Molecular Study on Some Virulence and Fluoroquinolone Resistance Genes of Pseudomonas aeruginosa Isolated from Naturally Infected Cultured Sea Bream Fish (Sparus aurata) in EgyptEman Khalifa1*, Mohamed Khallaf2 and Mahmoud Hashem3
Received Date: September 19, 2016; Accepted Date: October 16, 2016; Published Date: October 23, 2016
Citation: Khalifa E, Khallaf M, Hashem M (2016) Molecular Study on Some Virulence and Fluoroquinolone Resistance Genes of Pseudomonas aeruginosa Isolated from Naturally Infected Cultured Sea Bream Fish (Sparus aurata) in Egypt. J Infect Dis Preve Med 4:136. doi: 10.4172/2329-8731.1000136
Copyright: © 2016 Khalifa E, 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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This study carried out to investigate the presence of some virulence and fluoroquinolone resistant genes by PCR and comparing with antibiotic diffusion disc for isolated P. aeruginosa. A total number of 100 alive cultured Sea bream, showing clinical signs were collected from a private fish farm at Damietta, Egypt then subjected to clinical, postmortem (PM), bacteriological examination, biochemical and serological identification of the isolated bacteria, P. aeruginosa were more prevalent and subjected to VITEK2 and PCR for detection of outer membrane lipoprotein gene (oprL) at "504 bp" and exotoxin A gene (toxA) at "270 bp" which indicated the virulent isolates of P. aeruginosa and DNA gyrAse (gyrA) at "287 bp" and topoisomerase IV (parC) at "267 bp" for determining fluoroquinolone resistance genes compared with antibiotic sensitivity test by disc diffusion using 3 of fluoroquinolone members. The more prevalent isolates were P. aeruginosa (43.02%), of them 12 isolated showed by PCR the presence of oprL, toxA, gyrA and parC genes and confirmed with antibiogram resistance to tested 3 fluoroquinolone members. The present study explored that pathogenic and fluoroquinolone resistant isolates of P. aeruginosa were more prevalent that necessitate more rapid hygienic programs and narrow uses of antibiotics for Sea bream aquaculture fisheries in Egypt.
P. aeruginosa; PCR; Virulence; Fluoroquinolone; Sea bream
Sea bream (Sparus aurata) is straightaway one of the devastating consumable species around the globe. The world catch establishment of this species in 2004 and was around 10% of the total aquaculture yield . Fish is a principal inception of food for people and contributes around 60% of the worldwide's supply of protein. 60% of the producing countries decide 30% of their yearly protein from fish . Sea bream (Sparus aurata) fisheries were known starting late in Egypt and necessity for element progression especially in feeding and microbiological aspects .
Opportunistic diseases brought about by P. aeruginosa are a genuine medicinal issue, and quinolone resistant P. aeruginosa could be recovered from clinical cases. As of not long ago, the gyrA mutation in quinolone resistance has been examined in a few species of bacteria [4-6]. Albeit numerous studies have concentrated on E. coli  and S. aureus , concentrates on with P. aeruginosa are less cutting-edge. As of late,  cloned the wild kind of the gyrA gene in P. aeruginosa and watched the Thr-83-Ile, Asp-87-Asn, and Asp-87-Tyr mutations in gyrA from clinical isolates.
Fluoroquinolones are basic category of antimicrobial operators used as a part of the treatment of Pseudomonas infections. Fluoroquinolones are members from the quinolone family which act as bactericidal workers by limiting bacterial DNA gyrase and topoisomerase IV, therefore quelling DNA transcription and replication. DNA gyrase is ordinarily the target in Gram -ve microorganisms; Pseudomonas speedily makes resistance to these professionals, along these lines diminishing their effectiveness. The rule segments of resistance are mutations in the genes that encode DNA gyrase (gyrA) and topoisomerase IV (parC). Fluoroquinolone resistance can provoke treatment dissatisfaction in P. aeruginosa infection .
Regular transformations in fluoroquinolone resistant strains happened in codons 83 and 87 of gyrA gene . No mutations were found in parC. A novel transformation identified with fluoroquinolone resistance in codon 126 of the mexR gene, altering amino acid Val to Glu, was distinguished .
Infections are fortified by climatic changes that consider antagonistically the marine environment, which is a conventional media for different microbes and initiated by human interference that lead not simply to fabricate the damaging tendency of these pathogens also to the rise and advancements of new microorganisms not present some time as of late .
Septicemic bacterial infection with Pseudomonads, Vibrios, Photobacteria, Aeromonads, Streptococci and Staphylococci have been found in some fingerlings, juveniles, grown-ups and brood supplies of some marine fish species . This may to some degree recognized to the general existence of such pathogens in seawater, residue and furthermore digestive tract of marine fishes . Hence, the increase of rotted normal environments, similar to change in temperature or hardship of supplement would ordinarily drive them to effervescently beating fish .
The predominant parts of bacterial infection in fish are achieved by Gram -ve organisms including: Pseudomonas, Vibrio, Aeromonas, Citrobacter, Edwardsiella and Streptococcus as Gram +ve family. These species may be the vital causes of some fish disorders. Most of bacterial fish pathogens are specific inhabitants of the marine environment or fresh water .
New and developing ecological pathogens represent a portion of the best dangers to current aquaculture, a basic wellspring of nourishment protein worldwild. Likewise with other serious cultivating works on, expanding our comprehension of the science of diseases is vital to enhance creature welfare and farming .
In this investigation, a total number of 100 alive cultured Sea bream fish (Sparus aurata) with different body weight ranged from (50 g to 150 g) showing abnormal signs were collected from a private fish farm at Mahallat El-Diba region at Damietta governorate, Egypt. The sources of water in that region are numerous; drainage of canal originated from El-Manzalah Lake and El-headway downstream from Mediterranean Sea. Fish were clinically examined  and transferred in ice box soon to the laboratory located in the same farm where sampling occurs,. The freshly dead collected Sea bream fish were subjected to full postmortem (PM) examination .
Bacterial isolation and identification
Samples collected after fish surfaces swapped with 70% ethyl alcohol for surface sterilization and then inoculum were taken from liver, kidney, spleen and heart under complete aseptic condition and cultured on Trypticase Soya Broth+3% NaCl (Difco®) and incubated at 25oC for 24-48 hrs then subcultured on different types of media for primary isolation and identification; Trypticase Soya agar+3% NaCl (TSA); Pseudomonas base agar (Himdia®) with Cetrimide-Fucidin- Cephalosporin (CFC) supplement (Oxoid®), Thiosulphate citrate bile salt sucrose agar (TCBS) (Oxoid®), Blood agar media using 5% sheep RBCs for hemolytic activity, MacConkey bile salt lactose agar (Oxoid®), Mannitol salt agar medium (Oxoid®), Semi–solid 0.5% agar medium for preservation of bacterial isolates, Muller-Hinton agar (oxoid®) for antibiogram activity. The inoculated plates were incubated at 25oC for 24-48 hrs for bacteriological examination , biochemical identification by conventional test . Followed by serological identification of the isolated bacteria; the anti-sera for slide agglutination test used for serotyping of the bacterial isolates were obtained from (Deben Diagnostics Ltd, United Kingdom) and performed at Lab. of Fish Diseases at El Wafaa hatchery center – Ismailia governorate. Further confirmatory identification of the more prevalent isolated P. aeruginosa by automatically colorimetric biochemical confirmation VITEK2 COMPACT SYSTEM (BIOMERIUX, FRANCE) according to manufacturer’s instructions by matching the results of the bacterial isolates with those involved in its data base platform. After these, PCR assay performed as followed
Molecular characterization of isolated P. aeruginosa
• Extraction of DNA of isolated P. aeruginosa using the Qiagen Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions and was used as the DNA template for PCR.
• The oligonucleotide primers were synthesized and supplied by Metabion (Germany) which indicated the virulent isolates of P. aeruginosa, Table 1 and Table 2 were used.
• Conventional PCR used to explore the presence of DNA gyrAse (gyrA) at "287 bp" and topoisomerase IV (parC) at "267 bp"  for detection of fluoroquinolone resistance genes of P. aeruginosa isolated from naturally infected cultured Sea bream fish.
|Gene||Primer Sequence(5' - 3')||Size(bp)||Reference|
|toxA||F||CTGCGCGGGTCTATGTGCC||270||Stover et al. |
|oprL||F||ATG GAA ATG CTG AAA TTC GGC||504||Xu et al. |
|R||CCT CTT CAG CTC GAC GCG ACC|
Table 1: Oligonucleotide primer sequences for detecting toxA and oprL genes of p.aeruginosa isolated from Sea bream Fish.
|Gene||Primer Sequence(5' - 3')||Size (bp)||Reference|
|gyrA||F||GTGTGCTTTATGCCATGAG||287 bp||Gorgani et al. |
Table 2: Oligonucleotide primer sequences for detection of Fluoroquinolone resistance genes gyrA and parC of p.aeruginosa isolated from Sea bream Fish.
The reaction was conducted in a total volume of 50 μl in 0.5 ml microfuge tube as 25 μl Green Master Mix, 10 μl DNA template, 5 μl upstream primer, 5 μl downstream primer and 5 μl nuclease free water and the reaction mixture was over laid with 50 μl nuclease free mineral oil to prevent evaporation during thermocycling, DNA marker (100 bp DNA ladder) (Gene Ruler TM) was used as a DNA molecular weight marker for PCR product in agarose gel electrophoresis, followed by the use of ethidium bromide for staining nucleic acid .
Antibiogram activity of P. aeruginosa
Finally, PCR assay on fluoroquinolone resistance genes was compared with the antibiogram activity using antibiotic diffusion disc (Oxoid®) against 3 of fluoroquinolone members (Ciprofloxacin, Flumequine and Enrofloxacin), Gentamycin, Trimethoprim/ Sulphamethoxazole, Oxytetracycline, Amoxycillin and Erythromycin according to the method described .
Clinical signs and PM lesions of examined sea bream fish
Some of clinically examined Sea bream showed hemorrhages at pelvic and anal fins, moreover showed petechial hemorrhages at the operculum and around the eye (Figure 1), while others showed erythema in the mouth and severe exophthalmia, severe hemorrhagic patches at the body and severe hemorrhages at base of pectoral fins, severe hemorrhagic swollen protruded anal dopening (Figure 2), While by PM, some of examined Sea bream showed hemorrhages at gills, intestine and muscles (Figure 3), hemorrhages at heart (Figure 4), congestion and hemorrhages in the internal organs and distended gall bladder (Figure 5) others showed serous ascetic fluid tinged with blood dropping on behind paper upon opening the fish, while the others showed congestion of different degrees on the liver of examined fish.
Results of bacteriological examination of sea bream fish
Prevalence of Gram –ve and Gram +ve bacterial serotypes among naturally infected sea bream fish: Bacteriological examination of a total number of 100 naturally infected cultured Sea bream fish samples explored the isolation of 77 (89.53%) Gram –ve and 9 (10.47%) Gram +ve, totally; 86 (100.00%) bacterial isolates (Table 3). This confirmed with Saad who recorded in naturally infected fish Gram -ve bacteria were (78.00%) while Gram +ve bacteria were (22.00%). It has also been agreed that Gram -ve rods are the more causative agent in producing diseases in fish and to lesser extent Gram +ve cocci .
|Examined Fish Species||Bacterial Isolates|
|Gram -ve||Gram +ve||Total no. of Isolates|
Table 3: Prevalence of Gram +ve and Gram –ve bacterial serotypes among naturally infected Sea bream fish.
Colonial, morphological, conventional biochemical and VITEK2 identification of yielded bacterial isolate: Basic morphological and conventional biochemical characterization identified the yielded isolates into P. aeruginosa, V. alginolyticus, V. anguillarum serotype 01, Strept. iniae and Ph. damsela subsp. piscicida these agreed with Suanyuk, Eissa, Das [15,17,29]. All yielded isolates P. aeruginosa were identified with 100% probability by colorimetric VITEK2 COMPACT SYSTEM which used as a recent aid for more confirmation for identification of the more isolates of P. aeruginosa. Chatzigeorgiou et al.  have been concluded that VITEK2 GN card should be considered for identifying gram -ve non fermentative bacilli while Paim  found that VITEK2 COMPACT SYSTEM software version 5.03 is considered for identifying Gram +ve cocci. COMPACT SYSTEM software version 5.03 properly identified isolated Gram +ve cocci.
Prevalence of bacterial serotypes among naturally infected sea bream fish
According to Table 4, the more prevalent bacterial serotype in naturally infected cultured Sea bream fish was P. aeruginosa (43.02%), followed by V. alginolyticus (20.93%), V. anguillarum serotype 01 (16.28%), Strept. iniae (10.47%), and Ph. damsela subsp. piscicida (9.30%). These disagreed with Zorrilla  who found that Ph. damselae subsp. piscicida was more prevalent in Sea bream infection, but he recorded that Pseudomonas spp. and Vibrio spp. known as pathogenic bacteria for Sea bream fish and also agreed with Hossain  who isolated Strept. iniae from marine fish. Also agreed with Yiagnisis and Athanassopoulou  who isolated Ph. damsela subsp. Piscicida from diseased cultured Sea bream, El-Moghazy and Elham [1,35] also isolated Pseudomonas spp. from Sea bream fish. Also these results supported by Snoussi  reported that Vibrio found in tropical and temperate waters worldwide seaside water of the Mediterranean Sea. In addition, Moustafa  who said that Gram -ve bacteria isolates including V. alginolyticus, Pasteurella piscicida (P. damsela subspp piscicida) were the more isolated pathogens from marine fish species, while S. iniae was the most Gram +ve bacteria.
|No.of Examined Sea fish species||Bacterial isolates||No||%|
|100 sea bream||P. aeruginosa||37||43.02|
|V. anguillarum serotype 01||14||16.28|
|Ph. damsela subsp piscicida||8||9.30|
|Total no. of Isolates||86||100.00|
Table 4: Prevalence of different bacterial serotypes of infection in Sea Bream Fish.
The prevalence of bacterial isolates in the internal organs of the naturally infected sea bream fish
The incidence of bacterial isolates retrieved from different internal organs of naturally infected cultured Sea bream fish was illustrated in Table 5, in which; the more bacterial isolation recorded in liver (41.86%) followed by spleen (25.58%), heart (16.28%) and kidney (16.28%), these results agreed with Eissa  who reported that liver was the highest organ followed by kidney and spleen of marine fishes.
|Examined Sea fish species||Examined organs||Bacteriologically positive|
Table 5: The Prevalence of bacterial isolates in different organs of naturally infected Sea Bream Fish.
Total prevalence of bacterial serotypes retrieved from the different internal organs of naturally infected sea bream fish
The total prevalence of different bacterial serotypes retrieved from different internal organs of naturally infected cultured Sea bream fish was illustrated in Table 6 that showed liver infection was high incidence by P. aeruginosa (52.78%) followed by V. alginolyticus (19.4%), V. anguillarum serotype 01 (16.67%), Strept. iniae (8.33%) and Ph. damsela subsp. Piscicida (2.78%) while in heart, isolation of P. aeruginosa was also more prevalent (50.00%) but Ph. damsela subsp. piscicida, Strept. iniae and V. alginolyticus all isolated with the same percentage (14.29%) and the lowest was V. anguillarum serotype 01(7.14%). About kidney, V. alginolyticus was the more prevalently isolated (42.86%) followed by Ph. damsela subsp. piscicida (28.57%), Strept. iniae (14.28%) and both P. aeruginosa and V. anguillarum serotype 01 were isolated by the same percentage (7.14%). In spleen, P. aeruginosa was more prevalent (45.45%) followed by V. anguillarum serotype 01(27.28%), V. alginolyticus (13.64%), Strept. iniae (9.10%) and Ph. damsela subsp. piscicida (4.54%).
|V. anguillarum serotype 01||6||16.67||1||7.14||1||7.14||6||27.28|
|Ph. damsela subsp piscicida||1||2.78||2||14.29||4||28.57||1||4.54|
|Total no. of Isolates||36||100||14||100||14||100||22||100|
Table 6: The total Prevalence of bacterial serotypes isolated from different internal organs of naturally infected cultured Sea Bream Fish.
Results of PCR of the more prevalent isolates of P. aeruginosa from examined sea bream fish
Results of PCR for detection of virulence genes of P. aeruginosa
isolates from sea bream fish: In Figure 6, the primer sequence showed maximum identity with the sequence of virulence genes of all tested 12 isolates of P. aeruginosa with 100% homology to outer membrane lipoprotein gene oprL at "504 bp" and exotoxin A gene toxA at "270 bp" which indicated that these are virulent isolates of P. aeruginosa. These results agreed with those reported by De Vos, Pirnay, Xu, Deschaght and Billard-Pomares [24,38-41] who confirmed that PCR assay using the oprL gene was highly specific and more suitable than culture for detecting P. aeruginosa.
Figure 6: Agarose gel electrophoresis contacting the PCR products along with 100 bp DNA Ladder (lane L) While lane from 1 to 12 are the tested isolates of P. aeruginosa isolated from Sea Bream Fish in which all showed positive bands for both outer membrane lipoprotein gene(oprL) at 504 bp and exotoxin A gene (toxA) at 270 bp.
PCR for detection of fluoroquinolone resistance genes of P. aeruginosa isolates from sea bream fish
The primer sequence showed maximum identity with the sequence of fluoroquinolone resistance genes of all tested 12 isolates P. aeruginosa with 100% homology to DNA gyrAse (gyrA) at "287 bp" (Figure 7), and topoisomerase IV (parC) at "267 bp" (Figure 8), PCR amplified the correctly sized products which indicated that these are fluoroquinolone resistant isolates of P. aeruginosa.
Results of antibiogram activity of the more prevalent isolates of P. aeruginosa from examined sea bream fish
As shown in Table 7; P. aeruginosa isolates showed resistance to 3 of fluoroquinolone members: Ciprofloxacin, Flumequine and Enrofloxacin and these confirmed the result of positive detection of fluoroquinolone resistance genes by PCR, as well as P. aeruginosa isolates were resistant to Trimethoprim/Sulphamethoxazole, Oxytetracycline and Erythromycin while sensitive to Gentamycin and intermediately resistant to Amoxycillin. These agreed with Foti  who reported that P. aeruginosa resistant to oxytetracycline and amoxicillin and discussed these finding to contamination of marine water by polluted sewages residues of antibiotics can be spread and repetitive antibiotic usage. Also El-Moghazy and Aboutorkia [43,44] found that P. aeruginosa high sensitivity to Gentamicin and resistant to Moxifloxacin (fluoroquinolone member) and Trimethoprim/Sulfamethoxazole.
|Type of used antimicrobial disc||Disc code||Disc concentration(µg)||Sensitivity of isolated P. aeruginosa|
Table 7: Antibiotic sensitivity test of isolated P. aeruginosa from Sea Bream Fish by using different antimicrobial discs
The present study explored that Pseudomonas aeruginosa isolation is more prevalent in diseased cultured Sea bream fish and PCR is a rapid, sensitive, specific assay in determining its pathogenicity by detecting molecular characterization of some virulence genes by 24 hrs-work time and this necessitate more rapid hygienic programs to minimize the possible infection to optimize the Sea bream aquaculture fisheries quality in Egypt, as well as the haphazard usage of broad-spectrum antibiotics in Sea bream aquacultures is the cornerstone of emergence of highly resistant strains of P. aeruginosa which narrow the zone of effective antibiotics and this highlighted the focus on the importance of determining the antibiotic susceptibility pattern of P. aeruginosa so that this aquaculture pathogen can be treated by narrow-spectrum and target-specific antibiotics as well as to calculate the minimal inhibitory concentration (MIC) before using antibiotic in cure. Following further studies should be followed to investigate more about pathogenicity of isolated P. aeruginosa and clarify the correlation between a DNA gyrA mutation and quinolone resistance. It would be helpful for further union of new quinolones that are powerful in treatment of Sea bream fish.
Conflict of interest
All authors of this article have no conflict of interest.
This article does not contain any studies with animals performed by any of the authors.