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ISSN: 2155-9546
Journal of Aquaculture Research & Development
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Phenotypic and Genotypic Characterization of Some Pseudomonas sp. Associated with Burkholderia cepacia Isolated from Various Infected Fishes

Manal I El-Barbary1* and Ahmed M Hal2

1Fish disease Lab., National Institute of Oceanography and Fisheries

2Genetics and Genetic Engineering Lab., National Institute of Oceanography and Fisheries

*Corresponding Author:
Manal I El-Barbary
Fish Diseases Laboratory
National Institute of Oceanography and Fisheries (NIOF)
Egypt
Tel: 01006972324
E-mail: [email protected]

Received date: July 22, 2017; Accepted date: August 22, 2017; Published date: August 25, 2017

Citation: El-Barbary MI, Hal AM (2017) Phenotypic and Genotypic Characterization of Some Pseudomonas sp. Associated with Burkholderia cepacia Isolated from Various Infected Fishes. J Aquac Res Development 8: 499. doi: 10.4172/2155- 9546.1000499

Copyright: © 2017 El-Barbary MI, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

This study aims to characterize Pseudomonas species that had been isolated from various naturally diseased fresh and marine water fishes, Nile tilapia, catfish, gilt-head bream, and Sea bass using phenotypic method, morphology and biochemistry characters using API 20NE, and genotypic method (16S rRNA gene sequencing) with some of the histopathological characteristics. Six of seven presumptive Pseudomonas sp. were successfully identified by API 20NE method to level species; they were identified as 2 P. fluorescens, one P. putida, one Pseudomonas sp and 3 Burkholderia cepacia while genotypically with 16S rRNA gene sequencing that proved successful for all the four pseudomonas isolates (three as P. fluorescens and one P. putida). The results of phylogenetic analysis placed the isolates in the genus Pseudomonas based on 99% homology. Challenged test revealed that, P. fluorescens and P. putida are to be classified as pathogenic for O. niloticus and they also exhibited clinical signs and mortality rates up to 70% and showed histopathological changes of both liver and kidney which lead to death. The antibiogram study showed that no significant differences between P. fluorescens and P. putida which had intrinsically high sensitivity to nucleic acid synthesis inhibitors such as ciprofloxacin, norfloxacin, gentamycin, gatifloxacin, lomefloxacin and kanamycin. This study concluded that good overall agreement between phenotypic and genotypic identification procedures was found for the isolates with some minor differences in biochemical and physiological characteristics were observed between P. fluorescens strains, while the genotypic differences were significant observed between P. fluorescens and P. putida isolated from various fishes.

Keywords

Pseudomonas sp.; API 20NE; 16S rRNA gene; Antibiogram; Histopathology; Fish

Introduction

Bacterial pathogens are naturally present in fish environment and under some specific stress conditions they are the etiological agents of the most important disease problems in aquaculture that induces mortalities and severe economic losses to fish farms [1]. Pseudomonas putida and P. fluorescens have been recorded as serious bacterial pathogens of fish and were characterized by causing high mortalities and economic losses among fish [2-5].

Pseudomonas fluorescens infection is widely distributed in aquaculture industries and is considered as one of the primary causes of bacterial hemorrhagic septicemia in fish and appears to be a stress related disease of freshwater and salt-water fish throughout the world [2]. It also causes severe economic losses and decreases fish farms efficiencies especially under culture conditions [6-8] such as overcrowding, low temperature where the highest natural mortalities were at 15°C to 20°C, injuries as inappropriate handling and transportation and secondary pathogen of damaged fish tissues [9-14]. Austin [2] suggested the reason for the widespread incidence of Pseudomonas sp. in the aquatic environment may be due to its spread through the water, which acts as the major reservoir of infection.

Pseudomonas putida is a Gram-negative stain, slightly curved or straight rod bacterium, it also grows fast where oxygen is present [15]. The first isolation of P. putida was from ayu, and yellowtail fish [16-18], while, the first isolation from an infected common carp was in Turkey [19], where it caused ulcerative infection in fish.

Previous studies reported that P. putida is considered one of the serious bacterial pathogens that have endangered the aquaculture of various fishes such as rainbow trout, European eel, oyster toadfish and large yellow croaker [5,20-22]; thus, causing high mortality and resulting in severe economic loss [23].

On the other hand, Burkholderia cepacia previously known as Pseudomonas cepacia, is a Gram-negative rod usually found in soil, vegetation, and water [24,25]. P. cepacia was firstly described by Burkholder in 1950 [26]. Ramsey et al. [27] recorded that bacteria belonging to the genera Burkholderia are recognized as pathogenic in fish, however Kayis et al. [28] reported that Burkholderia cepacia was isolated from rainbow trout fish farms in Turkey (2006-2008) but was not a fish pathogen.

In Egyptian farms, the genus Pseudomonas has been described as a causative agent of diseases in fish where P. fluorescens, P. aeruginosa, P. putida and P. angulliseptica were identified in different species of fish as etiological agents of Pseudomonas septicemia [29-31]. The external changes related to infection by Pseudomonas sp. bacteria in different fish were fin rot, detached scales, hemorrhage and darkness of the skin, abdominal ascitis and exophthalmia [30,32]. The histopathological changes related to infection with Peudomonas sp. were observed in different organs as well, such as the liver, kidney, gills and skin of different infected fish [5,33-35].

The reliable and quick techniques for classification of pathogenic bacteria are important for successful diagnosis and control. Various studies have reported that the phenotypic identification methods are solely not enough for the classification of Pseudomonas sp. so, the genotypic identification systems are required to confirm the traditional identification of particularly potential dangerous Pseudomonads [36]. Molecular techniques using PCR-based methods allow fast, sensitive and exact identification of the bacteria that have been described for detection of fish diseases; 16S rRNA gene is one of these important methods, especially when used alongside phenotypic characteristics for microbial identification in the diagnostic laboratory [37].

The aim of this study was to isolate and identify Pseudomonas sp. during outbreaks among fresh and marine water fish farms in Damietta Governorate and to compare between their phenotypic and genotypic characterizations during studies the morphological and biochemical characters, pathogencity, antibiotic susceptibility and phylogenetic analysis of identified pseudomonas together with their histological effects on the liver and kidney of challenged O. niloticus.

Material and Methods

Isolation and characterization of bacterial isolates

Bacterial isolates have been isolated from naturally diseased fishes namely Gilt-head bream, (Sparus auratus) and Sea bass (Dicentracchus labrax) which are important marine fish farmed in Deeba Triangle located in Damietta Governorate, Egypt along with African cat fish (Clarias garipinus) and Nila tilapia (Oreochromis niloticus) which have a high market value. Twenty fish samples, five of each type of fish, were collected during a disease outbreak in March 2015 and were transported to the laboratory in an icebox and processed during 2 h after collection. Samples from liver and kidney of collected fishes were streaked onto Pseudomonas base agar plates and incubated at 37°C for 24 hr. Some growing colonies were picked up in pure form where the identification of selective colonies was done by morphological and biochemical characters according to Austin [3] by using API 20NE (Biomérieux) for Gram-negative fish pathogens and they were identified to the genus pseudomonas.

Bacteria genomic DNA purification protocol

DNA isolation from cultured bacteria: The pure isolates were incubated overnight in tryptic soy broth at 37°C, in order to isolate DNA. Bacterial genomic DNA was extracted using GeneJET Genomic DNA purification kit based on the manufacturer's instruction. The eluted DNA was used as a template for PCR detection of 16S rRNA gene.

PCR and 16S rRNA gene sequencing

Universal bacterial primers DG74 5′-AGGAGGTGATCCAACCGCA- 3′ and RW01 5′-AACTGGAGGAAGGTGGGGAT-3′ were used for detection of 16S rRNA [38]. The locations of primers were 1521- 1540 and 1170-1189 (respectively). PCR reactions were done using 12.5 μl of DreamTaq Green PCR Master Mix (2x) , 1 μl of each of DG74 and RW01 primers, 2 μl of template DNA PCR grade water to reach the final volume of 25 μl at room temperature. The amplification was performed using a thermal cycler with the following parameters: initial denaturation at 95°C for 5 min, followed by 40 cycles of amplification (denaturation at 95°C for 30s, annealing at 58°C for 30s, extension at 72°C for 30s) and a final extension step of 72°C for 10 min. After amplification, 10 μl of the PCR sample was loaded on a 1.5% agarose gel stained with ethidium bromide. PCR product was purified by QIAquick PCR purification kit and directly sequenced with a 3500/3500xL Genetic Analyzer (Applied Biosystems).

Phylogenetic analysis

The obtained sequences in this study were identified using BLASTN database. The 16S rRNA genes of the four identified strains with 25 related species, from the GenBank database, were used to generate a phylogenetic tree; their accession numbers are shown in Figure 1. So, the analysis involved 29 nucleotide sequences which was led in MEGA5 [39] and phylogenetic tree was performed using Neighbor-Joining method [40] with 1,000 replicates of bootstrap test [41]. Evolutionary distances were calculated using the maximum composite likelihood method [42]. All gaps and missing data were deleted.

aquaculture-research-development-Phylogenetic-tree

Figure 1: Phylogenetic tree of 16S rRNA gene sequences of P. fluorescens and P. putida isolated from various natural infected fishes with other related bacteria in GenBank database were constructed using the Neighbor-Joining method in MEGA5. The bacterial isolates of this study designated with an asterisk.

Pathogenecity test

An experimental infection study was carried out to examine the pathogenicity of four isolates of Pseudomonas sp. isolated from the liver of naturally infected fish for virulence to O. niloticus. Seventy healthy O. niloticus weighing 70 gm ± 2 gm were obtained from El-Manzala fish farm, transferred and kept in ten glass aquaria provided with dechlorinated water with aeration; the fish were acclimatized for 2 weeks. Fish were fed with commercial pellet feed twice a day. Aquaria were divided into 5 groups each contained 14 fish. Each group was injected intraperitoneally (i.p.) with 0.5 ml of a bacterial suspension from P. fluorescens (Pse, Psu1 and Pse172, groups B, C, D respectively) and P putida (Psa groups E) that contained 105 CFU/ml in Phosphate Buffered Saline (PBS). Fish in group A were injected with 0.5 ml of sterile PBS alone (as a control group) using the same procedure. The virulence of the Pseudomonas strains was classified based on the development of clinical signs and mortality rates of fish that had been observed daily for 7 days. According to the severity of the mortality the virulence was considered positive + or ++.

Antibiotic susceptibility test

Fifteen antimicrobial drugs were evaluated for efficiency against tested P. fluorescens and P. putida. The test was performed by the disc diffusion method in Muller Hinton Agar and incubated at 37°C for 24 h. There are fifteen antibiotics that include; two cell wall synthesis inhibitors [amoxicillin (AX) 20 μg and tazobactam/piperacillin (TPZ) 110 μg], seven protein synthesis inhibitors [erythromycin (E) 15 μg, gentamicin 10 μg, streptomycin (S) 10 μg, oxytetracycline (OTC) 30 μg, tobramycin (TOB) 10 μg, kanamycin (KA) 30 μg, azithromycin (AZM) 15] and six nucleic acid synthesis inhibitors [ciprofloxacin (CIP) 5 μg, gatifloxacin (GAT) 5 μg, sulfamethoxazole (SXT) 25 μg, lomefloxacin (LOM) 10 μg, nalidixic acid (NA) 30 μg and norofloxacin (NOR ) 10 μg] as listed in Table 1. Pseudomonas strains were characterized as sensitive, intermediate or resistant according to the size of the inhibition zones around the discs [43].

Histopathological examination of O. niloticus liver and kidney

Liver and kidney specimens were taken from both experimentally infected O. niloticus and control on the seventh day post infection. The samples were fixed in 10% formalin and the histopathological examination was performed according to Roberts [44] where the tissue sections were stained with hematoxylin-eosin (HE).

Results

Phenotypical characterization and hemolytic activity on blood agar

Some presumptive Pseudomonas colonies that were selected from PBA plates were identified morphologically as Gram-negative rod motile bacteria. Biochemically; bacteria were oxidase positive and unable to ferment glucose, thus characterized as Pseudomonas genus. Only seven of the presumptive Pseudomonas isolates were identified using API 20NE method. All isolates were positive for utilization of citrate, glucose, potassium gluconate, capric acid and malate but negative for indole production. Whereas the results of the arabinose, maltose, manose, manitol, phenyl acetic acid and gelatin liquefaction tests found variables among the isolates (Table 1).

The results of API 20NE tests were interpreted using the ‘apiweb’ program (BioMerieux). According to the standard tests and phenotypic characterization by the API 20NE identification system, similar to API 20NE code, the strains identified by API 20NE were of four different species; P. fluorescens (Pse, and Pse172), P. putida (Psa), Pseudomonas sp. (Psu1) and Burkholderia cepacia (P5, P6, P7) with different API 20NE code; 0556457, 0056555, 0140455, and 0046455 for Pseudomonas strains while Burkholderia cepacia showed 1477757, 0454557 and 0456547 codes (Table 1). The specific media, PAB, is specific for Pseudomonas sp. and sometimes Burkholderia cepacia grows on it. The method was successful for 6 out of the 7 tested isolates and Burkholderia cepacia was isolated only from marine fish Gilt-head bream and Sea bass.

Identification by 16S rRNA and sequencing

DG74 and RW01 primers were used to amplify of 16S rRNA of bacterial isolates, with amplicon size ∼ 370 bp. The four-bacterial species belonged to family Pseudomonadaceae. Four isolates were collected and identified by 16S rRNA (Pse, Pse1, Psu172 and Psa). The amplified nucleotide sequence showed 99% homology with the 16S rDNA sequence of P. fluorescens and P. putida. The results of 16S rRNA sequence correlated with phenotypic classification of the Pseudomonas strains. Overall good agreement between phenotypic and genotypic identification procedures was found for the 3 isolates (Pse, Pse172 and Psa). In fact, these isolates were genotypically identified as P. fluorescens and P. putida. However, discordance of identification between phenotypic methods and 16S rRNA sequence analysis was observed in strain Psu1 that was identified based on API 20NE as Pseudomonas spp. while, 16S rRNA sequencing identified as P. fluorescens (Table 2). The result of 16S rRNA sequence was deposited into GenBank under the accession number LC208785, LC208786, LC208787 (Pse, Psu1, and Pse172 as P. fluorescens respectively) and LC208784 (P. putida, Psa). The closer relationships among sequences of 16S rRNA were grouped with each other in phylogenetic tree, where phylotypes were distributed in the branches (Figure 1).

Test P. fluorescens P. putida P. species B. cepacia
Pse Pse 172 Psa Psu1 P5-P7
Utilization of NO3 - - - - +v
Indol production - - - - -
Glucose fermentation - - - - -
Arginine dihydrolase + - + - -
Urease - - - - -
β glucosidase + - - - +
Gelatin hydrolysis + + - - +
O-nitrophenyl-β-galactopyranoside - - - - +
Utilazition of sugar          
Glucose (GLU) + + + + +
Arabinose (ARA) - - - - +v
Manose (MNE) + + - + +
Manitol (MAN) + + - + +
N-Acetyl glucosamine (NAG) - + - - +
Maltose (MAL) - - - - +v
K gluconate (GNT) + + + + +
Capric acid (CAP) + + + + +
Adipic acid (ADI) - - - - -
Malate(MLT) + + + + +
Tri sodium citrate (CIT) + + + + +
Phenyl acetic acid (PAC) + - + - +
OX + + + + +
API 20NE code 556457 56555 140455 46455 1477757
454557
456547

Table 2: Phenotypic characteristics of Pseudomonasand strains.

The cluster of the Pseudomonas fluorescens harbored three bacterial isolates which were isolated from Sparus auratus, Clarias garipinus and O. niloticus (Pse, Psu1 and Pse172). They showed 99% of homology with P. fluorescens in GenBank (acc. nos. KF187316, KY190964 for Pse, KF776987, AB680223 for Psu1 and LN651257, KY073966 for Pse172, ). However, one bacterial isolate of P. putida (LC208784) was closely related with P. putida in GenBank (accession nos. KC478494 and KR054999).

Antibiotic sensitivity

Antibiotic susceptibility study showed that all isolates had intrinsically high sensitivity to ciprofloxacin, norfloxacin, gentamycin, gatifloxacin, lomefloxacin and kanamycin except P. putida (Psa) that reflected an intermediate reaction with gatifloxacin and lomefloxacin. However, these isolates were resistant to sulfamethoxazole, erythromycin, amoxicillin, tazobactam/piperacillin and nalidixic acid and were intermediate with oxytetracyclin. Other antibiotics such as tobramycin, streptomycin and azithromycin showed variation in their effects as antibacterial agents among bacterial isolates (Table 3).

Antibiotic Disc Response of bacterial strains to different antibiotics
P. fluorescensPse P. fluorescensPsu1 P. fluorescensPse172 P. putida
Psa
Norfloxacin S S S S
Cirprofloxacin S S S S
Gatifloxacin S S S I
Lomefloxacin S S S I
Nalidixic Acid R R R R
Gentamycin S S S S
Tobramycin I S I R
Kanamycin S S S S
Streptomycin I S S I
Azithromycin R I R I
Erythromycin R R R R
Oxytetracyclin I I I I
Sulfamethoxazole R R R R
Amoxicillin R R R R
Tazobactam/piperacillin R R R R
S: susceptible; I: intermediate susceptible; R: resistant.

Table 3: Antibacterial activity of different antibiotics against P. fluorescensandP. putida

Pathogenecity for O. niloticus

Fish groups injected with P. fluorescens (Pse, Psu1 and Pse172) and P. putida (Psa) revealed mortality ranging from 65% to 70% within 7 days (Table 4). Therefore, these isolates were pathogenic for O. niloticus and were classified as virulent for fish (+). They also exhibited external hemorrhage, skin ulceration and dark pigmentation while no mortality or clinical signs were observed during the experiment in control fish (group A).

Bacterial isolate (Groups) Fish mortality/isolate Pathogenicity %
Control(A) 0/14(-) 0.00
P. fluorescensisolate; Pse(B) 9/14(+) 65
P. fluorescensisolate; Psu1(C) 9/14(+) 65
P. fluorescens isolate;Pse172 (D) 9/14(+) 65
P. putidaisolate;Psa(E) 10/14 (+) 70

Table 4: Phenotypic differences among virulent bacteria isolates.

Histopathological examination

No histological changes were observed in the liver and kidney of control fish (A) (Plate 1a and 1d). The histopathological changes of the O. niloticus infected with pathogenic P. fluorescens (group B) were hemolysis between hepatocytes with cytoplasmic vacuolation and pyknotic nucleus in liver hepatocytes (Plate 1b). While O. niloticus injected with pathogenic P. putida (group E) showed severe lipid vacuoles with hepatocytes necrosis and severe vacuolar degeneration, between hepatocytes in liver (Plate 1c). Kidney of O. niloticus infected with pathogenic P. fluorescens had tubular degeneration with interstitial mononuclear cell infiltration in addition to, disconnection of renal tubules, hemorrhage between renal tubules, decrease of the glomerular component and degeneration and necrosis of renal tubular and dilation in Bowman’s space (Plate 1e). Similar lesions were observed in kidney of O. niloticus injected with P. putida which showed hemorrhage and necrosis area between renal tubules, dilation in Bowman’s space and decrease of the glomerular component (Plate 1f).

aquaculture-research-development-Histopathological-changes

Plates (1a-1f): Histopathological changes of the liver and kidney of O. niloticus groups (A, B, and D) stained with H & E; (a, d) A (control group) normal structure tissue of both liver and kidney (x400). (b) Group B hemolysis between hepatocytes, cytoplasmic vacuolation and pyknotic nucleus of the liver (x 400). (c) Group D severe lipid vacuoles and necrosis of hepatocytes with hemolysis (x400). (e) Group B tubular degeneration with interstitial mononuclear cell infiltration, disconnection of renal tubules, dilation in Bowman’s space and hemorrhage between renal tubules of the kidney (x400). (f) Group D necrosis area and hemorrhage between renal tubules, dilation in Bowman’s space and decrease of the glomerular component (x400). N: Necrosis; Hs: Hemolysis; V: Vacuolar degeneration; pk: Pyknosis; G: Glomerulus; BS: Bowman’s Space; If: Infiltration; RT: Renal Tubular; Rd: Renal tubular degeneration; He: Hemorrhage.

Discussion

During the spring of 2015 a disease outbreak occurred among some of the marine and freshwater fish farms where the fish died. The typical clinical signs were skin ulceration and hemorrhage. The causative bacteria isolated from these fish farms were identified as P. fluorescens and P. putida according to phenotypical characteristics and sequence of 16S rRNA. These findings agree with those of Eissa et al. and EL-Hady and Samy [30,45] who reported that P. fluorescens, Pputida with other species are an aquaculture pathogen that can infect various species of fish, including O. niloticus, Cyprinus carpio. In the current study, the clinical and postmortem findings for infected fishes that showed hemorrhages and ulcer on the skin, dark pigmentation and abdominal distention, similar lesions as hemorrhages over all of the fish body, tail and fins rot, scale separation, skin ulceration and abdominal swelling of naturally infected fishes were recorded by Eissa et al., Okaeme and Hanna et al. [30,32,35]. Some of these lesions caused by P. putida are similar to a disease caused by Flavobacterium columnare, F. psychrophilum and motile Aeromonas sp. [46-48], where the ulcers caused by P. putida can be observed almost exclusively on the dorsal surface of the fish [48]. This result may give a necropsy hint for differential diagnosis of the disease [5].

The results in Table 1 showed that, all isolates identified as Pseudomonas and B. cepacia along with all Pseudomonas were characterized by Gram-negative motile rods with cytochrome oxidase. These findings agree with those of Altinok et al., Nathan et al. [5,49] who had observed that Pseudomonas species are Gram-negative, oxidase positive and rod shaped. P. fluorescens showed variation in some phenotypic tests such as gelatin hydrolysis where it has an ability to hydrolyze gelatin in contrast to P. putida. This result agrees with the findings of Krieg and Holt [15] who reported that P. fluorescens can be discerned from P. putida through its ability to hydrolyze gelatin.

Isolates Fish species Identification method Accession
number
API 20NE 16S rRNA
Pse Gilthead bream(S. auratus)   P. fluorescens P. fluorescens LC208785
Psu1 Nile tilapia(O. niloticus)   Pseudomonassp P. fluorescens LC208786
Pse172 African cat fish (C. garipinus) P. fluorescens P. fluorescens LC208787
Psa Sea bass (D. s labrax) P. putida P. putida LC208784

Table 1: Comparison of API 20NE method and 16S rRNA identifications of Pseudomonasstrains.

In this study, the identification of all Pseudomonas species by API 20NE and phenotypic characters revealed good identification. Isolates Pse and Pse172 revealed profile of 0556457, 0056555 in the API 20NE systems and were identified as P. fluorescens. While Psa revealed profile of 0140455 of P. putida but the isolate of Pseudomonas Psu1 revealed a profile 0046455 and identified as Pseudomonas species, it was however identified as P. fluorescens by 16S rRNA gene. These results agree with those of Altinok et al. [5] who identified P. putida by API 20NE with a profile number of 0140455 but it was 0142457 in the study of Kayış and Er [50].

In addition, isolates were well identified at species level by API 20NE method and 16S rRNA sequences, and they showed good overall agreement. On the other hand, API 20NE identification gave incomplete result at species level that was identified as Pseudomonas sp. however; it was identified as P. fluorescens (Psu1) based 16S rDNA. This result agrees with Uğur et al. [51] who reported that in rare instances, (3/17), 16S rRNA sequences of two isolates were identified as P. fluorescens based on phenotypic characters, revealed that they were P. putida (96%), where these results indicated that the procedures for the identification of Pseudomonas sp. based on phenotypic characteristics should be additionally verified by the molecular methods to obtain results that are meaningful as well as accurate. Many studies have suggested that the phenotypic classification techniques are solely not adequate for identification of Pseudomonas sp., though, overall, the API 20NE was successful in the identification of all the four Pseudomonas strains that had been isolated from fish. That is in agreement with Wiedmann et al. [52], who reported that API 20NE resulted in the proper identification of Pseudomonas isolates to the species level.

The analysis of 16S rRNA gene presented fast and accurate identification of the detected bacteria [53-55]. Many groups of primers have been designed to amplify various regions of 16S rDNA and have been explained to have different specificities and susceptibility [38].

The present study used PCR technique targeting 16S rRNA for detection of bacterial isolates from infected fish through universal primers (RW01 and DG74), that was reported to be a sensitive screening technique to detect the bacterial communities' [38,56].

The phylogenetic analysis placed the bacterial isolates in the family Pseudomonadaceae based on 99% homology because >98.7% of 16S rRNA gene sequence similarity are not considered to be different species [57].

The results of the pathogenicity study exhibited that O. niloticus challenged with P. fluorescens and P. putida (Pse, Psu1, Pse172 and Psa) caused a mortality rate up to 70% within 7 days (Table 4). Therefore, the Pseudomonas strains were pathogenic for O. niloticus, these findings agree with Austin and Austin; Toranzo et al. and Altinok et al. [3-5], but disagree with some results of Eissa et al. [30] who observed that P. putida showed mortality rates of 86.66% in the injected fish. While P. fluorescens biovar I, II and III strains were nonpathogenic also, Altinok et al. [5] reported that mortality of fish injected with P. putida was 45%. The antibiotic susceptibility study recorded that P. fluorescens and P. putida exhibited sensitivity to ciprofloxacin, norfloxacin, gentamycin, gatifloxacin, lomefloxacin and kanamycin, that was found to be consistent with Eissa et al. [30] who reported that most strains of P. putida, P. anguilliseptica and P. aureginosa were sensitive to Avatryl and Amikicin, Novobiocin, Erythromycin, Gentamicin, and Sulfa-trimethoprime. Also, Altinok et al. [5] reported that P. putida has high resistance to Ampicillin, Erythromycin, Chloramphenicol, Tetracycline, Naladixic acid Rifampicin and Streptomycin. Thus, it is difficult to use these antibiotics to treat the fish infected with P. putida. In addition, P. fluorescens exhibited complete resistance to penicillin and erythromycin and susceptibility to oxytetracycline and amikacin more than kanamycin, neomycin and gentamicin [58]. Also, Darak and Barde [59] reported that P. fluorescens was very sensitive to kanamycin, nalidixic acid, gentamicin, neomycin, less sensitive to amikacin and tetracycline, and chlorophenicol and the least sensitive to oxytetracycline, erythromycin and penicillin.

The histological changes in the liver and kidney of the O. niloticus infected by pathogenic P. fluorescens and P. putida in the current study are similar to the findings of Amosu; Hanna et al. [34,35] who observed pathological lesions in the liver of African catfish, which were inoculated with P. aeruginosa, these lesions included widespread hepatic degeneration, focal area of cellular infiltration, disorganization of the hepatic cells and area of necrosis with hyperplesia in the wall of the blood vessels.

Altinok et al. [5] observed that, in rainbow trout the skin ulcer was initially described by sloughed off epithelia and epithelial necrosis while in the developed form of P. putida disease most of all the skin layers down to the epidermis were lost. Also, Kumaran et al. [60] reported that the infected Sea bass with Pseudomonas sp. showed irregularly shaped nuclei of hepatocytes and focal necrosis, eosinophilic granulocytes and erythrocytes.

Conclusion

In conclusion, API 20NE gave a good identification of Pseudomonas isolates. Using 16S rRNA gene sequencing proves that diagnosing fish's bacterial diseases is important for successful epidemiological studies and disease control. The results concluded that some minor differences in biochemical and physiological characteristics were observed between P. fluorescens strains, while the phenotypic and genotypic differences were observed between P. fluorescens and P. putida isolated from fish. Also, pathogenic P. fluorescens compared to P. putida is more sensitive to ciprofloxacin, norfloxacin, gentamycin, gatifloxacin, lomefloxacin and kanamycin.

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