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Antibiotic Resistance and Resistance Genes Among Tarda Isolated from

Ezzat Mahmoud1, Reham M. El-Tarabili1, Esawy A.M.2 and Nehal Elmasry 1 1Department of Bacteriology, Immunology, and Mycology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt. 2Animal Health Research Institute, Mansoura branch Corresponding author: (Reham M. El-Tarabili), [email protected], [email protected] https://orcid.org/0000-0001-8273-2747

Abstract Edwardsiella tarda is a common fish pathogen, causing significant septicemic diseases in fish. This study was carried out to investigate prevalence of E. tarda among Tilapia zilli at EL manzla lake, Dakahlia governorate and characterize the isolates phenotypically and genotypically in addition to detection of multi-drug resistance genes (B-lactames genes and plasmid-mediated quinolone resistant genes by PCR assay. Therefore, (100) samples of Tilapia zilli collected from different localities. Fish samples were subjected to clinical and post-mortem examination then bacteriological examination from liver, kidney and spleen. The suspected isolates were characterized by cultural and morphological characters, some conventional biochemical tests and API 20E system then by PCR assay. Eighteen % isolates were characterized as E. tarda. Furthermore, detection of antimicrobial resistance genes by PCR, the recovered isolates harbored blaSHV and blaOXA-1 with a prevalence of 50% and 25%, respectively with no detection of aac (6′)-Ib-cr and qepA genes in examined isolates. The results of sensitivity showed resistance to Nalidixic acid, Lincomycin, Amoxicillin and Norfloxacin and most of isolate were found to be sensitive to Florfenicol and Neomycin.55.6% of recovered isolates showed resistance to three antibiotic classes and 27.7% of recovered isolates showed resistance to four antibiotic classes and considered as MDR. The emergence of MDR-strains represents a threat-alarm and PCR is very rapid method for identification of E. tarda isolates which may be helpful in control of Edwardsiellosis. 172 Ezzat et al.

Keywords: Edwardsiell tarda, Tilapia zilli, PCR and Antibiotic resistance genes

Introduction blooded animals as human and Edwardsiella is enteric pathogen contains essential virulence includes strains of three species factors which increase bacterial (E. tarda, E. ictaluri and E. survival and disease in hosts hoshinae). E. tarda is primarily a (Castro et al., 2016 and Park et pathogen of fish associated with al., 2012). E. tarda strain possess fish gangrene, emphysematous several virulence and toxin putrefactive disease of catfish, secretion associated genes which red disease of (Evans et al., illustrate to some extent its 2011). E. tarda is mainly cause ability to survive within of a dangerous systemic disease, phagocytic cells and to infect a Edwardsiellosis which is one of wide range of hosts due to a the most important bacterial highly virulent and multidrug diseases in fish and it causes resistant strain (Verjan et al., mass mortality in the various 2013). Pathogenesis of E. tarda populations and age groups of is multifactorial and several fish in freshwater and marine potential virulence factors have of both farmed and wild been recognized to contribute to population all over the world the infection process have been (Enany et al., 2018). E. tarda is reported (Mohanty and Sahoo, a Gram-negative, short, rod- 2007). Also, it is well known that shaped bacterium of about 1cm diagnosis of a particular in diameter and 2-3 cm in length infection depends on detection (Evans et al., 2011). E. tarda and identification of its causative strains isolated from various agent (Das et al., 2014). geographical sources exhibit Nowadays, one of the main little variation in the phenotypic threats to public health is the characters (Austin and Austin, antibiotic resistance (WHO, 2007) and several studies have 2002). Multi-drug resistant demonstrated a wide degree of (MDR) was defined as resistance intraspecific diversity in the to only one agent in three or isolates from the different more antimicrobial classes. geographic regions and host (Basak, et al.,.2016). Several species (Nucci et al.,2002 and previous investigations revealed Wang et al., 2011). the emergence of multidrug- E.tarda has been recognized as a resistant bacterial pathogens normal guest of a wide range of from different origins especially animals, including fish, , fish that increases the need for crustaceans, chickens and warm- new natural immunostimulants SCVMJ, XXVI (1) 2021 173 and antimicrobial alternatives to internal lesions according to the commonly used old (Schaperclaus et al.,1992) The antimicrobial agents (EL-Sayed examined fish were placed on et al., 2019; Abouelmaatti et al., right side. Sterilization of the 2013; Algammal et al., 2020a; skin of fish by70% ethyl alcohol. Algammal et al., 2020b; The first cut was made in front of Algammal et al., 2020c; the anus through the abdominal Algammal et al., 2021). This wall with blunt sterile Scissors. work was carried out to isolate The second cut was made and characterize E. tarda from perpendicular to the first directly brackishwater fish Tilapia zilli in behind the branchial cavity and Dakahlia governorate, Egypt. In the third cut was from the anus to the current study we detect the the head parallel to middle line presence of E. tarda, determine where the abdominal wall was antibiotic sensitivity and removed and the internal organs screened the presence of become visible and examined for antimicrobial resistance genes of detection of any abnormalities as E. tarda isolated from diseased, change in color, size, dead and healthy fishes using hemorrhages ascetic fluid and molecular technique of other abnormalities. fish were conventional PCR. examined in a sterile manner using a three-line. Materials and methods Bacteriological examination Fish sampling: Samples from kidney, liver and A total number of 100 of spleen were aseptically brackishwater fish of Tilapia zilli inoculated on tryptic soya broth (healthy, diseased and morbid)) (Oxoid, UK) and incubated at were collected randomly from 25-30°C for 24 hrs. then EL-Manzla in Dakhlia inoculated on Salmonella- governorate, Egypt during the Shigella agar (SS agar; Oxoid period from April 2019 to April CM0099) incubated at 35°C for 2020. Samples were transported 24˗ 28 hrs. according to directly in plastic bags to the (Bergey's et al., 2005). For microbiological department of purification and further animal health research institute identification, recovered isolates in EL-Mansoura branch to be streaked on TSA slope and full clinical, postmortem and incubated at 37°C for 24 hrs. All bacteriological examination. recovered isolates Clinical and postmortem morphologically were detected examination with Gram’s stain and Fish were examined clinically biochemically according to. for the presence of external and (Austin&Austin.,2007) and by 174 Ezzat et al. using the analytical profile index Statistical analysis of API20E system The obtained findings were (Buller,2004). analysed using the Chi-square Antibiotic susceptibility test test with SAS software, version for E. tarda: 9.4, SAS Institute, Cary, NC, The sensitivity test was done on USA) (Significance-level; P < examined isolates of E. tarda 0.05). using disc diffusion method according to (Finegold and Results martin.1982) using various Clinical and postmortem antimicrobial agents: examination of fish Amoxicillin (10µg), Nalidixic Samples represented Tilapia zilli acid (30µg), Florfenicol (30µg), were randomly collected and Neomycin (30µg) Lincomycin analyzed. based on the type of (2µg) and Norfloxacin (10µg). fish, the most clinical signs The inhibition zone diameter observed in the examined was measured and interpreted Tilapia zilli were the appearance according to (NCCLS, 2007). of abdominal distension, Isolates showed resistance to hemorrhage on the surface of the more than two different body, congestion of the fins, skin antimicrobial classes were ulcers and congested vent. multiple drug resistant (MDR). Accumulation of the ascitic fluid Detection of antimicrobial and congestion of the internal resistance genes by polymerase organs, including the liver, chain reaction gallbladder, spleen and kidney, The DNA was extracted using occurred internally. As shown in QIAamp DNA mini-Kit Primers the (Fig. 1a,b). (Catalogue no. 239035),USA) Prevalence of E.tarda in the and used for the detection of fish and Their distributions in Edwardsiella tarda antibiotic Different Internal Organs resistance related genes to β- All examined strains were lactams (blaSHV and blaOXA1), reported as Edwardsiella tarda. Fluroquinolones(aac(6′)-Ib-cr Under microscope, the and qepA (Metabion, Germany) appeared as Gram-negative, as shown in (Table 1). short, rod-shaped bacterium. The Separation of amplified products bacteria grew well Salmonella by electrophoresis on 1.5% shigella agar appear as small agarose gel (Applichem, transparent with black center on Germany, GmbH) and TSA. All recovered isolates were photographing gels by gel oxidase negative and documentation system (Alpha biochemically homogenous. Innotech, Biometra). E.tarda were positive for Indole, SCVMJ, XXVI (1) 2021 175

Methyl Red, Catalase, H2s to different used antimicrobia l. production , glucose In present study, all recovered fermentation and reduction isolates showed resistance to nitrate to nitrite but negative for Nalidixic acid, Lincomycin, Lactose fermentation, sucrose, Amoxicillin and Norfloxacin Urease, Voges – Proskauer . and most of isolate were found to Also, the presence of E. tarda, be sensitive to, Florfenicol and identified using API20 E as Neomycin. Statistically, there is shown in Fig.(2). The results a significant difference (P < exhibited that 18 isolates 0.05) in the resistance and recovered from (zero from susceptibility of the recovered E. healthy fish ,10 from diseased tarda strains to various fish and 8 from mourbid fish ) as antimicrobial agents as shown in shown in table (2) and highest table (3). in the present study, distribution of E. tarda was in 55.6% of the recovered isolates liver (60%) then kidney (40%) showed resistance to three then spleen (0%). Statistically, antibiotic classes and 27.7% of there is no significant difference recovered isolates showed in distribution of E. tarda among resistance to four antibiotic different organs. classes were multidrug resistant Seasonal Variation of E. tarda. (MDR) Recovered strains of E. tarda Molecular characterization of were detected during spring, E. tarda isolates summer and autumn, while in Two isolates of representative winter not detected in any four isolates (2/4) showed samples. The summer season positive amplification of 392 bp showed the highest prevalence fragment specific for blaSHV with with 58.3% then the spring season a total percentage of 50%, one with 25%, finally autumn 16.7%. (1/4) isolate showed positive Statistically, there is no amplification of 619 bp fragment difference in seasonal variation specific for blaOXA1 with a total of E. tarda. percentage of 25%, no recovered Antibiotic Sensitivity of isolates detect aac(6)-Ib-cr and recovered E. tarda strains: qepA amplified at 113 and 403 E. tarda isolates vary in their bp fragment respectively, as antimicrobial sensitivity pattern shown in Fig (3,4,5,6)

176 Ezzat et al.

Table (1): Oligonucleotide primers sequences of genes among recovered isolates

Actual cycle Amplified Gene Sequence Reference 35 cycles product Denaturation:94˚C/30sec aac(6′)- CCCGCTTTCTCGTAGCA Lunn et Annealing:52˚C/30sec 113 bp Ib-cr al., 2010 TTAGGCATCACTGCGTCTTC Extension:72˚C/30sec AGGATTGACTGCCTTTTTG Denaturation:94˚C/30sec blaSHV Annealing:54˚C/40sec 392 bp ATTTGCTGATTTCGCTCG Extension:72˚C/40sec Colom et Denaturation:94˚C/30sec al., 2003 ATATCTCTACTGTTGCATCTCC blaOXA-1 Annealing:54˚C/40sec 619 bp AAACCCTTCAAACCATCC Extension:72˚C/45sec Denaturation:94˚C/30sec CGTGTTGCTGGAGTTCTTC Cattoir et qepA Annealing:54˚C/40sec 403 bp CTGCAGGTACTGCGTCATG Extension:72˚C/45sec al., 2008

Table 2. prevelance of Edwardsiella tarda in accordance to fish status:

Fish Apparently healthy Diseased morubid Total No of examined 20 30 50 100 sample No of isolated zero 10 8 18 E.tarda

Table 3. Antibiotic sensitivity of recovered E. tarda strains

Interpretation Specific tested Sensitive Intermediate Resistance antibiotic N % N % N % Amoxicillin 0 0 2 11.2 16 88.8 Neomycin 11 61.1 2 11.2 5 27.7 Norofloxacin 4 22.2 4 22.2 10 55.6 Nalidixic acid 3 16.6 8 44.4 7 38.9 Lincomycin 4 22.2 2 11.2 12 66.6 Florfenicol 11 61.6 3 16.6 4 22.2

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Figure 1a. Naturally infected Tilapia zilli with E. tarda showing, external hemorrhages and congested fins and gills.

Figure 1 b. Naturally infected Tilapia zilli with E. tarda showing hemmorhage with congested liver

Figure 2. Biochemical identification of the isolates by using API 20E.

178 Ezzat et al.

Figure 3. Agarose gel electrophoresis showing MDR gene of E. tarda isolates using primer set for blaSHV gene (392bp). LaneL: 100-1000bp ladder. P: control positive. N: control negative. Lanes3, 4: E. tarda isolates Positive the blaSHV gene. Lanes1, 5: E. tarda isolates Negative the blaSHV gene

Figure 4. Agarose gel electrophoresis showing MDR gene of E. tarda isolates using primer set for blaOXA-1 gene (619bp). LaneL: 100-1000bp ladder. P: control positive. N: control negative. Lanes 1: E. tarda isolates Positive for the blaOXA-1 gene Lanes 3, 4,5: E. tarda isolates Negative for the blaOXA-1 gene.

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Figure 5. Agarose gel electrophoresis of MDR gene of E. tarda isolates using primer set for aac(6-)-Ib-cr gene (113bp). LaneL: 100-1000bp ladder. P: control positive. N: control negative. Lanes1, 3, 4, 5: E. tarda isolates Negative for the aac(6-)-Ib-cr gene.

Figure 6. Agarose gel electrophoresis of MDR gene of E. tarda isolates using primer set for qepA gene (403bp). LaneL: 100-1000bp ladder. P: control positive. N: control negative. Lanes1, 3, 4, 5: E. tarda isolates Negative for the qepA gene.

Discussion E. tarda infection among the Edwardsiellosis is one of the examined Tilapia zilli was 18% most important bacterial agree with results of (Korni et diseases in fishes causing al., 2012) who recorded that massive mortalities in the prevalence of Edwardsiellos is various populations and age among the cultured Nile Tilapia groups of fish consequently high in spring season at Beni-Suef economic losses (Plumb, 1993 governorate was 13.33 % and and Jun and Yin, 2006). In the higher than (Ali et al., 2008) who current study, the prevalence of reported that incidence of E. 180 Ezzat et al. tarda among the diseased veterinary, agricultural and Tilapia zilli at Beni-Suef aquacultural settings as Governorate was 3.7%. Our prophylactic measures and results also were disagreed with growth promoters have resulted (Abd El-Mageed et al, 2002) in proliferation of antibiotic who recorded that incidence of resistant genes in horizontal gene E. tarda in Tilapia zilli collected pool (Meervenne et al., from different localities in Egypt 2012).Our results detected that was 0% The difference in the recovered isolates showed prevalence of E. tarda may be resistance to nalidixic acid, attributed to the difference in Lincomycin, amoxicillin and water temperature, stocking Norfloxacin and this agree with density, water quality and/or those reported by (Noor ELDeen location of the study. et al., 2017 ;Nagy et al., 2018) Examination of Tilapia zilli and most of isolate were found to infected with E. tarda revealed be sensitive to, florfenicol and number of the clinical signs as neomycin. (Ahamad et al., abdominal distension, 2013; Anyanwu et al.,2014; hemorrhages on the body Thangapalam Jawahar surface, congestion of the fins, Abraham et al.,2015; Pankaj presence of skin ulcers and Kumar et al., 2016). These congested vent. Internally, there resistance results may be were accumulation for the ascitic attributed to mutations in the fluid and congestion of internal gyrase or to poisomerase organs including liver, spleen antibiotic genes. resistance genes and kidney. These clinical signs or by horizontal gene transfer of and post-mortem lesions were antibiotic resistance similar to those reported by determinants (Poole,2004) and (Kubota et al., 1981; Eissa and (Sorum,2006). Yassien 1994; Galal et al, 2002; Plasmid mediated quinolone Saad El-Deen et al., 2005; El- resistance (PMQR) has been Deeb et al., 2006; Ramadan et shown to play an important role al., 2009; Yu et al., 2009 and in resistance not only to Hashiem and Abd El-Galil quinolones, but also B-lactamase ,2012). and aminoglycosides. In fact, Multidrug resistance could be quinolones resistant genes partly attributed to the represent one of the most inadequate dose, extensive use important PMQR mechanisms and sub-active concentration of and frequently carried along with the drug used in fish farms. B-lactamase on the same Furthermore, widespread use of plasmids also aac (6-)-Ib-cr antibiotics in medical, genes. qepA was identified SCVMJ, XXVI (1) 2021 181

PMQR gene encoding efflux intrinsic resistance, often pump (Yamane et al.,2007) was chromosomal mediated and detected in Edwardsiella isolates transferable to new generations resistant to quinolones. DNA (Kümmerer, 2009). on the other sequencing of qepA revealed that hand, blaOXA-1 was present in one the gene includes three alleles of isolate only with a percentage of qipao (qepA1, qepA2, qepA3), (25%) and our results detect (Cattoir et al.,2008 and wang et blaSHV with a percentage of al., 2015). In the present work (50%) disagree with (Kees et al., qepA gene could not be 2008). Who recorded 0% and identified in any isolates of 25%, respectively in E. tarda edwardsiella and disagree with while disagree with (Goudarzi et (Liu et al., 2011) who found al., 2013) who could not detect qepA gene in isolates of E. tarda. blaSHV genes in isolates and Quinolone resistance genes are (Shahcheraghi et al., 2010) widely distributed among found blaSHV genes among 6% of bacteria (Flach et al.,2013). In 200 isolates. this study aac(6-)-Ib-cr genes were investigated using specific Conclusion primers and PCR, could not be Edwardsiellosis infection leads detected in any one of the to high morbidity and mortality isolates and this results are in rate resulting great economic disagreement with these results losses. The total prevalence of E. recorded by(Sudu et tarda in brackishwater fishes is al.,2018)who isolate 11 out of 30 high may be due to pollution and isolates of E.tarda from fish in stress factors so that japan, (Huang et al., 2012 and overcrowded, bad environmental Yu et al.,2012)who identified condition, bad water quality and this gene in one isolate and high organic matter in fish farms. mentioned that this gene was E. tarda has also public health located on large plasmid. While significance in people engaged (Liu et al., 2011) mentioned that in fishery industry and those quinolones resistant genes were depend on fish products for their acquired from chromosomal annual income. Overuse or miss genes in bacteria and are usually use of antibiotics increase associated with mobilizing or Edwardsiella resistance to most transposable elements on antibiotics. PCR method can use plasmids. as an important technique in the The high levels of resistance to diagnosis of antibiotics the β-lactam antibiotics in resistance genes of MDR several Gram-negative bacteria E.tarda isolates aac(6-)-Ib-cr, has been attributed to their qnrA, blaSHV, blaOXA-1,qepA - 182 Ezzat et al. based techniques are used Staphylococcus aureus (MRSA): increasingly in food- One Health Perspective microbiology research as they Approach to the Bacterium are well developed and when Epidemiology, Virulence applied as culture confirmation Factors, Antibiotic-Resistance, tests, they are reliable, fast and and Zoonotic Impact. Infect sensitive which measure Drug Resist.;13: 3255-3265. epidemics occurrence and https://doi.org/10.2147/IDR.S27 subsequently decreasing the 2733. economic losses. Algammal, A. M., Mabrok, M., Sivaramasamy, E., Youssef, F. Refrences M., Atwa, M. H., El-Kholy, A. Abd El-Mageed, A. A.; Khalid, W., ... & Hozzein, W. N. A. M. and Shaheen. (2002). 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المقاومة للمضادات الحيوية والجينات المقاومة للمضادات الحيوية االدواردسيال تاردا المعزوله من السمك

يعتبر ميكروب االدواردسيييين مل اكريكرو ال اكتس ب يييبل مرييياخط مر ييييث ير وب ر علس اكثرو اك يييركيث . وك كه ب هذ اكهراسيييث اكس بالهيه االهيييا ث ا اكرر اك يييار وخيفيث عزل اكريكروب اكر ييييييبل كث وبالهيه اكجينال اكرقاومث كلر يييييييادال اكالي يث مل اكرعزوالل وك كه قه بم عزل ميكروب االدواردسييييييين مل عهد 100سييييييركث ل ى واكتس جرعت مل اليره اكرنزكه رالافظث اكهق ليث فس اكفتر مل ا رايط 2019اكس ا رايط 2020 واظ رل اكهراسييث عزل ميكروب االدواردسييين 18% وقه بم اجراء اكفال هييال اكظا ريث واكترييييييرياليييث واكبكتيري ك جيييث و بم ا يي اكعينييال مل اك الييال واككبييه واككلس كلفال اكبيكتري ك جس. فقه ظ ر علس االسييراا اكربييا ث زيى علس سيي م اكج ييم يرييرط ووج د بقرحال علس عض االسيرااكخرا ببيل وج د ق زيفيث فس اكع ينل اكخارجيث اال يافث اكس ظ ر حاالل مل االسييييييت ييييييقاء وا تفاي فس اكب ل حي ظ ر اكت اب و احتقا اككلس واك الال اكهماء وب رم فس اككبه ووج د عنمال زيفيث وبم اجراء ا تبار اكال يييييياسيييييييث كلر ييييييادال اكالي يث اكرختلفث كتله اكعترال وخا ت اكنتايع ا معظم اكعترال ح يييييياسييييييث فل روفينيك ل واكني مي يييييييل ينريا اب ييييييم ا معظم اكعترال مقياوميث االم خ ييييييي ييييييليل واكن رفل خ ييياسييييل واكلينك مي ييييل و يلهيك ييييه و55.6% كتريا م ييياده كعهه م يييادال اكالي يث. وبم اجراء ا تبار اكبلرر اكجزيئيث علس 4 مل اكرعزوالل اكتس بم عزك ا اسيتخهام اكبييادا اكرتخبيييييي ككييط جيل مل اكجينييال اكرقيياومييث كلر يييييييياد اكالي يييث رعزوالل االدواردسييييييين)qepA aac(6-)-Ib-cr اكخاهييييييث اككين ك ال - اكخاص اكبيتا الختام blaOXA, blaSHV(وحيي اظ رل اكنتيايع ب اجه م فس اكعينيال اكرعزوكث اكن يييييييل االبيث:blaOXA%25) blaSHV(%50 وكم يظ ر فىqepA aac(6-)-Ib-cr