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

Sequenciamento e resistência antimicrobiana múltipla de Pseudomonas fluorescens isoladas de tilápia-do-nilo no Egito

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).

Keywords:
16SrDNA gene; Pseudomonas fluorescens; multiple antimicrobial-resistant; nile tilapia fish

Resumo

Pseudomonas fluorescens é uma das principais causas de doenças septicêmicas em peixes de água doce, causando graves perdas econômicas e diminuindo a eficiência da fazenda. Assim, esta pesquisa teve como objetivo investigar a ocorrência de P. fluorescens em peixes de tilápia-do-nilo (O. niloticus) no Egito, sequenciamento do gene 16S rDNA e suscetibilidade antimicrobiana. Cepas de P. fluorescens foram detectadas em 32% (128\400) de peixes tilápia-do-nilo aparentemente saudáveis ​​(9%; 36\400) e doentes (23%; 92\400). A maior prevalência foi observada nas brânquias dos peixes, 31,3%, seguida pelo intestino 26,9%, fígado 24,2% e rins 17,6%. Os resultados da PCR para o gene 16SrDNA de P. fluorescens mostraram o gene 16SrDNA em 30% dos isolados examinados. Além disso, a homogeneidade e uma forte relação entre cepas de P. fluorescens foi confirmada usando sequências de 16SrDNA. Além da responsabilidade do gene 16SrDNA na virulência de P. fluorescens. Os resultados dos testes de suscetibilidade antimicrobiana revelaram que todas as cepas foram resistentes à piperacilina (100%), seguida pela ceftazidima (29,7%) e cefepima (25,8%). As cepas de P. fluorescens foram altamente sensíveis à cefotaxima (74,2%), seguida pela ceftriaxona e levofloxacina (70,3% cada). Curiosamente, 29,7% das cepas de P. fluorescens eram multirresistentes a antimicrobianos (MAR).

Palavras-chave:
gene 16SrDNA; Pseudomonas fluorescens; multirresistente a antimicrobianos; tilápia-do-nilo

1. Introduction

Pseudomonas fluorescens (P. fluorescens) is a Gram-negative, rod shape organism, motile by polar flagella, and could produce fluorescent pigment (fluorescein) (Darak and Barde 2015DARAK, O. and BARDE, R.D., 2015. Pseudomonas fluorescens associated with bacterial disease in Catla catla in Marathwada region of Maharashtra. International Journal of Advanced Biotechnology and Research, vol. 6, no. 2, pp. 189-195.). 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., 2007SWAIN, P., BEHURA, A., DASH, S. and NAYAK, S., 2007. Serum antibody response of Indian major carp, Labeo rohita to three species of pathogenic bacteria; Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens. Veterinary Immunology and Immunopathology, vol. 117, no. 1-2, pp. 137-141. http://dx.doi.org/10.1016/j.vetimm.2007.02.010. PMid:17383016.
http://dx.doi.org/10.1016/j.vetimm.2007....
; Wang et al., 2009WANG, H.-R., HU, Y.-H., ZHANG, W.-W. and SUN, L., 2009. Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine. Vaccine, vol. 27, no. 30, pp. 4047-4055. http://dx.doi.org/10.1016/j.vaccine.2009.04.023. PMid:19501788.
http://dx.doi.org/10.1016/j.vaccine.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 term of Red Skin Disease which described as related stress disease that causes high economic losses in the fish farm during culturing (Zhang et al., 2009ZHANG, W.-W., HU, Y.-H., WANG, H.-L. and SUN, L., 2009. Identification and characterization of a virulence-associated protease from a pathogenic Pseudomonas fluorescens strain. Veterinary Microbiology, vol. 139, no. 1-2, pp. 183-188. http://dx.doi.org/10.1016/j.vetmic.2009.04.026. PMid:19464828.
http://dx.doi.org/10.1016/j.vetmic.2009....
). It occurs throughout the year, especially at low temperatures (Swain et al., 2007SWAIN, P., BEHURA, A., DASH, S. and NAYAK, S., 2007. Serum antibody response of Indian major carp, Labeo rohita to three species of pathogenic bacteria; Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens. Veterinary Immunology and Immunopathology, vol. 117, no. 1-2, pp. 137-141. http://dx.doi.org/10.1016/j.vetimm.2007.02.010. PMid:17383016.
http://dx.doi.org/10.1016/j.vetimm.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., 2010EISSA, N., EL-GHIET, E., SHAHEEN, A. and ABBASS, A., 2010. Characterization of Pseudomonas species isolated from tilapia “Oreochromis niloticus” in Qaroun and Wadi-El-Rayan lakes, Egypt. Global Veterinaria, vol. 5, no. 2, pp. 116-121.). The changes in histopathology of different infected fish appear in organs as the liver, kidney, gills, and skin (Khalil et al., 2010KHALIL, S., KHALIL, R., SAAD, T. and SAFAA, M., 2010. Studies on Pseudomonas septicemia among cultured Oreochromus niloticus. Journal of the Arabian Aquaculture Society, vol. 5, no. 1, pp. 55-64.; Magdy et al., 2014MAGDY, I., EL-HADY, M., AHMED, H., ELMEADAWY, S. and KENWY, A., 2014. A contribution on Pseudomonas aeruginosa infection in African catfish (Clarias gariepinus). Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol. 5, no. 5, pp. 575-588.).

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., 2004PANICKER, G., VICKERY, M.C. and BEJ, A.K., 2004. Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish. Canadian Journal of Microbiology, vol. 50, no. 11, pp. 911-922. http://dx.doi.org/10.1139/w04-085. PMid:15644908.
http://dx.doi.org/10.1139/w04-085...
; Raghunath et al., 2007RAGHUNATH, P., PRADEEP, B., KARUNASAGAR, I. and KARUNASAGAR, I., 2007. Rapid detection and enumeration of trh‐carrying Vibrio parahaemolyticus with the alkaline phosphatase‐labelled oligonucleotide probe. Environmental Microbiology, vol. 9, no. 1, pp. 266-270. http://dx.doi.org/10.1111/j.1462-2920.2006.01145.x. PMid:17227431.
http://dx.doi.org/10.1111/j.1462-2920.20...
; Rajwar and Sahgal, 2016RAJWAR, A. and SAHGAL, M., 2016. Phylogenetic relationships of fluorescent pseudomonads deduced from the sequence analysis of 16S rRNA, Pseudomonas-specific and rpoD genes. 3 Biotech, vol. 6, no. 1, pp. 80. http://dx.doi.org/10.1007/s13205-016-0386-x. PMid:28330150.
http://dx.doi.org/10.1007/s13205-016-038...
). 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., 2000DAHLLÖF, I., BAILLIE, H. and KJELLEBERG, S., 2000. rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity. Applied and Environmental Microbiology, vol. 66, no. 8, pp. 3376-3380. http://dx.doi.org/10.1128/AEM.66.8.3376-3380.2000. PMid:10919794.
http://dx.doi.org/10.1128/AEM.66.8.3376-...
; Crosby and Criddle, 2003CROSBY, L.D. and CRIDDLE, C.S., 2003. Understanding bias in microbial community analysis techniques due to rrn operon copy number heterogeneity. BioTechniques, vol. 34, no. 4, pp. 790-794. http://dx.doi.org/10.2144/03344rr01. PMid:12703304.
http://dx.doi.org/10.2144/03344rr01...
). Some studies have identified organisms with identical 16SrRNA gene sequences with significant sequence divergence in protein-encoding genes (Pernthaler and Pernthaler, 2005PERNTHALER, A. and PERNTHALER, J., 2005. Simultaneous fluorescence in situ hybridization of mRNA and rRNA for the detection of gene expression in environmental microbes. Methods in Enzymology, vol. 397, pp. 352-371. http://dx.doi.org/10.1016/S0076-6879(05)97021-3. PMid:16260302.
http://dx.doi.org/10.1016/S0076-6879(05)...
).

Pseudomonas species are naturally resistant to beta-lactam 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., 2012BENITO, N., MIRELIS, B., LUZ GÁLVEZ, M., VILA, M., LÓPEZ-CONTRERAS, J., COTURA, A., POMAR, V., MARCH, F., NAVARRO, F., COLL, P. and GURGUÍ, M., 2012. Outbreak of Pseudomonas fluorescens bloodstream infection in a coronary care unit. The Journal of Hospital Infection, vol. 82, no. 4, pp. 286-289. http://dx.doi.org/10.1016/j.jhin.2012.09.008. PMid:23103246.
http://dx.doi.org/10.1016/j.jhin.2012.09...
; Düyüncü and Ulusoy, 2019DÜYÜNCÜ, D. and ULUSOY, S., 2019. Pseudomonas fluorescens isolation from green salads and antibiotic susceptibilities of isolates. Akademik Gıda, vol. 17, no. 4, pp. 444-449.). 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., 2016WOAPPI, Y., GABANI, P., SINGH, A. and SINGH, O.V., 2016. Antibiotrophs: the complexity of antibiotic-subsisting and antibiotic-resistant microorganisms. Critical Reviews in Microbiology, vol. 42, no. 1, pp. 17-30. http://dx.doi.org/10.3109/1040841X.2013.875982. PMid:24495094.
http://dx.doi.org/10.3109/1040841X.2013....
; Düyüncü and Ulusoy, 2019DÜYÜNCÜ, D. and ULUSOY, S., 2019. Pseudomonas fluorescens isolation from green salads and antibiotic susceptibilities of isolates. Akademik Gıda, vol. 17, no. 4, pp. 444-449.).

Fish represent very high nutrition value and cheap protein for everyone in Egypt. Thus, this study was carried out to detect the occurrence of P. fluorescens in Nile Tilapia (O. niloticus) fish in Egypt using conventional and molecular methods and gene sequencing of 16SrDNA gene. In addition, the antimicrobial susceptibility of P. fluorescens strains were investigated.

2. Material and Methods

2.1. Samples collection

A total of 400 (O. niloticus) fish of various sizes (300 diseased and 100 apparently healthy) was collected from different fish markets and farms in Damietta and Kafr-Elsheikh Governorates, Egypt, from March 2019 to June 2020. Fishes were separately taken in sterile plastic bags, kept in icebox and transferred immediately to the laboratory for further examination. Fish were inspected for clinical and postmortem lesions as observed by Eissa et al. (2010)EISSA, N., EL-GHIET, E., SHAHEEN, A. and ABBASS, A., 2010. Characterization of Pseudomonas species isolated from tilapia “Oreochromis niloticus” in Qaroun and Wadi-El-Rayan lakes, Egypt. Global Veterinaria, vol. 5, no. 2, pp. 116-121..

2.2. 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)EISSA, N., EL-GHIET, E., SHAHEEN, A. and ABBASS, A., 2010. Characterization of Pseudomonas species isolated from tilapia “Oreochromis niloticus” in Qaroun and Wadi-El-Rayan lakes, Egypt. Global Veterinaria, vol. 5, no. 2, pp. 116-121..

2.3. 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., 2013MACHADO, S.G., BAZZOLLI, D.M.S. and VANETTI, M.C.D., 2013. Development of a PCR method for detecting proteolytic psychrotrophic bacteria in raw milk. International Dairy Journal, vol. 29, no. 1, pp. 8-14. http://dx.doi.org/10.1016/j.idairyj.2012.09.007.
http://dx.doi.org/10.1016/j.idairyj.2012...
). 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).

2.4. 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)THOMPSON, J.D., HIGGINS, D.G. and GIBSON, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, vol. 22, no. 22, pp. 4673-4680. http://dx.doi.org/10.1093/nar/22.22.4673. PMid:7984417.
http://dx.doi.org/10.1093/nar/22.22.4673...
. Maximum likelihood were used to carry out Phylogenetic analyses, neighbor-joining and maximum parsimony in MEGA6 (Tamura et al., 2013TAMURA, K., STECHER, G., PETERSON, D., FILIPSKI, A. and KUMAR, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, vol. 30, no. 12, pp. 2725-2729. http://dx.doi.org/10.1093/molbev/mst197. PMid:24132122.
http://dx.doi.org/10.1093/molbev/mst197...
).

2.5. 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.

2.6. 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. The sensitivity of P. fluorescens isolates against seventeen antibiotic disks (Oxoid, Ltd.) was detected with the consider of their medical need in human and veterinary field; as following: amoxicillin-clavulanic acid (AMC) (30 μg); ceftriaxone (CRO) (30 μg); imipenem (IPM) (10 μg); meropenem (MEM) (10 μg); doxycycline (Do) (30 μg); ceftazidime (CAZ) (30 μg); cefotaxime (CTX) (30 μg); cefipime (FEP) (30 μg); ciprofloxacin (CIP) (5 μg); levofloxacin (LEV) (5 μg); ofloxacin (OFX) (5 µg); pipracillin (PRL) (100); erythromycin (E) (15 µg); cefoperazone (CEP) (75); tobramycin (TOB) (10 µg); pepracillin\tazobactam (TPZ) (110 µg); and gentamicin (GN) (10 μg). The inhibition zone of antibiotic disc with strains was detected by measuring its diameter and noted as sensitive, intermediate sensitive or resistant as recorded by Foysal et al. (2011)FOYSAL, M., RAHMAN, M. and ALAM, M., 2011. Antibiotic sensitivity and in vitro antimicrobial activity of plant extracts to Pseudomonas fluorescens isolates collected from diseased fish. International Journal of Natural Sciences, vol. 1, no. 4, pp. 82-88. http://dx.doi.org/10.3329/ijns.v1i4.9733.
http://dx.doi.org/10.3329/ijns.v1i4.9733...
.

3. Results

3.1. Prevalence of P. fluorescens strains in Nile tilapia fish

P. fluorescens strains were detected in 32% (128\400) of apparently healthy (9%; 36\400) and diseased (23%; 92\400) Nile tilapia fish. Regarding the occurrence of P. fluorescens in organs and tissues of examined fish (gills, liver, kidneys and intestine) with a prevalence 22.75% (364\1600), the highest incidence was observed in gills of fish 31.3% (114/364) followed by intestine 26.9% (98/364), liver 24.2% (88\364), and kidneys 17.6%(64\364) as illustrated in Table 1.

Table 1
P. fluorescens in different organs of O. niloticus.

3.2. 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.

Figure 1
Phylogenetic relatedness of the 16SrDNA gene. Maximum-likelihood unrooted tree generated indicated clustering of the tested strain with different P. fluorescens strains.

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.

Figure 2
Sequence distance of the 16SrDNA gene of the tested strain (generated by lasergene software) showing identity range of 99.2-100% with the P. fluorescens strains included in the analysis.

3.3. Antibiotic sensitivity test results

The antibiotic sensitivity test was carried out on 364 strains of P. flourescens obtained from different organs of Nile tilapia fish. The results of In-vitro sensitivity test as shown in Table 2 demonstrated that the highest resistance of P. flourescens strains was observed to piperacillin (100%), followed by ceftazidime (29.7%), and cefepime (25.8%). Absolute moderate sensitivity was observed to tobramycin, gentamicin (100%), followed by doxycycline (92.3%), amoxicillin\clavulanic acid (89%), peperacillin\tazobactam (85.2%), erythromycin (78%), and cefepime (74.2%). The strains of P. flourescens were highly sensitive to cefotaxime (74.2%), followed by ceftriaxone and levofloxacin (70.3% each).

Table 2
The antibiotic resistance of P. fluorescens strains isolated from O. niloticus different organs.

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).

Table 3
Multiple Antimicrobial Resistance of P. fluorescense isolates.

4. 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, 2015DARAK, O. and BARDE, R.D., 2015. Pseudomonas fluorescens associated with bacterial disease in Catla catla in Marathwada region of Maharashtra. International Journal of Advanced Biotechnology and Research, vol. 6, no. 2, pp. 189-195.). In Egyptian farms, P. fluorescens, was identified in different fish species as the main etiological agents of Pseudomonas septicemia (Swain et al., 2007SWAIN, P., BEHURA, A., DASH, S. and NAYAK, S., 2007. Serum antibody response of Indian major carp, Labeo rohita to three species of pathogenic bacteria; Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens. Veterinary Immunology and Immunopathology, vol. 117, no. 1-2, pp. 137-141. http://dx.doi.org/10.1016/j.vetimm.2007.02.010. PMid:17383016.
http://dx.doi.org/10.1016/j.vetimm.2007....
; El-Sayyad et al., 2010EL-SAYYAD, H.I., ZAKI, V.H., EL-SHEBLY, A.M. and EL-BADRY, D.A., 2010. Studies on the effects of bacterial diseases on skin and gill structure of Clarias gariepinus in Dakahlia Province, Egypt. Annals of Biological Research, vol. 1, no. 4, pp. 106-118.; Khalil et al., 2010KHALIL, S., KHALIL, R., SAAD, T. and SAFAA, M., 2010. Studies on Pseudomonas septicemia among cultured Oreochromus niloticus. Journal of the Arabian Aquaculture Society, vol. 5, no. 1, pp. 55-64.). In this study, P. fluorescens overall prevalence was 32% in apparently healthy and diseased fish. P. fluorescens incidence in different fish organs was 22.75%. This result was consistent with Eissa et al. (2010)EISSA, N., EL-GHIET, E., SHAHEEN, A. and ABBASS, A., 2010. Characterization of Pseudomonas species isolated from tilapia “Oreochromis niloticus” in Qaroun and Wadi-El-Rayan lakes, Egypt. Global Veterinaria, vol. 5, no. 2, pp. 116-121., who reported 30.83% Pseudomonas infections, with 23.3% P. fluorescens among the examined fish. In contrast, El-Hady and Samy (2011)EL-HADY, M.A. and SAMY, A., 2011. Molecular typing of Pseudomonas species isolated from some cultured fishes in Egypt. Global Veterinaria, vol. 7, no. 6, pp. 576-580. identified 55.3% of Pseudomonas species with 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., 2015YOUNES, A., MOHAMED, L.A., EIDA, M. and GAAFAR, A., 2015. Characterization and pathogen challenge of Pseudomonas species from Oreochromis niloticus in Egypt. Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol. 6, no. 1, pp. 312-317.; Abd El Tawab et al., 2016ABD EL TAWAB, A.A., MAAROUF, A.A. and AHMED, N.M., 2016. Detection of virulence factors of Pseudomonas species isolated from fresh water fish by PCR. Benha Veterinary Medical Journal, vol. 30, no. 1, pp. 199-207. http://dx.doi.org/10.21608/bvmj.2016.31364.
http://dx.doi.org/10.21608/bvmj.2016.313...
).

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., 2016ABD EL TAWAB, A.A., MAAROUF, A.A. and AHMED, N.M., 2016. Detection of virulence factors of Pseudomonas species isolated from fresh water fish by PCR. Benha Veterinary Medical Journal, vol. 30, no. 1, pp. 199-207. http://dx.doi.org/10.21608/bvmj.2016.31364.
http://dx.doi.org/10.21608/bvmj.2016.313...
).

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., 2004SCARPELLINI, M., FRANZETTI, L. and GALLI, A., 2004. Development of PCR assay to identify Pseudomonas fluorescens and its biotype. FEMS Microbiology Letters, vol. 236, no. 2, pp. 257-260. http://dx.doi.org/10.1111/j.1574-6968.2004.tb09655.x. PMid:15251205.
http://dx.doi.org/10.1111/j.1574-6968.20...
; Tsai et al., 2012TSAI, M.A., HO, P.Y., WANG, P.C., E, Y.J., LIAW, L.L. and CHEN, S.C., 2012. Development of a multiplex polymerase chain reaction to detect five common Gram‐negative bacteria of aquatic animals. Journal of Fish Diseases, vol. 35, no. 7, pp. 489-495. http://dx.doi.org/10.1111/j.1365-2761.2012.01372.x. PMid:22571515.
http://dx.doi.org/10.1111/j.1365-2761.20...
). 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., 2015YOUNES, A., MOHAMED, L.A., EIDA, M. and GAAFAR, A., 2015. Characterization and pathogen challenge of Pseudomonas species from Oreochromis niloticus in Egypt. Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol. 6, no. 1, pp. 312-317.; Okasha et al., 2016OKASHA, L., AMMAR, A., EL-HADY, M., SAMIR, A., SAMY, A., ABDELMEGEID, M. and EL-JAKEE, J., 2016. Identification of common fish bacterial pathogens in Kafr El Sheikh governorate, Egypt using PCR. International Journal of Biology, Pharmacy and Allied Sciences, vol. 5, pp. 522-537.; El-Gamal et al., 2018EL-GAMAL, A., EL-GOHARY, M. and GAAFAR, A., 2018. Detection and molecular characterization of some bacteria causing skin ulceration in cultured nile tilapia (Oreochromis niloticus) in Kafr El-Sheikh Governorate. International Journal of Zoological Research, vol. 14, no. 1, pp. 14-20.).

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)ANZAI, Y., KIM, H., PARK, J.-Y., WAKABAYASHI, H. and OYAIZU, H., 2000. Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. International Journal of Systematic and Evolutionary Microbiology, vol. 50, no. 4, pp. 1563-1589. http://dx.doi.org/10.1099/00207713-50-4-1563. PMid:10939664.
http://dx.doi.org/10.1099/00207713-50-4-...
and Yamamoto et al. (2000)YAMAMOTO, S., KASAI, H., ARNOLD, D.L., JACKSON, R.W., VIVIAN, A. and HARAYAMA, S., 2000. Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes. The GenBank accession numbers for the sequences determined in this work are: gyrB, D37926, D37297, D86005–D86019 and AB039381–AB039492; rpoD, D86020–D86036 and AB039493–AB039624. Microbiology, vol. 146, no. 10, pp. 2385-2394. http://dx.doi.org/10.1099/00221287-146-10-2385. PMid:11021915.
http://dx.doi.org/10.1099/00221287-146-1...
. The sequence of amplified nucleotides appears 99.2-100% identity with the P. fluorescens 16SrDNA sequence described by (Radisic et al., 2020RADISIC, V., NIMJE, P.S., BIENFAIT, A.M. and MARATHE, N.P., 2020. Marine plastics from Norwegian west coast carry potentially virulent fish pathogens and opportunistic human pathogens harboring new variants of antibiotic resistance genes. Microorganisms, vol. 8, no. 8, pp. 1200. http://dx.doi.org/10.3390/microorganisms8081200. PMid:32784594.
http://dx.doi.org/10.3390/microorganisms...
). 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., 2013CHEN, L., JIA, R.-B. and LI, L., 2013. Bacterial community of iron tubercles from a drinking water distribution system and its occurrence in stagnant tap water. Environmental Science. Processes & Impacts, vol. 15, no. 7, pp. 1332-1340. http://dx.doi.org/10.1039/c3em00171g. PMid:23702591.
http://dx.doi.org/10.1039/c3em00171g...
), BMS 2 (MT752968) has 99.3% identity with K10 (KT767974) isolated from milk in China (Xin et al., 2017XIN, L., MENG, Z., ZHANG, L., CUI, Y., HAN, X. and YI, H., 2017. The diversity and proteolytic properties of psychrotrophic bacteria in raw cows’ milk from North China. International Dairy Journal, vol. 66, pp. 34-41. http://dx.doi.org/10.1016/j.idairyj.2016.10.014.
http://dx.doi.org/10.1016/j.idairyj.2016...
) and BMS 3 (MT753009) has 99.8% identity with PF74 (MF838661) which isolated from dairy product in Italy (Rossi et al., 2018ROSSI, C., SERIO, A., CHAVES-LÓPEZ, C., ANNIBALLI, F., AURICCHIO, B., GOFFREDO, E., CENCI-GOGA, B.T., LISTA, F., FILLO, S. and PAPARELLA, A., 2018. Biofilm formation, pigment production and motility in Pseudomonas spp. isolated from the dairy industry. Food Control, vol. 86, pp. 241-248. http://dx.doi.org/10.1016/j.foodcont.2017.11.018.
http://dx.doi.org/10.1016/j.foodcont.201...
).

Table 4
Overview of the different P. fluorescens 16SrDNA genes detected in nucleotide sequences of the isolates with percent of identity%.

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, 2009EL-HADY, M. and EL-KHATIB, N., 2009. Isolation and characterization of some pathogenic agents causing wobbling syndrome in cultured Oreochromis niloticus. Mediterranean Aquaculture Journal, vol. 2, no. 1, pp. 35-44. http://dx.doi.org/10.21608/maj.2009.2668.
http://dx.doi.org/10.21608/maj.2009.2668...
; Darak and Barde, 2015DARAK, O. and BARDE, R.D., 2015. Pseudomonas fluorescens associated with bacterial disease in Catla catla in Marathwada region of Maharashtra. International Journal of Advanced Biotechnology and Research, vol. 6, no. 2, pp. 189-195.; Younes et al., 2015YOUNES, A., MOHAMED, L.A., EIDA, M. and GAAFAR, A., 2015. Characterization and pathogen challenge of Pseudomonas species from Oreochromis niloticus in Egypt. Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol. 6, no. 1, pp. 312-317.; Abd El Tawab et al., 2016ABD EL TAWAB, A.A., MAAROUF, A.A. and AHMED, N.M., 2016. Detection of virulence factors of Pseudomonas species isolated from fresh water fish by PCR. Benha Veterinary Medical Journal, vol. 30, no. 1, pp. 199-207. http://dx.doi.org/10.21608/bvmj.2016.31364.
http://dx.doi.org/10.21608/bvmj.2016.313...
). The resistance against penicillin group in P. fluorescens was common worldwide (Ghosh et al., 2011GHOSH, A., DAS, B.K., ROY, A. and CHANDRA, G., 2011. Antibiotic resistance and herbal treatment of bacterial fish pathogens causing epizootic ulcerative syndrome. Journal of Herbs, Spices & Medicinal Plants, vol. 17, no. 1, pp. 47-51. http://dx.doi.org/10.1080/10496475.2011.560082.
http://dx.doi.org/10.1080/10496475.2011....
; Younes et al., 2015YOUNES, A., MOHAMED, L.A., EIDA, M. and GAAFAR, A., 2015. Characterization and pathogen challenge of Pseudomonas species from Oreochromis niloticus in Egypt. Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol. 6, no. 1, pp. 312-317.; Abd El Tawab et al., 2016ABD EL TAWAB, A.A., MAAROUF, A.A. and AHMED, N.M., 2016. Detection of virulence factors of Pseudomonas species isolated from fresh water fish by PCR. Benha Veterinary Medical Journal, vol. 30, no. 1, pp. 199-207. http://dx.doi.org/10.21608/bvmj.2016.31364.
http://dx.doi.org/10.21608/bvmj.2016.313...
); 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., 2014MAGDY, I., EL-HADY, M., AHMED, H., ELMEADAWY, S. and KENWY, A., 2014. A contribution on Pseudomonas aeruginosa infection in African catfish (Clarias gariepinus). Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol. 5, no. 5, pp. 575-588.; Roy et al., 2014ROY, R.P., BAHADUR, M. and BARAT, S., 2014. Studies on antibiotic resistant activity of Pseudomonas sp., isolated from fresh water loach, Lepidocephalichthys guntea and water sample of river Lotchka, Darjeeling, India. Journal of Environmental Biology, vol. 35, no. 1, pp. 237.). As well, their resistance to the cephalosporine group was previously observed (Abd El Tawab et al., 2016ABD EL TAWAB, A.A., MAAROUF, A.A. and AHMED, N.M., 2016. Detection of virulence factors of Pseudomonas species isolated from fresh water fish by PCR. Benha Veterinary Medical Journal, vol. 30, no. 1, pp. 199-207. http://dx.doi.org/10.21608/bvmj.2016.31364.
http://dx.doi.org/10.21608/bvmj.2016.313...
). On the contrary, P. fluorescens strains isolated from water in Paris were resistant to carbapenems (Girlich et al., 2010GIRLICH, D., POIREL, L. and NORDMANN, P., 2010. Novel Ambler class A carbapenem-hydrolyzing β-lactamase from a Pseudomonas fluorescens isolate from the Seine River, Paris, France. Antimicrobial Agents and Chemotherapy, vol. 54, no. 1, pp. 328-332. http://dx.doi.org/10.1128/AAC.00961-09. PMid:19901091.
http://dx.doi.org/10.1128/AAC.00961-09...
). Maja et al. (1996)MAJA, M., MARINA, R., SNEZANA, C. and LEVNAIC, D., 1996. Massive death of silver carp (Hypophthalmichthys molitrix Val.) and big head (Aristichthys nobilis Rich.) caused by Pseudomonas fluorescens bacteria. Veterinarski Glasnik-Beograd, vol. 50, pp. 761-766. 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 29.7% of P. flourescens strains. Godebo et al. (2013)GODEBO, G., KIBRU, G. and TASSEW, H., 2013. Multidrug-resistant bacterial isolates in infected wounds at Jimma University Specialized Hospital, Ethiopia. Annals of Clinical Microbiology and Antimicrobials, vol. 12, no. 1, pp. 17. http://dx.doi.org/10.1186/1476-0711-12-17. PMid:23879886.
http://dx.doi.org/10.1186/1476-0711-12-1...
detected 19.3% multidrug resistant strains of P. aeruginosa isolated from wounds in Ethiopia. On the other hand, Düyüncü and Ulusoy (2019)DÜYÜNCÜ, D. and ULUSOY, S., 2019. Pseudomonas fluorescens isolation from green salads and antibiotic susceptibilities of isolates. Akademik Gıda, vol. 17, no. 4, pp. 444-449. 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.

5. 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.

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Publication Dates

  • Publication in this collection
    16 Mar 2022
  • Date of issue
    2024

History

  • Received
    09 Oct 2021
  • Accepted
    08 Feb 2022
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