Molecular Identification and Pathogenicity of Citrobacter And

Egyptian Journal of Aquatic Research xxx (2017) xxx–xxx

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Egyptian Journal of Aquatic Research

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Full length article Molecular identiﬁcation and pathogenicity of Citrobacter and Serratia species isolated from cultured Oreochromis niloticus ⇑ Manal I. El-Barbary a, , Ahmed M. Hal b a Fish Diseases Lab, National Institute of Oceanography and Fisheries, Egypt b Genetics and Genetic Engineering Lab, National Institute of Oceanography and Fisheries, Egypt article info abstract

Article history: This study aimed to isolate and characterize some pathogenic bacterial strains belonging to the family Received 5 August 2017 Enterobacteriaceae. They had been isolated from gills, liver, kidney and skin of naturally infected Revised 19 September 2017 Oreochromis niloticus and had been identified by biochemical test and 16S rRNA gene using four universal Accepted 25 September 2017 primers. Additionally, the isolates were tested for antimicrobial susceptibility, histopathological alter- Available online xxxx ations of liver, kidney and gills and the pathogenicity of the identified isolates for O. niloticus. The results of phylogenetic analysis placed the isolates in the family Enterobacteriaceae (genera Serratia and Keywords: Citrobacter) based on 99% homology. The primer pair (17F and 1390R) is the most appropriate pair of uni- Citrobacter sp. versal primers employed for the identification of 16S rRNA gene as a reason behind fish disease in Serbia Serratia sp. Phylogenetic analysis it covers as much as possible of the variable regions (Vs). V1 and V2 regions of 16S rRNA gene presented Histology weak evidence of the diversity of the genera Serratia. The mortality rate was 40–60% after challenging O. Antibiotic sensitivity niloticus by identified isolates, which revealed its sensitivity to ciprofloxacin and norfloxacin. Histological Oreochromis niloticus changes showed dilation in sinusoids with severe vacuolar degeneration in the liver, tubular degeneration and hemorrhage between renal tubules with pyknotic nuclei in the kidney, epithelial hyperplasia, aneurism and evident epithelium interstitial edema in gills of O. niloticus. This study concluded that these isolates should be considered as an opportunistic pathogen of O. niloticus. The study also states that the sequencing of 16S rRNA is an important tool for the identification of unknown bacterial species of fish pathogen. Ó 2017 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction tilapia and mullet (Hassan et al., 2012). While, the first report of pathogenicity of Citrobacter braakii in fish was in Bursa (Altun Bacteriosis in fish has a worldwide distribution that causes et al., 2013). On the other hand, Serratia marcescens has been asso- either rapid or low mortality in an acute or chronic form (Baeck ciated with bacterial septicemias and mortalities in salmonids and et al., 2009; Austin et al., 1992). Bacterial Enterobacteriaceae has were recognized as emerging pathogens (Austin and Gibb, 1993). It been reported as an opportunistic pathogen in fish and has been is a common microorganism present in soil and fresh water. It is considered a marker of sewage pollution (Rajasekaran, 2008). classified as a proteobacteria that produces biosurfactant which Serratia and Citrobacter are bacterial genera belonging to the contributes to the pathogenesis (Horng et al., 2002) where several Enterobacteriaceae family, where Citrobacter freundii is one of the virulence factors were found within the genome of S. marcescens infectious agents that have been recognized as an opportunistic which were isolated from infected tilapia (Chan et al., 2013). pathogen in veterinary and medical sciences (Chuang et al., Numerous studies used nucleic acid-based methods to identify 2006); it was first mentioned as an emerging fish pathogen from the fish pathogenic bacteria (Buller, 2004), such as the 16S rRNA sunfish (Mola mola) in Japan (Sato et al., 1982). Jeremic et al. gene in which its value lies in slow evolution (Page and Holmes, (2003) recorded the first confirmation of C. freundii as a reason 1998). In addition, 16S rRNA contains a variable of highly behind fish disease in Serbia and also in Egypt. It was isolated from conserved regions that can be specific to a genus (Buller, 2004), therefore, 16S rRNA gene sequencing is a strong technique for mea- suring the genetic diversity of biological samples and microbial Peer review under responsibility of National Institute of Oceanography and taxonomy (Van Berkum et al., 2003). Of the primers described as Fisheries. ⇑ Corresponding author. common, many with the analysis of 16S rDNA sequence have been E-mail address: [email protected] (M.I. El-Barbary). used to elucidate prokaryotic biodiversity (Baker et al., 1999). https://doi.org/10.1016/j.ejar.2017.09.004 1687-4285/Ó 2017 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Based on different parts of 16S rRNA gene, recombination events bacterial DNA template and was filled up to 50 lL of PCR grade among species may sometimes cause a few incoherencies among water. The PCR started with a denaturation at 95 °C for 1 min fol- phylogenies (Young and Haukka, 1996), so longer sequences of lowed by 35 cycles of denaturation at 95 °C for 40 s, annealing at 16S rRNA give more accurate results concerning the classification 58 °C for 1 min and elongation at 72 °C for 2 min. Finally, the PCR of a new species. Citrobacter freundii isolated from tilapia finger- was extended for an additional 10 min at 72 °C (BioRad, T100 Ther- lings, showed symptoms such as tail necrosis and hemorrhage on mal Cycler). The results of amplification were analyzed on a 1.2% the skin (Thanigaivel et al., 2015). In addition, C. freundii caused agarose gel stained with ethidium bromide. PCR products were systemic hemorrhages and gastroenteritis in a number of different purified with a QiaQuick PCR purification kit (QIAGEN) and were fish (Austin et al., 1992; Sato et al., 1982). Histological changes in directly sent to Macrogen Inc. for sequencing of 16S rRNA using fish infected by C. freundii were recorded in rainbow trout fry, high throughput Applied Biosystems 3730XL sequencer. cyprinids and Catfish (Jeremic et al., 2003; Pádua et al., 2014). In Egypt, the studies of effects of C. freundii, C. braakii and S. marces- Phylogenetic analysis cens on fish are rare. The aims of this study were characterize and diagnose the bacterial strains of Citrobacter and Serratia species To find related species, the sequences obtained in this study isolated from O. niloticus based on the sequence of 16S rRNA gene were compared to those in the BLASTN database (http://www. as well as to study the mismatching rates of 4 universal primers ncbi.nlm.nih.gov/blast). The 16S rRNA genes of 24 related species and the variance of different regions of 16S rDNA gene of these iso- were used from the GenBank database to generate a phylogenetic lates. Also, antimicrobial susceptibility and pathogenicity of iso- tree. Their accession numbers are shown in (Fig. 1) and were con- lates and their histopathological alterations on liver, kidney and ducted in MEGA6 (Tamura et al., 2013), performed using the gills of O. niloticus were studied. Neighbor-Joining method (Saitou and Nei, 1987) and inferred from 1000 replicates of the datasets using the bootstrap test (Felsenstein, 1985). Evolutionary distances were calculated using Material and methods the Maximum Composite Likelihood method (Tamura et al., 2004). Multiple alignments of sequences were done using Clustal Culture, isolation and characterization Omega (http://www.ebi.ac.uk/Tools/msa/clustalo). Thirty naturally infected O. niloticus were sampled and taken from El-Serw fish farm located on El Manzala Lake, Egypt. Infected Antibiotic susceptibility tests fish were characterized by typical hemorrhagic septicemia on the skin and internal organs. Live fish were transferred directly to Sensitivity analysis was carried out to 12 antibiotics by the dif- the experimental laboratory of fish diseases in the same farm to fusion method in Mueller Hinton Agar (Oxoid) and incubated at 37 detect the pathogenic bacteria. Swabs from inside each liver, kid- °C for 48 h. The tested antibiotics (Oxoid) were showed in Table 3. ney, skin and gills under aseptic precautions were inoculated into Incubation zone diameters were measured after 48 h of incubation trypticase soy broth (TSB; Oxoid) at 32 ± 1 °C for 24 h. A loopful of and classified as sensitive or resistant according to the size of the the obtained broth culture was streaked onto MacConky agar zone of the bacteria growth inhibition (CLSI, 2010). (Oxoid) and incubated at 32 ± 1 °C for 24–48 h. Morphologically similar and dominant bacterial colonies were selected and Pathogenicity assay streaked onto nutrient agar (NA) plates for 24 h at 32 ± 1 °Cin order to obtain pure isolates. After purification, isolates were re- Isolates of C. freundii,C. braakii, S. marcescens and Serratia sp. cultured on NA slants and stored at 4 °C for further identification. (OS-1, OS-2, OS-3 and OS-4, respectively) were inoculated in TSB All the bacterial isolates were characterized morphologically by and incubated at 32 ± 1 °C for 24 h. A number of 60 apparently applying Gram staining and by testing the motility of each isolate. healthy O. niloticus 80 ± 2 g was used in experiments. Fish were Hemolytic activity was evaluated by the standard diffusion method using tryptic soy agar (Oxoid) supplemented with 5% sheep blood. Table 2 Phenotypic profiles of Citrobacter and Serratia species isolated from liver of O. Bacterial DNA purification and PCR amplification niloticus. Test OS-1 OS-2 OS-3 OS-4 The pure isolates were incubated overnight in trypticase soy Oxidase test ° broth (TSB) at 32 C, in order to isolate the bacterial DNA. The Red pigment + genomic DNA of bacteria was purified according to Das and Dash Motility ++ (2015). The universal bacterial primers have been used to amplify Lactose fermentation + + + the 16S rRNA genes as they have been included in Table 1. With L-Lysine ++ different combinations of forward and reverse primers, a different Arginine dihydrolase +++ product size for each sample can be amplified. The 50 lL PCR reac- L-Ornithine l l Trisodium citrate ++++ tion mixture contained 1 L(1 M) of each primer (forward and Sodium thiosulphate + + reverse), 10 lLof5 Taq master mix (Jena Bioscience), 2 lLof Urease + L-tryptophane Indole Table 1 Sodium pyruvate + Universal 16S rRNA primers used in this study. D-Glucose ++++ 0 0 Name Sequences (5 –3 ) Ref. D-Mannitol ++++ Inositol ++ 27F (8F) AGAGTTTGATYMTGGCTCAG Ludwig et al. (1993) ++++ 519R (536R) GTATTACCGCGGCKGCTG Ruff-Roberts et al. (1994) D-Sorbitol 519F CAGCMGCCGCGGTAATAC Burggraf et al. (1997) L-Rhamnose ++ + 1390R (1406R) ACGGGCGGTGTGTRCAA Brunk and Eis (1998) Hemolysis + ++

Y: C or T, R: A or G, M: A or C and K: T or G. + positive; negative.

Please cite this article in press as: El-Barbary, M.I., Hal, A.M. Molecular identification and pathogenicity of Citrobacter and Serratia species isolated from cultured Oreochromis niloticus. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.004 M.I. El-Barbary, A.M. Hal / Egyptian Journal of Aquatic Research xxx (2017) xxx–xxx 3 divided into five groups (A, B, C, D and E, in 10 aquarium), each specimens were performed according to Roberts (2004). Tissue group contained 12 fish and was maintained in two aquariums sections were stained with Hematoxylin-Eosin (HE) and pho- containing 50 L (70 40 30 cm) with aerated fresh water and tographed using an ICC50 HD camera and a Leica LAS EZ feeding on commercial pellets, which included 30% protein by rat microscope. 3% of body weight for two weeks to adaptation on laboratory con- ditions. Fish were anesthetized in a clove oil solution (Hamackova Results et al., 2006). Each of the 12 fish in each group were injected intraperitoneally (IP) with 0.2 mL of a suspension containing 105 Isolation and identification of bacteria CFU mL 1 (measured by McFarland suspension) in phosphate buffered saline (PBS). Group A, injected with 0.2 mL of sterile PBS In total, 26 isolates of Enterobacteriaceae bacteria (Gram- alone, served as a control group. The observation time was 14 days negative bacillus and oxidase negative) were obtained from the to record any clinical signs or mortality. The pathogenicity test was samples, including the suspected 11 Citrobacter and Serratia spe- considered positive when the mortality rate was more than 50% of cies that were isolated from O. niloticus. These isolates were the injected fish. obtained from the skin, liver, gills and kidney (2, 4, 3 and 2 respectively). The phenotypic and the biochemical characters of the pre- Histological examination of internal organs sumptive Citrobacter and Serratia species that had been isolated only from O. niloticus liver were showed in Table 2. The four iso- This took place after 15 days of having the injection, samples of lates are oxidase negative and Gram-negative rods. According to liver, kidney and gills fixed in buffered formalin solution (10%). The the schemes of biochemical reactions provided in Bergey’s Manual

Serratia plymuthica AS9 (NR102827) Serratia plymuthica DSM4540 (NR114579) Serratia liquefaciens ATCC27592 (NR122057) Serratia ficaria NCTC12148 (NR114750) Serratia grimesii LMG7883 (NR114576) Serratia quinivorans 4364 (NR037112) Serratia proteamaculans DSM4543 (NR025341) Serratia entomophila DSM12358 (NR025338) Serratia glossinae C1 (NR116808)

Serratia fonticola LMG7882 (NR114577) Serratia Serratia odorifera PADG1073 (NR037110) Serratia symbiotica CWBI-2.3 (NR117512) Serratia marcescens LB21 (KC335216) Serratia marcescens ZC06-1 (KT438729) Serratia marcescens OS-3 (KY404159)* Serratia sp. OS-4 (KY404160)* Serratia nematodiphila H1 (KJ149817) Citrobacter freundii K6 (KX156769) Citrobacter freundii OS-1 (KY404157)* Citrobacter braakii OS-2 (KY404158)* Citrobacter braakii Hb-7 (KC139411) Citrobacter koseri LMG5519 (NR117751) Citrobacter diversus CDC3613-63 (AF025372) Citrobacter farmeri (AF025371) Citrobacter Citrobacter farmeri CCB19B (FN433016) Citrobacter amalonaticus ARB01 (FR773713) Citrobacter sedlakii CIP105037 (KM515973) Citrobacter rodentium ICC168 (NR074903)

Fig. 1. Phylogenetic relationships of 16S rRNA genes of strains of bacteria isolated from diseased O. niloticus (designated with an asterisk) with other related bacteria in the GenBank database, were constructed by the neighbor-joining method. Numerals at nodes indicate bootstrap percentages derived from 1000 replications. Accession numbers of 16S rRNA genes are shown in brackets.

Please cite this article in press as: El-Barbary, M.I., Hal, A.M. Molecular identification and pathogenicity of Citrobacter and Serratia species isolated from cultured Oreochromis niloticus. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.004 4 M.I. El-Barbary, A.M. Hal / Egyptian Journal of Aquatic Research xxx (2017) xxx–xxx of Systematic Bacteriology for the identification of genus or species under the accession No. KY404157 for C. freundii (OS-1), level, the obtained results indicated that C. freundii and C. braakii, KY404158 for C. braakii (OS-2), KY404159 for S. marcescens (isolates OS-1 & OS-2) produced acid from glucose, mannitol, sor- (OS-3) and KY404160 for Serratia sp. (OS-4). bitol and rhamnose, but did not produce acid from inositol. In addition, they were found to be ornithine decarboxylase negative, and Antibiotic susceptibility did not produce indole, urease and tryptophan, while isolate OS-2 was discovered to be arginine dihydrolase positive. In addition, iso- The results of the antimicrobial tests revealed that all the tested lates OS-3 & OS-4 that were identified as S. marcescens and Serratia isolates were susceptible to ciprofloxacin and norfloxacin except sp did not produce indole and formed glucose, mannitol, inositol OS-1 and OS-3 which were susceptible to nalidixic acid. Gen- and sorbitol while, Serratia marcescens (OS-3) produces red pig- tamycin proved effective against all strains except OS-1, but OS-3 ment and it is Voges-Proskauer positive and ferment lactose was susceptible to sulfamethoxazole, streptomycin and clar- (Table 2). Hemolytic test showed that all isolates except C. braakii ithromycin. In addition to this, all isolates were found to be resis- b (OS-2) were hemolytic positive. tant to the other test antibiotics (Table 3).

Identiﬁcation by 16S rRNA Macroscopic ﬁndings

Primers used in the present study targeted the variable regions Isolates OS-1, OS-3 and OS-4 were pathogenic to O. niloticus of 16S rRNA of bacterial community. In the PCR reaction, no ampli- where the mortality rate was 60% in Groups B, D, E in which fish fication was observed using universal primers (519F and 519R) for were injected IP with of those bacteria. It was 40% in Group C that some bacterial isolates. However, a pair of universal primers (17F had been injected IP with OS-2 isolate (Table 4). The infected fish and 1390R) succeeded to amplify the 16S rRNA gene in the PCR showed fin erosions, body surface hemorrhage, skin shallow ulcer reaction and the resulting sequences covered variable regions 1 with liver, spleen, kidneys and gall bladder congestion with (V1) to 7 (V7) of 16S rRNA in bacterial isolates in order to accu- enlargement and fatty gut. No mortality was observed in control rately identify the bacterial species. Comparing the nucleotide fish (Group A). sequences of 16S rRNA gene using BLASTN, showed that the similarity of studied bacterial isolates was a 99% match with that of C. Table 3 freundii (accession No. KX156769), while C. braakii in this study, Antibiotic sensitivities of bacteria isolates. showed 99% of homology with C. braakii (accession No. KC139411). The similarity of S. marcescens with S. marcescens in Antibacterial agents The inhibition zones (mm) GenBank reached 99% (accession Nos. KT438729 and KC335216). OS-1 OS-2 OS-3 OS-4 One of the studied isolates was close to Enterobacter sp. and Uncul- Ampicillin (AP) 10 lg 0.0 0.0 0.0 0.0 tured gamma proteobacterium clone, but was found to be more Cefazolin (CZ) 30 lg 0.0 0.0 0.0 0.0 l related to Serratia sp. based on BLASTN. The phylogenetic tree Ciprofloxacin (CIP) 10 g 20203035 Erythromycin (E) 15 lg 0.0 0.0 0.0 0.0 showed that the genera of Citrobacter harbored C. freundii and C. Fusidic acid (FA) 10 lg 0.0 0.0 0.0 0.0 braakii of the identified bacteria while Serratia sp. and S. marcescens Gentamycin (GN) 120 lg 12192830 were clustered in the genera of Serratia as shown in Fig. 1. On the Nalidixic Acid (NA) 30 lg 22 0.0 20 15 l other hand, this study showed multiple alignments of sequences of Norfloxacin (NOR) 10 g 22223530 l Serratia sp (OS-4) with other Serratia isolates from GenBank (Fig. 2) Oxytetracycline (OTC) 30 g 10 7 0.0 0.0 Streptomycin (S) 10 lg 10 7 18 10 which showed their similarity in V1 and V2 based on the regions Sulfamethoxazole (SXT) 25 lg 0.9 0.0 20 10 map of the 16S rRNA gene (Baker et al., 2003). The 16S rDNA Tobramycin (TOB) 10 lg10101110 sequences resulting from this study were deposited into GenBank

Fig. 2. Sequence alignment of 16S rRNA of Serratia sp.* (in this study) with S. nematodiphila, S. marcescens and Serratia sp. (accession Nos. KJ149817, KT438729 and LT009511 respectively), was done using Clustal Omega. The dotted line is variable regions (V1 and V2) and the continuous line is conserved regions.

Table 4 Histopathological analysis Phenotypic differences among virulent and avirulent bacteria isolates.

Bacterial isolate Groups Pathogenicity % No histopathological alterations were observed in any of the Control A 0.00 studied organs (liver, kidney and gills) of control ﬁsh (A) (Plates C. freundii B601–3a). The histopathological changes in the organs of the O. niloti- C. braakii C 40 cus infected with OS-1 (group B) were severe dilation in sinusoids S. marcescens D60with severe vacuolar degeneration in the liver, while the kidney Serratia sp. E 60 showed a dilated Bowman’s space in the glomeruli with intersti- + isolates positive; Isolates negative; ± Denote weak but not negative. tial mononuclear cell inﬁltration. Epithelial hyperplasia and

Plate 1. Histopathological changes in the liver of O niloticus groups (A–E) stained with H&E.; Group A (control) (a) normal structure (200). Group B (b) severe dilation in sinusoids with vacuolar degeneration (300). Group C (c) slight vacuolation of the hepatic cells and slight congestion with dilation in sinusoids (200). Group D (d,e); lipid vacuoles and hemosiderin accumulation around pancreatic acinus (d 200). (e) cytoplasmic vacuolation and pyknotic nucleus with necrosis (300). Group E (f) severe pyknotic nucleus (200). h = Hepatocyte, s = sinusoids, v = vacuolation, lv = lipid vacuoles, hs = hemosiderin, cv = cytoplasmic vacuolation, n = necrosis, pk = pyknotic nucleus.

Please cite this article in press as: El-Barbary, M.I., Hal, A.M. Molecular identification and pathogenicity of Citrobacter and Serratia species isolated from cultured Oreochromis niloticus. Egyptian Journal of Aquatic Research (2017), https://doi.org/10.1016/j.ejar.2017.09.004 6 M.I. El-Barbary, A.M. Hal / Egyptian Journal of Aquatic Research xxx (2017) xxx–xxx aneurism with evident epithelium interstitial edema were the hemosiderin accumulation around pancreatic acinus, cytoplasmic main histological alterations observed in the gills (Plates 1–3b). vacuolation with a clearly pyknotic nucleus. The kidneys also Oreochromis niloticus infected with OS-2 (group C) showed slight showed disconnection of the renal tubules and hemorrhage congestion with dilation in sinusoids along with slight vacuola- between them with pyknotic nuclei, decrease in the glomerular tion of the hepatic cells. The kidney showed tubular degeneration component and degeneration with necrosis of renal tubular. As with interstitial mononuclear cell infiltration. In addition to that, for the gills, they exhibited epithelial lifting and hyperplasia, the gills exhibited congestion and hemorrhage in primary lamel- aneurism, congestion and collapsed secondary lamellae (Plates lae, curling and shortening in the length of secondary lamellae 1–3,d,e). Fish infected with OS-4 (group E) showed severe hepatic and proliferation of the epithelium cells (Plates 1–3c). The necrotic cells, in addition to pyknotic nuclei in the tubules, tubu- histopathological alterations in the O. niloticus infected with lar degeneration with interstitial mononuclear cell infiltration and S. marcescens were lipid vacuoles between hepatocytes, slight necrosis in the kidney while curling of secondary lamellae,

Plate 2. Histopathological changes in the kidney of O niloticus groups (A-E) stained with H&E.; Group A (control) (a) normal structure (300). Group B (b) dilation of Bowman’s space and interstitial mononuclear cell infiltration with tubular degeneration (200). Group C (c) tubular degeneration with interstitial mononuclear cell infiltration (200). Group D (d,e) hemorrhage between renal tubules with pyknotic nuclei and disconnection of renal tubules (d 200). (e) showed decrease of the glomerular component and tubular degeneration with necrosis (200). Group E (f) showing pyknotic nuclei in the tubules, tubular degeneration with interstitial mononuclear cell infiltration and necrosis (200), G = glomerulus, BS = Bowman’s space, If = infiltration, RT = Renal tubular, pk = pyknosis, He = hemorrhage.

Plate 3. Histopathological changes in the gills of O niloticus groups (A–E) stained with H&E.; Group A (control) (a) normal appearance of gill ﬁlaments and lamellae (200). Group B (b) epithelial hyperplasia, aneurism and evident epithelium interstitial edema (200). Group C (c) congestion and hemorrhage in primary lamellae, curling and shortening in the length of secondary lamellae and proliferation of the epithelium cells (200). Group D (d, e) epithelial lifting hyperplasia and aneurism of secondary lamellae and congestion (d 200). (e) showing collapsed secondary lamellae (200). Group E (f) curling of secondary lamellae, an intense lamellar epithelium lifting and lamellar fusion (200). Si = secondary lamellae, Pi = primary lamellae, El = epithelial lifting, E = edema Cu = curling of secondary lamellae, An = aneurism Sh = shortening in the length, Con = congestion. intense lamellar epithelium lifting and lamellar fusion were Voges-Proskauer positive and formed D-sorbitol, but OS-3 and recorded in the gills (Plates 1–3f). OS-4 did not produce indole; these results are in agreement with those of the previous study conducted by Farmer et al. (2007) and Discussion Donnenberg (2005). In the present study, the phenotypic and the biochemical characters of the presumptive Citrobacter and Serratia The obtained results showed that OS-1 and OS-2 formed manni- species that had been isolated only from O. niloticus liver were tol, sorbitol and rhamnose but did not form acid from inositol or attributed to the previous study of Hassan et al. (2012), the men- produce indole, urease and tryptophan. While ornithine decarboxy- tioned study had discovered that C. braakii and C. freundii were iso- lase and lysine tests were negative, some of those ﬁndings agreed lated from tilapia and the results showed that the highest number with those of Austin et al. (1992) and Jeremic et al. (2003) who of isolates was recovered from liver. The phylogenetic analysis found that there are strains of C. freundii that weakly decompose placed the bacterial isolates in the family Enterobacteriaceae based carbohydrates and ones that do so in a stronger manner. It was on 99% homology, because bacteria with >98.7% of 16S rRNA gene also found that S. marcescens (OS-3) products red pigment, sequence similarity were not considered to be different species

(Schleifer, 2009), while this percentage increased to 99% in specific interstitial hemorrhage and hydropic degeneration (Pádua et al., probes based on their 16S rRNA sequences (Brooks et al., 1992). 2014). Reports of the effects of S. marcescens on fish histology are Comparing the variable regions (V1 and V2) showed high similarity rare, Camus et al. (2013) studied the histological effects of bonnet among the studied bacteria of phylum Enterobacteriaceae. However, head shark infected with S. marcescens. the ability of V3 to identify bacterial 16S rRNA was different among strains (Mackie et al., 2000). The importance of the V1 and V2 Conclusion regions was also different in 16S rRNA gene of lactic acid bacteria, which provided strong evidence for identification (Balcázar et al., The results concluded that C. freundii, S. marcescens and Serratia 2007). Overall, 16S rRNA full sequence is desired for the character- sp. should be considered as opportunistic pathogens for fish, caus- ization of a new species (Young and Haukka, 1996) and the longer ing a 60% mortality rate in the experimental challenge test of O. sequences give more accurate taxonomic assignments (Liu et al., niloticus with severe clinical signs and histological alterations in 2007). This requires a combination of primers that are used as a part liver, kidney and gills. The antibiogram test of all identified iso- of DNA to be amplified, they can then be sequenced for identifica- lates, OS-1, OS-2, OS-3 and OS-4, revealed sensitivity to ciproflox- tion (Buller, 2004). Also, mismatch of universal primers, 519 F/R acin and norfloxacin. It was also found that sequencing of 16S rRNA could be illustrated through the observation of Mao et al. (2012) gene is a vital tool for the characterization and identification of that the differences were <5% for all of the universal primers except unknown bacteria species of fish pathogen. Additionally, the pair 519F at the phylum level. In addition, two mismatches of primer of primers (27F/1390R) was recognized as the most appropriate 519F were observed (Mori et al., 2014), when it covered eukaryotic for identification of 16S rRNA genes to cover as much as possible rRNA genes. These previous notes with the present observation of the variable regions. explain the failure of PCR amplification based on 519F/R primers with some bacterial isolates. Citrobacter and Serratia species were isolated from liver, kidney, gills and skin O. niloticus. Citrobacter fre- References undii was isolated as the most likely causative agent from the kidney, liver, spleen, gills and eroded skin of sunfish, Mola mola, Altun, S., Duman, M., Buyukekiz, A., Ozyigit, M., Karatas, S., 2013. First isolation of (Sato et al., 1982). Also, C. braakii and C. freundii were isolated from Citrobacter braakii from rainbow trout (Oncorhynchus mykiss). Isr. J. Aquacult. tilapia and their incidence was 19%, 8.1%, respectively. The highest Bamid., 1–9 Austin, B., Gibb, A., 1993. Emerging bacterial fish pathogens and their likely incidence was recovered from liver (Hassan et al., 2012). The results significance for aquaculture. In: Proceedings of the first international of antimicrobial tests in the current study revealed that all the symposium on aquaculture technology and investment opportunities. tested isolates were susceptible to ciprofloxacin and norfloxacin Ministry of agriculture and water, Riyadh, Saudi Arabia, pp. 410–425. Austin, B., Stobie, M., Robertson, P., 1992. Citrobacter freundii: the cause of gastro- that had frequently used antibiotics in aquaculture. 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