrhages and ulcers on the body and on the operculum, occasional scale loss, and fin congestion and rot, especially on the tail fin (Fig. 1A and B). Furthermore, congestion of the liver, spleen, and kidney were observed during the postmortem examination (Fig. 1C).

Fig. 1
figure 1

Clinical signs of P. glacincola infection of some red sea fishes: A Rhabdosargus haffara hemorrhagic body ulcer; B Cheilinus lunulatus hemorrhagic body ulcer; C Rhabdosargus haffara liver congestion

Full size image

Prevalence of P. glacincola infection

The clinical examination of 180 Red Sea fish revealed a total prevalence of 6.7% of P. glacincola infection among all examined fish. Furthermore, the prevalence rates were 16.7%, 10%, and 13.3% among blackspot snapper (Lutjanus ehrenbergii), Haffara seabream (Rhabdosargus haffara), and broomtail wrasse (Cheilinus lunulatus) fish, respectively (Table 2).

Table 2 Occurrence of P. glacincola among six marine fish species of the Red Sea
Full size table

Bacteriological identification and morphological characteristics of the colonies

The colonies of these isolates were diplococci, cream-colored to unpigmented, smooth, and opaque with a buttery consistency after incubation at 22 °C for up to 48 h on tryptone soy agar. These microorganisms were Gram-negative non-motile diplococci and did not result in hemolysis of the blood agar, while they formed yellowish colonies on the MacConkey agar. Colonies also were able to grow at 1.5%, 3%, 7%, and 10% NaCl and at 4 °C, 22 °C, and 37 °C (supplemental file).

Biochemical identification

As shown in Table 3, the 12 isolates were biochemically homogeneous and positive for cytochrome oxidase, catalase, and citrate utilization (CIT) and variable for urease (URE) and Voges Proskauer (VP) tests; all strains were negative for O-nitrophenyl-β-D-galactopyranoside (ONPG), arginine dihydrolase (ADH), lysine decarboxylase (LDC), ornithine decarboxylase (ODC), indole production (IND), H2S production (H2S), tryptophane deaminase (TDA), gelatin liquefaction tests, and acids from all sugars (glucose [GLU], mannitol [MAN], inositol [INO], sorbitol [SOR], rhamnose [RHA], sucrose [SUC], melibiose [MEL], amygdaline [AMY], and arabinose [ARA]).

Table 3 Biochemical characteristics of the recovered strain in the present study of P. glacincola
Full size table

Molecular identification

The nucleotide sequences of the 16S rRNA gene could detect the isolates of bacteria at the species level according to levels of homology compared with the GenBank database. The recovered strain was identified and named MRB62. The draft genome sequence of strain MRB62 was deposited in National Center for Biotechnology Information (NCBI) and assigned an accession number (MZ413384.1). Interestingly, high similarity of the 16S rRNA gene sequences of the MRB62 isolate to that of P. glacincola T (Accession No. AB334769.1) with 100% identity is a remarkable finding. Furthermore, a closed genetic relationship was detected for the recovered strain (the strain MRB62 of the genus Psychrobacter) in the present study with some other strains in GenBank. Thus, an identity of 99.71%, 99.64%, 99.43%, and 98.86% was reported with that of P. glacincola LMG 21274 T (Accession No. AJ 430,830.1), P. glacincola DSM 12194 T (Accession No. NR 042,076.1), P. adeliensis DSM 15333 T (Accession No. 117634.1), and P. immobilis NBRCT 15,733 (Accession No. AJ NR113805.1), respectively. The matched sequences from GenBank were aligned using CLC Sequence viewer7 for similarity analysis. The phylogenetic tree was constructed using the MEGA 7 software (Fig. 2).

Fig. 2
figure 2

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, showing the phylogenetic position of strain MRB63 and related members within the genus Psychrobacter, showing the evolutionary history. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches

Full size image

Pathogenicity test

The P. glacincola experimentally infected Haffara seabream (Rhabdosargus haffara) showed a 23.3% mortality and clinical signs similar to those recorded in the naturally infected fish (Fig. 3). These clinical signs included skin hemorrhages, scale loss, tail fin rot and congestion, and liver congestion (Fig. 4). P. glacincola was also isolated and identified from the internal organs of the experimentally infected fish. The bacterial pathogen was re-isolated from the kidneys of infected fish.

Fig. 3
figure 3

Kaplan–Meier survival curve of Haffara seabream (Rhabdosargus haffara) fish after challenged with P. glacincola AMP-FB1 isolate for 14 days; each fish was intraperitoneally injected with 0.1 mL of bacterial suspensions (3 × 107 CFU/mL)

Full size image
Fig. 4
figure 4

Clinical signs of Haffara seabream (Rhabdosargus haffara) experimentally challenge with P. glacincola, showing (A) scale loss and tail fin rot; B tail fin rot; C ulcer on the body; and (D) liver congestion

Full size image

Antibiotic sensitivity test

In accordance with the antibiotic sensitivity test, the recovered isolates of P. glacincola were sensitive to amikacin, streptomycin, ciprofloxacin, gentamycin, chloramphenicol, tobramycin, and ofloxacin and resistant to tetracycline, cephalothin, cefotaxime, erythromycin, oxolinic acid, trimethoprim/sulfamethoxazole, clindamycin, flucloxacillin, and amoxicillin/clavulanic acid.

Histopathological examinations

A photomicrograph of the posterior kidney sections from Rhabdosargus haffara fish experimentally infected with Psychrobacter glacincola (Fig. 5) shows congested vein, hyperplasia of the melanomacrophage centers, glomerular atrophy, necrosis and detachment of renal tubular epithelium, and interstitial mononuclear inflammatory cellular infiltration. Photomicrograph of liver sections from Rhabdosargus haffara fish experimentally infected with Psychrobacter glacincola shows hepatic tissue showing necrosis and dissociation from its cord arrangement, congested veins, and dilated portal vein engorged with blood (Fig. 6).

Fig. 5
figure 5

A photomicrograph of the posterior kidney sections from Rhabdosargus haffara fish experimentally infected with Psychrobacter glacincola shows congested vein, hyperplasia of the melanomacrophage centers, glomerular atrophy, necrosis and detachment of renal tubular epithelium, and interstitial mononuclear inflammatory cellular infiltration

Full size image
Fig. 6
figure 6

Photomicrograph of liver sections from Rhabdosargus haffara fish experimentally infected with Psychrobacter glacincola shows hepatic tissue showing necrosis and dissociation from its cord arrangement, congested veins, and dilated portal vein engorged with blood

Full size image

Discussion

In the Egyptian national income structure, aquaculture and fisheries represent an important sector [33]. However, this sector is challenged by a wide range of opportunistic pathogens that result in high mortalities and considerable economic losses. Bacterial pathogensare naturally present in the fish environment, and under some stressful conditions, they become the pathogens of the most important diseases in aquaculture [34]. 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]. Salinity levels in the Red Sea are higher than most saline water bodies in the world, which is between 36 and 38% [35]. P. glacincola is able to grow at up to 10% NaCl. Clearly, together with the investigation of the pathogen characteristics, providing updates and baseline information regarding the occurrence of Psychrobacter spp. infection among marine fishes is crucial for the implementation of the appropriate measures to prevent and control the infection. Reviewing the available literature, no previous study has reported elucidating the novel pathogenic strain of P. glacincola infection in fish either at a national or international level. However, P. glacincola as a novel strain was identified from sea ice cores in Antarctica [22], mud of the Shetland Islands [23], processed fresh edible sea urchin in Tokyo [24], and red tanner crab as reported by several previous studies [16]. Additionally, other previous studies [23, 36] registered P. glacincola in NCBI following isolation from marine environments and sea urchins. Through the isolation, identification, and characterization of the bacterium by bacteriological, biochemical, and molecular methods, 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.

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. Conversely, P. glacincola could not be detected among Lethrinus borbonicus, Siganus rivulatus, and Scarus ghobban. Reviewing the available literature, no previous study has reported for P. glacincola infection among fishes worldwide, 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 postmortem lesions as a result of P. glacincola infection. 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, hemorrhages and ulcers on the body and operculum, scale loss, fin congestion, and rot, especially in the tail fins, combined with congestion of the liver, spleen, and kidney.

It is noteworthy to state that there is no available literature that reported the clinical signs of P. glacincola infection. This study’s results corroborate to some extent with that of Hisar et al. [15], who found skin discoloration, gill paleness, abnormal swimming, and 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 behavior of the diseased fish, leading to loss of condition and weakness [39,40,41]. The diffuse hemorrhages on the fish body could be attributed to the secretion of some enzymes such as elastase and hemolysin that damage the blood vessels, leading to blood leakage [42]. Moreover, the clinical signs and postmortem lesions of the diseased fishes may be attributed to the extracellular products of Psychrobacter spp. such as proteases and hyaluronidase, which are involved in the development of clinical pathology and lesions [43, 44].

Isolation and identification of the causative agents remain one of the main interventions for infection control [45]. To identify and characterize bacterial pathogens, phenotyping is commonly used in combination with genotyping [45,46,47]. Likewise, biochemical characterization has been proven to be a valuable method for the typing and differentiation of several bacterial fish pathogens [48,49,50]. In this study, the phenotyping of the recovered isolates showed that the morphological characteristics of the colonies were cream-colored, unpigmented, smooth, and opaque with a buttery consistency. Additionally, yellowish colonies were formed 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) [22] and Garcia-Lopez et al. (2014) [51], who reported similar biochemical reactions with P. glacincola. Therefore, variations in any biochemical characteristics may be attributed to the presence or absence of plasmid(s) or mobile genetic elements that control its metabolic traits [52].

In accordance with the molecular methods, the phylogenetic analysis based on 16S rRNA gene sequence is an important tool, which confirms the genetic relatedness and stands alongside the biochemical tests and bacteriological tests for accurate and quick identification of bacteria [53,54,55]. The 16S rRNA gene sequence “alongside the biochemical tests” provides an accurate and rapid identification of the bacterial pathogen [4, 54, 56], and the phylogenetic analysis of 16S rRNA gene allows and confirms the identification of unknown bacterial isolates [57]. In this study, the phylogenetic analysis identified the recovered strain as P. glacincola (MRB62) based on the 16S rRNA gene sequence. Comparing the 16S rRNA gene sequence of the present strain (P. glacincola MR B62) with the known 16S rRNA gene sequences of Psychrobacter spp. on GenBank databases revealed a close similarity of 100% with P. glacincola T (Accession No. AB334769.1) [24], and the partial 16S ribosomal RNA gene sequence of this strain was deposited in NCBI and assigned accession number MZ413384.1.

Based on these results, this is the first study to report 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 21274 T (Accession No. AJ 430,830.1) [22], P. glacincola DSM 12194 T (Accession No. NR 042,076.1) [22], P. adeliensis DSM 15333 T (Accession No. 117634.1) [58], and P. immobilis NBRCT 15,733 (Accession No. AJ NR113805.1) [59], respectively. Bacteria with an identity of more than 98.7% in the 16S rRNA gene sequence are considered to be the same species which confirms the hypothesis of the present study [60].

The pathogen was isolated and identified from the experimentally challenged Haffara seabream (Rhabdosargus haffara) fish to fulfill Koch's postulates in accordance with the results of the pathogenicity test. The present study proved that the present P. glacincola isolate was pathogenic to Haffara seabream and 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 resistant to tetracycline, cephalothin, cefotaxime, erythromycin, oxolinic acid, trimethoprim/sulfamethoxazole, clindamycin, flucloxacillin, and amoxicillin/clavulanic acid. Some of our results concur with a previous study [5], where P. glacincola was sensitive to streptomycin and gentamycin and resistant to tetracycline and ampicillin. Generally, the high variations in the antibiotic sensitivity test results may be due to the dramatic antimicrobial resistance growth and the bacterial isolate variations.

Conclusions

Given the above information, the present study reported for the first time a novel pathogenic bacterial isolate named P. glacincola from naturally diseased Snubnose emperor (Lethrinus borbonicus), Haffara seabream (Rhabdosargus haffara) and Broomtail wrasse (Cheilinus lunulatus) marine fishes. The isolated strains were identified by their morphological and biochemical characteristics. In addition, the phylogenetic analysis of the 16S rRNA gene sequence of the present study MR B62 isolate revealed 100% identity with P. glacincola T (Accession No. AB334769.1) the recovered strain, P. glacincola, was also pathogenic to Haffara seabream (Rhabdosargus haffara) and sensitive to amikacin, streptomycin, ciprofloxacin, gentamycin, chloramphenicol, tobramycin, and ofloxacin. The present data suggest large-scale surveys of P. glacincola infection in the fish sector in Egypt, which might be helpful for the implementation of effective control strategies for combating this infection.