مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Genetic Relationship Between Heavy Metals Resistance and β- Lactamase Production In E. Coli and Staphylococcus Aureus A.S . Husein Department of Biology, College of Science, university of Al-Mustanseryia Received in : 26 April 2011 Accepted in : 11 Ochober 2011 Abstract This study is a trail to know if the genes controlling some of heavy metals resistance ( lead, zinc, cadmium, cromium) in two types of pathogenic bacteria E. coli as gram negative bacteria and S. aureus as gram positive bacteria, present on the β-lactamase plasmid. Ten isolates of each bacterial types which produced β-lactamase enzyme, were cultivated in the presence of acridine orange. The growing in the presence of acridine orange resulted in loss of the β-lactamase genes in S. aureus and E. coli, and loss of the heavy metals resistance in S. aureus, while the resistance of E. coli against heavy metals still without any change. The results indicate that the genes for heavy metals resistance exist on the β-lactamase plasmid in S. aureus only, while in E. coli the genes that controlling heavy metals resistance are not on β- lactamase plasmid. Key words: heavy metals resistance, betalactamase and resistance, heavy metals and betalactamase. Introduction Most of pathogenic bacteria produce enzymes that inhibit the antibiotics as a resistance mechanism, one of these enzymes is the β-lactamase. β-lactamase enzymes of gram-positive bacteria such as staphylococcus aureus differ from that in gram-negative such as E. coli in an important point, they are an extra cellular enzymes in the first group, in the other words bacteria secrete the enzyme to the culture media where that the antibiotic lyse take, while in the gram-negative they are cell-bound enzymes, i.e. the β-lactamase antibiotics lyses inside of bacteria such as β-lactamase of E. coli [1]. Bacteria resistant to antibiotics and other antibacterial agents is an increasing problem in today's society. Products such as disinfectants, sterilants and heavy metals used in industry and in household products are, along with antibiotics, creating a selective pressure in the environment that lead to the mutations in microorganisms that will allow them better to survive and multiply [2]. In this study we will try to know if the gene controlling heavy metals resistance in two types of pathogenic bacteria presents on the β-lactamase plasmid. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Materials and Methods 1-Culture media: blood agar, MacConky agar, manitol salt agar, eosin methyline blue(EMB), nutrient agar, brain heart infusion broth, muller hinton agar 2- Solution of standard rapid iodometric method for β-lactamase detecting a- 1% starch solution b- Iodin solution c- Penicillin G solution The solution were prepared according to (Perret,1994) [3]. 3- Heavy metals solutions Salts of heavy metals were used to prepare certain heavy metals concentrations. a- Lead nitrate Pb(No3)2 . b- Cadmium chloride CdCl. c- Zinc chloride ZnCl2 . d- Chromium Oxide CrO3. These salts were used to prepare lead, cadmium, zinc, chromium solutions respectively. 4- Acridin orange 1-Sample collection and identification 25 nasal swabbed samples were obtained from young adults to isolates S. aureus bacteria. 30 samples were obtained from the mid stream urine from patients with urinary tract infections to isolates E. coli bacteria. The samples were cultured on the blood agar and macconky agar as a differential medium then incubated at 37ċ for 24 hr. Bacterial isolates identification included: microscopic exam, cultural, morphological, and biochemical characteristics of each isolate [4,5]. 2- β-lactamase detection ( rapid iodometric method) Twenty-four hours bacterial growth on nutrient agar was prepared for each isolates then 4- 5 colonies were transported to an appendrof tubes containing 100 µl of penicillin G solution and incubated at 37ċ for 30 minutes. Then 50 µl starch solution was added to each tube and mixed with the other content. After that 20 µl of iodin solution was added, a dark blue color will appear immediately due to starch-iodin interaction. The positive result was recorded if the blue color change to white within one minute [6]. 3- Detection of bacterial isolates resistance against some heavy metals The resistance of bacterial isolates against heavy metals was detected by adding 5mM of each heavy metals ( Pb(No3 )2, Cdcl, Zncl2, Cro3 ) separately to muller hinton medium after cooling to 45-50 ċ. The mixture was mixed immediately after heavy metals adding and then seeded on plates and kept at 4ċ for 24 hr. after incubation, 5µl of all bacterial isolates were spread on plates which contain 5mM of each heavy metals. The plates were left in room temperature, to dry, then incubated at 37ċ for 18-24 hr [7]. The bacteria that showing good growth on the medium with 5mM of the used heavy metals was considered as a heavy metal resistant isolates. 4- Curing of the β-lactamase plasmid state Small inocula of β-lactamase positive strains for each bacterial isolates were inculate in nutrient broth containing 12.5 µg/ml of acridin orange for 24 hr at 37 ċ [8]. Mutants that lost the β-lactamase genes were detected by streaking appropriate dilutions of the nutrient broth culture on to nutrient agar medium. After incubation for 24 hr the colonies were sufficiently large to be tested by the rapid iodometric method. The isolates that had lost the capacity to produce β-lactamase were tested for resistance to ( Pb(No3)2,Cdcl, Zncl2 and Cro3). مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Results and Discussion 1-β-lactamase production ( rapid iodometric method ) Table (1) shows the results of β-lactamase production for staphylococcus aureus and E. coli isolates. The results illustrate that all isolates of two bacterial types were producing β- lactamase enzyme except two isolates of E. coli did not show production of β-lactamase, because in some gram-negative bacteria the quantity of β-lactamase enzyme is too little to be detected by using rapid iodometric method [6]. 2- Bacterial resistance against heavy metals Table (2) shows the results of bacterial resistance to some heavy metals. The results illustrate that most of isolates were resistant to all heavy metals, because the bacteria have evolved several types of resistance mechanisms. These mechanisms include the efflux of metal ions outside the cell, accumulation and complexation of the metal ions inside the cell and reduction of the heavy metal ions to a less toxic state [8,9]. 3- β-lactamase production and heavy metals resistance of staphylococcus aureus and E. coli in the presence of acridin orange The results shows that all the isolates of staphylococcus aureus and E. coli lost the ability of β-lactamase production while the resistance of S. aureus to the heavy metals changed and the bacteria became sensitive to the used heavy metals. In E. coli isolates the resistance to the heavy metals stilled without any change. The growth in the presence of acridin orange resulted in lossing of β-lactamase production and heavy metals resistance in S. aureus because the β-lactamase plasmid in S. aureus also carries genes determining resistance to several metal ions. For example, in cadmium resistant S. aureus, two resistant determinants were found on penicillinase-plasmid pI258, called cadA (drives cadmium ion across the membrane by using energy from ATP hydrolysis to confer cadmium resistance) and cadB [10]. In the case of E. coli isolates the growth of bacteria in the presence of acridin orange resulted in losing of the β-lactamase production while the resistance of E. coli isolates to the heavy metals still without any change, i.e the genes controlling resistance to these heavy metals are not on the β-lactamase plasmid but existing on the other determinants like zntA chromosome which responsible for transporting Zn+2 and Cd+2 in the presence of ATP in E. coli . So, the resistance to these heavy metals was not always associated with β-lactamase production in E. coli [11] [12]. Conclusion Resistant determinants to the heavy metals are carrying on the β-lactemase plasmid in S. aureus only. While in E. coli the resistant determinants are carrying on chromosomal genes. References 1. Mediaros, A.A. (1997). Evolution and dissemination of β-lactamase accelerated by generations of β-lactamase. Clini. Infec. Dis. 24: 519-525. 2. Baquero, F., Negri, MC., Morosini, MI., and Blazquez, J. (1998). Antibiotics selective environments. Clinical Infection Diseases. 27: S5-S11. 3. Perret, C.J. (1999). Nature. Iodometric assay of penicillinase. 174:1012. 4. Holt, J.J, Krieg, N.R., Seneath, B.H., Staley, J.T., and Williams, S.T. (1994). Bergys manual determinative bacteriology 9 th edition. William & Wilken. Baltimore. P: 175-248. 5. Jawetz, E.M., Adelberg, E.A.(1998). Medical Microbiology, 25 th edition. Appleton and L ange. USA. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 6. WHO. (2002). Techniques for the detection of β-lactamase producing strains of Neisseria gonorrhoeae.616: 137-143. 7. Nies, D.H. (1999). Microbial heavy metal resistance. Appl. Microbiol Biotechnol. 51: 730- 750. 8. Jack N. Baldwin, Robert H. Strickland, and Marilyn F. Cox. (2000). β-lactamase genes in Staphylococcus epidermidis. Applied microbiology. 18: 628-630. 9. Bouanchaud, D.H., M.R. Scavizzi, and Y.A. Chabbert. (1999). Elimination by ethidium bromide of antibiotic resistance in enterobacter and staphylococci. J. Gen. Microbiol. 54: 417- 425. 10. Butaye, P., Cloeckaert, A., and Schwarz, S. (2003). Mobile genes coding for efflux mediated antimicrobial resistance in Gram-positive and Gram-negative bacteria. Int J AntimicrobAgents. 22: 205-210. 11. Otitoloju, A. A.; Rogers, G. B.; Bury, N. R. and Bruce, K. D. (2009). Chromosomal genes conferring tolerance to heavy metal (Ag) toxicity. The Enviromeny. Vol 29: 85-92. 12. Piddock, L. V. (2006). Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin. Microbiol. Rev. 19: 382-402 Table (1): β-lactamase production of staphylococcus aureus and E. coli s. aureus β-lactamase production E. coli β-lactamase production S1 + E1 + S2 + E2 * S3 + E3 + S4 + E4 + S5 + E5 + S6 + E6 + S7 + E7 * S8 + E8 + S9 + E9 + S10 + E10 + Key:(+): positive results, (*) : unknown results Table (2): the results of S . aureus resistance against four types of heavy metals S. aureus Pb(No3)2 Cdcl Zncl2 Cro3 S1 R * R R S2 * R R R S3 R R * R S4 R R R * S5 R R R * S6 R R R R S7 R R R R S8 * R R R S9 R R R R S10 R R R R Key :R= resistant (good growth), (*) =few growth مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Table (3):the results of E. coli resistance against four types of heavy metals E. coli Pb(No3)2 Cdcl Zncl2 Cro3 E1 R * R R E2 * R R R E3 * R R R E4 R R R R E5 * R R R E6 R R R R E7 R R R R E8 * R R R E9 R R R R E10 R R R R Key :R= resistant (good growth), (*) =few growth Table (4): the results of β-lactamase production and heavy metal resistance of S. aureus in the presenceof acridin orange S. aureus β-lactamase production Pb(No3)2 Cdcl Zncl2 Cro3 S1 - * * * * S2 - * * * * S3 - * * * * S4 - * * * * S5 - * * * * S6 - * * * * S7 - * * * * S8 - * * * * S9 - * * * * S10 - * * * * Key :( - )= no β-lactamase production , (*) = no growth of bacterial colonies Table (5): the results of β-lactamase production and heavy metal resistance of E. coli in the presence of acridin orange E. coli β-lactamase production Pb(No3)2 Cdcl Zncl2 Cro3 E1 - R ** R R E2 * ** R R R E3 - ** R R R E4 - R R R R E5 - ** R R R E6 - R R R R E7 * R R R R E8 - R R R R E9 - R R R R E10 - R R R R Key :( * ) = unknown results , (**) = few growth of bacterial colonies مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 العالقة الوراثیة بین مقاومة المعادن الثقیلة وإنتاج إنزیم البیتاالكتامیز في بكتریا E. coli و Staphylococcus aureus أنسام صفاء حسین الجامعة المستنصریة ، كلیة العلوم ،قسم علوم الحیاة 2011 تشرین االول 11: قبل البحث في 2011 نیسان 26:استلم البحث في لخالصةا ھذه الدراسة ھي محاولة لمعرفة فیما اذا كان الجین المسیطر على المقاومة لبعض المعادن الثقیلة الرصاص، الزنك، بكتریا موجبة ) S. aureus(بكتریا سالبة لصبغة كرام و ) E. coli(الكادمیوم و الكروم في نوعین من البكتریا المرضیة عشر عزالت من كل نوع بكتیري والمنتجة للبیتاالكتامیز قد تم تنمیتھا . غة كرام، موجود على البیتاالكتامیز بالزمیدلصب ج acridine orangeالتنمیة بوجود ال . acridine orangeبوجود ال نتج عنھا فقدان للجینات المسؤولة عن انتا فقط ، بینما S. aureus وفقدان المقاومة للمعادن الثقیلة في ال E. coli والS.aureusمن ال " البیتاالكتامیز انزیم في كال . للمعادن الثقیلة بقیت نفسھا دون أي تغییرE. coliمقاومة بكتریا ال یلة موجودة على البیتاالكتامیز بالزمید في بكتریا ھذه النتائج تدل على أن الجینات المسؤولة عن المقاومة للمعادن الثق ر E. coli فقط ، بینما في بكتریا ال S. aureusال فالجینات المسیطرة على مقاومة البكتریا للمعادن الثقیلة فھي غی .موجودة على نفس البالزمید المسؤول عن انتاج البیتالكتامیز انزیم زیم البیتاالكتامیزمقاومة المعادن الثقیلة ، إنزیم البیتاالكتامیز، المعادن الثقیلة وإن: الكلمات المفتاحیة مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012