Sequencing and multiple antimicrobial resistance of Pseudomonas fluorescens isolated from Nile tilapia fish in Egypt

Pseudomonas fluorescens (P. fluorescens) is a Gram-negative, rod shape organism, motile by polar flagella, and could produce fluorescent pigment (fluorescein) (Darak and Barde 2015). P. fluorescens is a common aquaculture opportunistic psychrotrophic microorganism, cause pseudomoniasis in a vertebrate as fresh and salts water fish, and invertebrates as shrimp, wide-spreading in soil, water, plants, and animals grows, and can grow at 25-30 °C (Swain et al., 2007; Wang et al., 2009). Pseudomoniasis; the disease caused by P. fluorescens, is usually characterized by ulcerations on the body among fishes, septicemia, petechial hemorrhages, skin darkness, and scales detachment, abdominal ascites and exophthalmia. These pathogenic signs carry the medical Abstract Pseudomonas fluorescens is one of the main causes of septicemic diseases among freshwater fish, causing severe economic losses and decreasing farm efficiency. Thus, this research was aimed to investigate the occurrence of P. fluorescens in Nile Tilapia (O. niloticus) fish in Egypt, gene sequencing of 16SrDNA gene, and antimicrobial susceptibility. P. fluorescens strains were detected in 32% (128\400) of apparently healthy (9%; 36\400) and diseased (23%; 92\400) Nile tilapia fish. The highest prevalence was observed in gills of fish, 31.3% followed by intestine 26.9%, liver 24.2%, and kidneys 17.6%. The PCR results for the 16SrDNA gene of P. fluorescens showed 16SrDNA gene in 30% of examined isolates. Moreover, Homogeny and a strong relationship between strains of P. fluorescens was confirmed using 16SrDNA sequences. Beside the responsibility of 16SrDNA gene on the virulence of P. fluorescens. The results of antimicrobial susceptibility tests revealed that all strains were resistant to piperacillin (100%), followed by ceftazidime (29.7%), and cefepime (25.8%). The strains of P. fluorescence were highly sensitive to cefotaxime (74.2%), followed by ceftriaxone and levofloxacin (70.3% each). Interestingly, 29.7% of strains of P. fluorescens were multiple antimicrobial-resistant (MAR).


Isolation and identification of P. fluorescens
A total of 4 samples (gills, liver, kidneys, and intestine) from each fish were taken for bacteriological analysis. Each sample was inoculated onto MaCconkey ҆ s agar and Bacto Pseudomonas Agar F (Oxoid) and incubated aerobically at 25°C for 24 h. Suspected colonies of P. fluorescens were examined morphologically (Gram ҆ s staining), motility, and biochemically (oxidase, catalase, triple sugar iron, and citrate utilization tests) as identified by Eissa et al. (2010).

Genotypic detection of 16SrDNA gene in P. flourescens
A total of 50 representative P. flourescens strains were subjected to a polymerase chain reaction for detection of 16SrDNA gene which is responsible for bacterial virulence. The amplification and purification of P. fluorescens16SrDNA gene was carried out using the QIAamp DNA mini kit (QIAGEN) Catalogue no.51304. The 16SrDNA from the strains was amplified using the primers F (5´-TGCATTCAAAACTGACTG-3´) and R (5´-AATCACACCGTGGTAACCG -3´) (Machado et al., 2013). PCR was carried out in a total volume of 25 μl containing 12.5 μl of Emerald Amp GT PCR mastermix (Takara) (2x premix), 1 μl of each primer (20 pmol), 6 μl Template DNA and 4.5 μl PCR grade water. The cycling conditions consisted in a first denaturation step at 94 °C for 5 min followed by 35 cycles of amplification (denaturation at 94 °C for 30 sec, annealing at 48 °C for 40 sec and extension at 72 °C for 1 min) followed by a final extension at 72 °C for 10 min. PCR products were electrophoretically separated in 1% agarose gels and visualized in a Gel Documentation (Biometra, Germany).

Sequencing and analysis of the 16SrDNA in P. fluorescens
Purified PCR product from three representative strains was sequenced in the forward and/ or reverse directions on an Applied Biosystems 3130 automated DNA Sequencer (ABI, 3130, USA) using a ready reaction Bigdye Terminator V3.1 cycle sequencing kit (Perkin-Elmer/Applied Biosystems, Foster City, CA). On GenBank the defrentiation among sequences of our strains and other strains was done by applying PSI-BLAST research (NCBI) and BLAST 2.0. Program of multiple sequence alignment was performed to demonstrate and analyze the sequences, using Pairwise software of Lasergene DNA Star, version 1.83 module of MegAlign, which was designed by Thompson et al. (1994). Maximum likelihood were used to carry out Phylogenetic analyses, neighbor-joining and maximum parsimony in MEGA6 (Tamura et al., 2013).

Nucleotide accession number
The deposition of nucleotide sequences of P. fluorescencs 16SrDNA gene were done in GenBank taking MT752964, MT752968 and MT753009 as accession numbers.

Antibiotic sensitivity test
In vitro sensitivity test for P. fluorescens isolates was done as recorded by the Clinical and Laboratory Standards Institute using technique of standard diffusion disk. term of Red Skin Disease which described as related stress disease that causes high economic losses in the fish farm during culturing . It occurs throughout the year, especially at low temperatures (Swain et al., 2007). Pseudomonas in fish leading to skin darkness, fin rot and detachment in scales. Infected fish also suffer from ascites, hemorrhages and exophthalmia (Eissa et al., 2010). The changes in histopathology of different infected fish appear in organs as the liver, kidney, gills, and skin (Khalil et al., 2010;Magdy et al., 2014).
Biochemical and physiological identification need more time than polymerase chain reaction (PCR) as molecular techniques aid in rapidly and accurately identifying Pseudomonas isolates (Panicker et al., 2004;Raghunath et al., 2007;Rajwar and Sahgal, 2016). Furthermore, the sequencing of 16SrRNA genes could identify an organism by reconstructing its phylogeny, along with the possibility of storing sequences in databases, resulting in the rapid adoption of the 16SrRNA gene by microbiologists. It is also created by multiple heterogeneous copies of the 16SrRNA gene within a genome (Dahllöf et al., 2000;Crosby and Criddle, 2003). Some studies have identified organisms with identical 16SrRNA gene sequences with significant sequence divergence in protein-encoding genes (Pernthaler and Pernthaler, 2005).
Pseudomonas species are naturally resistant to betalactam group antibiotics and can quickly develop resistance to antibiotics depending on their various properties. Although there are a limited number of studies about the opportunistic pathogen as P. fluorescens, it is also important to investigate the antibiotic resistance of P. fluorescens isolates, as they are one of the species frequently isolated from environmental samples and are closely related to public health (Benito et al., 2012;Düyüncü and Ulusoy, 2019). The overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, including foodborne pathogens, making it difficult to treat infections caused by these pathogens (Woappi et al., 2016;Düyüncü and Ulusoy, 2019).

Genotypic identification and sequence analysis of the 16SrDNA of P. fluorescens
PCR technique for 16SrDNA gene was applied on 50 representatives P. fluorescens strains isolated from different organs (liver, gills, kidneys, and intestine) of diseased Nile tilapia fish. The PCR results for 16SrDNA gene of P. fluorescens showed 16SrDNA gene in 30% (15\50) of examined isolates. The 16SrDNA gene was amplified, giving product of 850 bp. Additionally, 16SrDNA sequencing analyses and confirms that P. fluorescens isolates are closely related, besides the responsibility of 16SrDNA gene on the virulence of P. fluorescens. Three isolates (BMS1, BMS2 and BMS3) were subjected to collected 16S rRNA P. fluorescens sequences analysis. The close relation and comparison between Studied isolates and other P. fluorescens strains were represented phylogenetic tree found in Figure 1.
The sequences of 16SrDNA gene isolates were assembled by statistics program and GenBank deposition numbers. The nucleotide sequences of three sequence-analysed isolates were deposited in GenBank under submitted numbers MT752964, MT752968 and MT753009 as in Figure 2.
Interestingly, the strains of P. fluorescens resistant to more than two antibiotic classes were considered multiple antimicrobial-resistant strains, and characterized by Multiple Antimicrobial Resistant phenomena (MAR). A total of 108 out of 364 (29.7%) strains of P. fluorescens isolates were considered multiple antimicrobial-resistant, as in Table 3. All tested strains are resist to pepracillin (penicillin groups), and ceftazidime (Cephalosporins).

Discussion
P. fluorescens is one of the most pathogenic microorganism pathogens where it was found to be associated with various freshwater fish diseases throughout the world (Darak and Barde, 2015). In Egyptian farms,  P. fluorescens, was identified in different fish species as the main etiological agents of Pseudomonas septicemia (Swain et al., 2007;El-Sayyad et al., 2010;Khalil et al., 2010). In this study, P. fluorescens overall prevalence was 32% in apparently healthy and diseased fish.   55.4% P. fluorescence from naturally infected O. niloticus in different localities of Egypt. Overall, a wide range of P.fluorescens incidence was previously reported from 18.8-60% in diseased Nile tilapia fish in Egypt (Younes et al., 2015;Abd El Tawab et al., 2016). In this investigation, regarding type of organ samples, the highest incidence was observed in gills (31.3%) followed by the intestine (26.9%), liver (24.2%), and kidneys (17.6%). On the contrary, the previous study demonstrated that the high occurrence of P. fluorescens was in kidneys (50.0%), followed by liver (33.3%), gill (16.7%) and (0.0%) from skin (Abd El Tawab et al., 2016).
Morphological and biochemical methods as conventional methods for diagnosing bacterial infections in fish are complex and time-consuming for reaching a definitive diagnosis. Consequently, polymerase chain reaction (PCR) has been used to rapidly diagnose P. fluorescens as a member of Pseudomonas group (Scarpellini et al., 2004;Tsai et al., 2012). In the current study, PCR was performed on P. flourescens isolates to detect 16SrDNA gene that is specific for P. fluorescens and responsible for bacterial virulence. The 16SrDNA gene was detected in 30% of examined strains of P. fluorescens. Similarly, previous studies amplify 16SrDNA gene of P. fluorescens strains isolated from diseased fish (Younes et al., 2015;Okasha et al., 2016;El-Gamal et al., 2018). In this research, 16SrDNA sequencing is important for confirmation the strong connections between P. fluorescens strains. Besides the responsibility of 16SrDNA gene on the virulence of P.fluorescens. Databases of sequencing explaining the close matching between P. fluorescens strains of P. fluorescens complex as stated by Anzai et al. (2000) and Yamamoto et al. (2000). The sequence of amplified nucleotides appears 99.2-100% identity with the P. fluorescens 16SrDNA sequence described by (Radisic et al., 2020). 16SrRNA sequence results were related with the phenotype identification of the Pseudomonas strains. Generally, the 3 isolates (BMS1, BMS2 and BMS3) were recorded due to high agreement and good phenotypic matching with genotypic identification procedures. Genotypically these isolates were identified as P. fluorescens.
Accession number in GenBank and statistics of gene sequencing of 16SrDNA gene of P. fluorescens isolates are represented in Table 4. In this study, BMS 1(MT752964) has 99.5% identity with SX001 (HM631729) isolated from water and soil in China (Chen et al., 2013), BMS 2 (MT752968) has 99.3% identity with K10 (KT767974) isolated from milk in China (Xin et al., 2017) and BMS 3 (MT753009) Table 4. Overview of the different P. fluorescens 16SrDNA genes detected in nucleotide sequences of the isolates with percent of identity%.

Strains
Other isolates of Identity has 99.8% identity with PF74 (MF838661) which isolated from dairy product in Italy (Rossi et al., 2018). The antimicrobial characteristics of pathogenic P. fluorescens isolates passed with many resistance steps to different classes of antobiotics. P. fluorescens. The results of in-vitro sensitivity test for the P. fluorescens strains revealed high resistance to piperacillin (100%), followed by ceftazidime (29.7%), and cefepime (25.8%), meanwhile high sensitivity was observed to the most examined antibiotics, particularly cefotaxime ceftriaxone, and levofloxacin. The sensitivity of P. fluorescens to different classes of antibiotics as aminoglycosides, quinolones and chloramphenicol were previously detected (El-Hady and El-Khatib, 2009;Darak and Barde, 2015;Younes et al., 2015;Abd El Tawab et al., 2016). The resistance against penicillin group in P. fluorescens was common worldwide (Ghosh et al., 2011; Younes et al., 2015;Abd El Tawab et al., 2016); thus, this antibiotic group cannot be used as therapeutic agents for treatment. Also, previous investigations detected the resistance of P. fluorescens to amoxicillin, cephalothin, erythromycin, lincomycin, nitrofurantoin and sulphamethoxazole-trimethoprim (Magdy et al., 2014;Roy et al., 2014). As well, their resistance to the cephalosporine group was previously observed (Abd El Tawab et al., 2016). On the contrary, P. fluorescens strains isolated from water in Paris were resistant to carbapenems (Girlich et al., 2010). Maja et al. (1996) noted the susceptibility of P. fluorescens to gentamicin, kanamycin and neomycin, as well as their resistance to chloramphenicol, erythromycin, penicillin and sulphonamides.
The emergence of multidrug-resistant strains against multiple antibacterial classes threatens humans and animals worldwide. In this study, MAR was detected in  and Research, vol. 6, no. 2, pp. 189-195. DÜYÜNCÜ, D. and ULUSOY, S., 2019. Pseudomonas fluorescens isolation from green salads and antibiotic susceptibilities of isolates. Akademik Gıda,vol. 17,no. 4, EISSA, N., EL-GHIET, E., SHAHEEN, A. and ABBASS, A., 2010.  (2019) found that all examined P. fluorescens isolates were multidrug-resistant. Development of mechanisms of antimicrobial resistance, failing control of infection, ineffective treatment, prolonged suffering and may lead to death.

Conclusion
In this study, P. fluorescens strains was isolated from Nile tilapia (O. niloticus) organs (gills, liver, kidney and intestine), Biochemical identification was done and confirmed using PCR and gene sequencing with deposition of studied strains in Genbank. Also Antibiotic sensitivity test was carried out with estimation of multiple antimicrobial resistance. We detected that the prevalence of P. fluorescens as a common opportunistic bacterium threatens industrial aquaculture and affects income of fish culturing in Egypt. The emergence of MAR strains of P. fluorescens in freshwater fish is alarming. As a rule, precautions of good hygiene must be performed to control infection with P. fluorescens in freshwater fish. Also, treatment assays implemented to deal with antibiotic usage should be considered in Egyptian fish farms.