ISSN:1991-8178 Australian Journal of Basic and Applied Sciences Journal home page: www.ajbasweb.com Isolation and Molecular Differentiation of Brucella Isolates in Some Foods in Egypt 1 Safaa Ali, 2 Nadia Soliman, 1 Salah Abdel-Rahman 1 Nucleic Acid Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt. 2 Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt. A R T I C L E I N F O Article history: Received 28 August 2015 Accepted 15 September 2015 Available online 15 October 2015 Keywords: Brucella, Foods, Contamination, Antibiotics, RAPD-PCR. A B S T R A C T This study was conducted to examine the frequency of contamination of some foods such as Cocos nucifera, Arachis hypogaea, Ceratonia siliqua and Hyphaene thebaica with Brucella. Samples were collected from two different sources (perfumery and market shops) in Egypt, and then cultured in specific medium for Brucella. The isolated bacteria showed an ability to grow in Mackonky agar and gave gram negative sense. Using RAPD-PCR technique, 19 Brucella isolates were captured in tested samples: Cocos nucifera, Arachis hypogaea, Ceratonia siliqua and Hyphaene thebaica at ratio 4: 4: 6: 5, respectively, with prevalence to market (60%) than perfumery shops. Both antibiotic and temperature sensitivities were checked and resistance to wide range of antibiotics was observed. Resistance rates to Amoxycillin/clavulanic acid, Ampicillin, Cefotaxime, Ceftriaxone, Metronidazole, Penicillin and Sulphamethoxazole/trimethoprim were 100%, while, the resistance rates to Erythromycin, Oxacillin, Rifampin and Trimethoprim/sulphamethoxazole were 50-95%. On the other hand, the sensitive rates to Ciprofloxacin, Doxycycline, Gentamicin, Ofloxacin and Streptomycin were 100%, while, high sensitivity ranged ~80-90% was recorded towards Azithromycin, Cefoperazone/sulbactam, Chloramphenicol, Spiamycin, Tetracycline and Vancomycin. Concerning to temperature sensitivity, a great drop near zero% in bacterial growth after 10 min exposure to boiling temperature was found. Consequently, boiling for 10 min can be applied as a precautionary step to prevent microbial growth of Brucella in tested samples as a recommendation before usage. 2015 AENSI Publisher All rights reserved. To Cite This Article: Safaa Ali, Nadia Soliman, Salah Abdel-Rahman., Isolation and Molecular Differentiation of Brucella Isolates in Some Foods in Egypt. Aust. J. Basic & Appl. Sci., 9(31): 103-109, 2015 INTRODUCTION Brucellosis is one of the most widespread zoonoses worldwide; it has high morbidity for both humans and animals that causing economic loss and public health problems in many developing countries (Colmenero et al., 1996). Brucellosis is a zoonotic infection transmitted to humans by contact with fluids from infected animals or derived food products such as un-pasteurized milk and cheese (Bosilkovski et al., 2009). In general, Brucella (B) melitensis causes more severe infection than Brucella abortus (Young, 1995, Troy et al., 2005) and in particular, Brucella suis can be as severe as B. melitensis in human (WHO, 2006). In a small series, patients infected with B. suis did not have a more severe clinical course comparing with those infected with B. abortus (Buchanan et al., 1974). Brucellosis is a systemic infection with a broad clinical spectrum ranging from asymptomatic disease to severe and/or fatal illness (Colmenero et al., 1996). Clinical and laboratory features vary widely (Bosilkovski et al., 2010). The main presentations are acute febrile illness with or without signs of localization and chronic infection. Infection among children is generally more benign than in adults with respect to likelihood and severity of complications and response to treatment (Pappas et al., 2005). Brucellosis in pregnancy is associated with risk of spontaneous abortion, premature delivery, miscarriage and intrauterine infection with fetal death (Khan et al., 2001, Doganay and Aygen, 2003, Gotuzzo et al., 2004). The aim of the current study is to investigate the frequency of contamination of some foods such as Cocos nucifera, Arachis hypogaea, Ceratonia siliqua and Hyphaene thebaica with Brucella in perfumery and market shops in Egypt using specific medium for Brucella. Corresponding Author: Salah Abdel-Rahman, Nucleic Acid Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt. Ph: +201112727468; E-mail: salahmaa@gmail.com
104 Safaa Ali, et al, 2015 MATERIALS AND METHODS Samples: Cocos nucifera, Arachis hypogaea, Ceratonia siliqua, and Hyphaene thebaica samples were collected in 2014 from perfumery and market outlets existing in Egypt. Microbiological analyses: The samples were prepared by weighing one gram then dissolved in 50 ml water and then incubated under shaking for 5 hrs at room temperature. One ml of sample suspension was inoculated in Brucella agar selective medium (acumedia, Michigan) and incubated overnight at 37 C. Phenotypic Characterization: Microscopic Examination: Examination the isolated bacteria that could be Brucella isolates by gram stain technique which is the most important differential stain used for diagnostic identification of various organisms. Further, confirmation was carried out by growing the tested isolates on Mackonky (Oxid) which is specific only for gram negative bacteria. Temperature Sensitivity: The tested isolates were allowed to grow at different temperatures 37, 40, 45, 50, 60, and 65 C in agar plates, then the data were recorded as positive or negative. Antibiotic sensitivity: Antibiotic sensitivity was conducted by Disc Diffusion-Method modified by Kirby-Bauer-method (Onanuga et al., 2005). The tested isolates were allowed to grow overnight in 4-5 ml nutrient broth at 37 C and vortex before spreading over the plates. The dried surface of a Muller-Hinton agar (Oxid) plate was inoculated by streaking the swab over the entire sterile agar surface. Just quickly pick up the disc and move it to the appropriate place with the sterile forceps. Incubate upside down and incubate at 37 o C for 24 hrs. The diameters of zones of complete inhibition were measured, including the diameter of the disc. Antibiotic disk used Ciprofloxacin (CIP 5μg), Doxycycline (DO 30μg), Gentamicin (CN 10μg), Streptomycin (S 10μg), Ofloxacin (OFX 5 μg), Cefotaxime (CTX 30μg), Metronidazole (MTZ 50μg), Penicillin (GP 10 unit), Sulphamethoxazole/trimethoprim 19:1 (SXT 25 μg), Ceftriaxone (CRO 30μg), Ampicillin (AMP 10 μg), Amoxycillin/clavulanic acid (AMC 3μg), Chloramphenicol (C 30μg), Azithromycin (AZM 15μg), Cefoperazone/sulbactam 2:1 (SCF 105μg), Clarithromycin (CLR 15μg), Erythromycin (E 15μg), Tetracycline (TE 30μg), Trimethoprim/sulphamethoxazole (SXT 25μg), Vancomycin (VA 30μg), Oxacillin (OX 5μg), Spiamycin (Sp 100μg) and Rifampin (Ra 5μg). Genotypic characterization: DNA Preparation: The genomic DNA was isolated by a modified method of (Mullis et al., 1986). Cells from overnight LB cultures of the selected isolates were collected by centrifugation then re-suspended in 500µl TEN buffer, and 25µl lysozyme (10 mg/ml). After incubation at 37 C for 1h, 75µl of 10% SDS were added and mixed gently tell complete lysis. Then the mixture was extracted several times with phenol/chlorophorm to remove protein and once with chloroform to remove phenol traces. 100µl of NaCl (5M) were added then the DNA was precipitated using 0.7 volume of isopropanol and washed with 70% ethanol. The DNA was dried and dissolved in 10mM tris HCl (ph 8.0) and stored at - 20 0 C. The isolated chromosomal DNA was checked on 1% agarose gel electrophoresis. RAPD-PCR for isolates differentiation: Four primers (16sF 785 (18mer) 5ꞌ- GGATTAGATACCCTGGTA-3ꞌ; 16SF start (20mer) 5ꞌ-AGAGTTTGATCMTGGCTCAG-3ꞌ; 16sR end (21mer) 5ꞌ-TACGGYACCTTGTTACGACTT-3ꞌ; 16sR 1100 (15mer) 5ꞌ-GGGTTGCGCTCGTTG-3ꞌ) were used to amplify DNA fragments (Sambrook et al., 1989). PCR was carried out in a reaction volume of 50μl containing 5μl of 10X PCR buffer, 1μl of 10mM dntps, 2μl template DNA, 4μl of 25mM MgCl 2, 3μl of 10 pmol of each primer and 0.5μl Taq DNA polymerase (Dream). Thermal cycling (MyGene Series Peltier Thermal Cycler) was carried out by initial denaturation at 94 C for 4 min, followed by 25 cycles each at 94 C for 1 min, annealing temperature at 30 C for 1 min, polymerization temperature at 72 C for 1 min and final extension at 72 C for 10 min, then the samples were held at 4 C. The amplified DNA fragments were separated on 0.8-2.5% agarose gel (Mullis et al., 1986), stained with ethidium bromide, visualized on a UV Transilluminator and photographed by Gel Documentation system (Alpha Imager M1220, Documentation and Analysis System, Canada). Cluster analyses: Cluster analysis of the bacterial isolates under investigation was carried out according to the results of antibiotic sensitivity and test for temperature using past program. As well, cluster analyses based on RAPD-PCR results was carried out using the same program. Boiling effect: To test the boiling effect on bacterial growth of isolated Brucella (19 isolates), the isolates were allowed to grow individually in NB overnight at 37 C, then equivalent amounts of each bacterial cells were added to each other in a consortium for each
105 Safaa Ali, et al, 2015 treatment. Seven treatments were applied using boiling for 2, 4, 6, 8, 10, 15 and 20 min. Equivalent amounts of mixed treated cells were sub-cultured again in NB and allowed to grow again at 37 C overnight. Total bacterial count (%) was performed in each sample compared to reference (the untreated bacteria) by measuring the optical densities at 600 nm. RESULTS AND DISCUSSION In the current study, we focus on the attention to detect the presence of Brucella in Cocos nucifera, Arachis hypogaea, Ceratonia siliqua and Hyphaene thebaica which were obtained from perfumery and market shops in Egypt. Using selective medium for Brucella, the primary results indicated that the presence of this genus in the collected samples. Where, 19 isolates appeared in the tested samples: Cocos nucifera, Arachis hypogaea, Ceratonia siliqua and Hyphaene thebaica with ratio 4: 4: 6: 5, respectively. The majority of isolates (~60%) were isolated from market shop as a source of these tested foods. To verify these results, the isolates were cultured in Mackonky and blood agar media which is specific for gram negative. As well a routine differential Gram staining was carried out for isolates which showed an ability to grow in the former media. Gram smear revealed that all the selected bacteria (19 isolates) were Gram-negative, coccobacilli, non-spore-forming and non-motile. Further investigation for obtained 19 isolates was carried out by testing their sensitivity to different antibiotics (23 kinds) and temperatures (37-65 C). Both of the antibiotic and temperature sensitivities were recorded individually and in percentages. The results revealed that all selected Brucella isolates were showed 100% resistant to: Cefotaxime (CTX 30μg), Metronidazole (MTZ 50μg), Penicillin (GP 10 unit), Sulphamethoxazole/trimethoprim 19:1 (SXT 25μg), Ceftriaxone (CRO 30μg), Ampicillin (AMP 10μg) and Amoxycillin/clavulanic acid (AMC 3μg). In general, the tested isolates were 100% sensitive to Ciprofloxacin (CIP 5μg), Doxycycline (DO 30μg), Gentamicin (CN 10μg), Streptomycin (S 10μg) and Ofloxacin (OFX 5μg). However, the interaction towards the other tested antibiotics Chloramphenicol (C 30μg), Azithromycin (AZM 15μg), Cefoperazone/sulbactam 2:1 (SCF 105μg), Clarithromycin (CLR 15μg), Erythromycin (E 15μg), Tetracycline (TE 30μg), Trimethoprim/sulphamethoxazole (SXT 25μg), Vancomycin (VA 30μg), Oxacillin (OX 5μg), Spiamycin (Sp 100μg) and Rifampin (Ra 5μg) ranged 5.72-94.74% sensitivity (Figure 1). Current recommended treatment regimens involve the use of two or more antibiotics in order to avoid relapses occurring and to prevent prolonged use of these drugs, which may lead to problems of drug resistance arising. Fig. 1: Antibiotic sensitivity profile of Brucella bacterial pathogens isolated from Cocos nucifera, Arachis hypogaea, Ceratonia siliqua and Hyphaene thebaica samples. Temperature sensitivity of isolated Brucella at temperatures 37-65 C revealed that the optimum temperature for growth at 37-40 C, but the sensitivity to high temperatures were varied. Temperature sensitivity was recorded as percentage as shown in Figure 2, where the recorded growth (%) 80, 60, 50, 40, 32 at temperatures ( C) 45, 50, 55, 60, 65, respectively. Based on recorded individual results of high temperature and antibiotic sensitivities, cluster analyses for Brucella isolates
106 Safaa Ali, et al, 2015 were carried out (Figure 3). Figure 3 explains the presence of three clusters (I, II and III), each cluster sub-divided into two sub-cluster including isolate numbers: ((10,11,12,13,4) (15,18)), ((7,19,1,14,16) (9,2,3,5)) and ((17) (6,8)), respectively. As well clustering of Brucella isolates based on RAPD-PCR was carried out (Figure 4 a, b, c and d). Where, four primers (16sF 785, 16sF start, 16sR 1100 and 16sR end ) were used and the results were recorded in Fig 5 (a, b, c and d, respectively). Cluster view was created based on data analysis of RAPD-PCR results to find the kind of genetic relationship among the examined isolates (19 isolates). Fig. 2: Temperature sensitivity profile of Brucella bacterial pathogens isolated from Cocos nucifera, Arachis hypogaea, Ceratonia siliqua, and Hyphaene thebaica samples. Fig. 3: Clustering of Brucella isolates based on combined results of antibiotics and temperature sensitivities.
107 Safaa Ali, et al, 2015 Fig. 4 a, b. c and d: PCR amplifications obtained using RAPD-primers 16sF 785 (a), 16sF start (b), 16sR 1100 (c) and 16sR end (d). Lane M is a molecular weight marker and lanes 1-19 are tested Brucella isolates. Fig. 5: Dendogram shows the relationship among Brucella isolates based on RAPD-PCR profile results using four primers. Finally the boiling effect on Brucella (isolated bacteria) was studied and expressed the results as percentage compared to the control untreated samples (see Fig. 6). It is easily recognized a great drop near zero% in bacterial growth after 10 min exposure to boiling temperature. Consequently, boiling for 10 min can be applied as a precautionary step to prevent microbial growth of Brucella in tested samples as a recommendation before usage.
108 Safaa Ali, et al, 2015 Fig. 6: Effect of treatment boiling on bacterial growth. Conclusion: For detection of the pathogen in some foods, Cocos nucifera, Arachis hypogaea, Ceratonia siliqua and Hyphaene thebaica samples were collected from perfumery and market shops in Egypt. 19 Brucella isolates were captured in tested previous samples at ratio 4:4:6:5, respectively. Both antibiotic and temperature sensitivities were checked. Resistance rates to Amoxycillin/clavulanic acid, Ampicillin, Cefotaxime, Ceftriaxone, Metronidazole, Penicillin and Sulphamethoxazole/trimethoprim were 100%, while, the resistance rates to Erythromycin, Oxacillin, Rifampin and Trimethoprim/sulphamethoxazole were 50-95%. Sensitive rates to Ciprofloxacin, Doxycycline, Gentamicin, Ofloxacin and Streptomycin were 100%, while, high sensitivity ranged ~80-90% was recorded towards Azithromycin, Cefoperazone/sulbactam, Chloramphenicol, Spiamycin, Tetracycline and Vancomycin. The isolates were sensitive for high temperature, where, 10 min exposure to boiling temperature resulted to a great drop near zero% in bacterial growth. REFERENCES Bosilkovski, M., M. Dimzova and K. Grozdanovski, 2009.Natural history of brucellosis in an endemic region in different time periods. Acta Clinica Croatica, 48: 41-46. Bosilkovski, M., L. Krteva, M. Dimzova, I. Vidinic, Z. Sopova and K. Spasovska., 2010. Human Brucellosis in Macedonia 10 Years of Clinical Experience in Endemic Region. Croatian medical journal, 51: 327-336. Buchanan, T.M., L.C. Faber and R.A. Feldman, 1974. Brucellosis in the United States, 1960-1972. An abattoir-associated disease. Part I. Clinical features and therapy. Medicine (Baltimore), 53: 403-413. Colmenero, J.D., J.M. Reguera, F. Martos, D. Sánchez-De-Mora, M. Delgado, M. Causse, A. Martín-Farfán and C. Juárez, 1996. Complications associated with Brucella melitensis infection: a study of 530 cases. Medicine (Baltimore), 75: 195-211. Corbel, M.J., 2006. Brucellosis in humans and animals. Geneva: World Health Organization (WHO). pp: 89. Doganay, M. and B. Aygen, 2003. Human brucellosis: an overview. International Journal of Infectious Diseases, 7: 173-182. Fiori, P.L., S. Mastrandrea, P. Rappelli and P. Cappuccinelli, 2000. Brucella abortus infection acquired in microbiology laboratories. Journal of Clinical Microbiology, 38: 2005-6. Gotuzzo, E. and C. Carillo, 2004. Brucella. In: Infectious Diseases, 3rd Ed, Gorbach SL, Bartlett JG, Blacklow NR (Eds), Lippincott Williams & Wilkins, Philadelphia. p: 1717. Khan, M.Y., M.W. Mah and Z.A. Memish, 2001. Brucellosis in pregnant women. Clinical Infectious Diseases, 32: 1172-1177. Mantur, B.G., S.K. Amarnath and R.S. Shinde, 2007. Review of clinical and laboratory features of human brucellosis. Indian Journal of Medical Microbiology, 25: 188-202. Mullis, K., F. Faloona, S. Scharf, R. Saiki, G. Horn and H. Erlich, 1986. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor symposia on quantitative biology, 51: 263-273. Onanuga, A., A.R. Oyi, B.O. Olayinka and J.A. Onaolapo, 2005. Prevalence of communityassociated multi-resistant Staphylococcus aureus
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