ة مجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or 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 De partment of Biology, College of Science, unive rsity of Al-Mustanseryia Received in : 26 April 2011 Accepte d in : 11 Ochober 2011 Abstract This st udy is a trail to know if the genes controllin g some of heavy metals resistance ( lead, zinc, cadmium, cro mium) in two typ es of p athogenic bacteria E. coli as gram ne gative bacteria and S. aureus as gram p ositive bacteria, p resent on the β-lactamase p lasmid. Ten isolates of each bacterial typ es which p roduced β-lactamase enzy me, were cultivated in the p resence of acridine oran ge. The growin g in the p resence of acridin e oran ge 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 st ill without any chan ge. The results indicate that the genes for h eavy metals r esistance exist on the β-lactamase p lasmid in S. aureus only, while in E. coli the genes that controlling heavy metals resist ance are not on β- lactamase p lasmid. Key words: heavy metals resistance, betalactamase and resistance, heavy metals and betalactamase. Introduction M ost of p athogenic bacteria p roduce enzy mes that inhibit the antibiotics as a r esistance mechan ism, one of these enzy mes is the β-lactamase. β-lactamase enzy mes of gram-positive bacteria such as staphylococcus aureus differ from that in gr am-negative such as E. coli in an imp ortant p oint, they are an extra cellular enzy mes in the first group , in the other words bacteria secrete the enzy me to the culture media where that the antibiotic lyse take, while in the gram-negative they are cell-bound enzy mes, 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 incr easin g p roblem in today's society. Products such as disinfectants, sterilants and heavy metals used in industry and in household p roducts are, alon g with antibiotics, creating a selective p ressure in the environment that lead to the mutations in microorganisms that will allow them better to survive and multiply [2]. In this st udy we will try to know if the gene controlling heavy metals resistance in two types of p athogenic bacteria presents on the β-lactamase plasmid. ة مجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 Materials and Methods 1-Culture media: blood agar, M acConky agar, manitol salt agar, eosin methy line blue(EM B), nutrient agar, brain heart infusion broth, muller hinton a gar 2- Solution of st andard rapid iodometric method for β-lactamase detecting a- 1% st arch solution b- Iodin solution c- Penicillin G solution The solution were prep ared accordin g to (Perret,1994) [3]. 3- Heavy metals solutions Salts of heavy metals were used to p rep are certain heavy metals concentrations. a- Lead nitrate Pb(No3)2 . b- Cadmium chloride CdCl. c- Zinc chloride ZnCl2 . d- Chromium O xide CrO3. These salts were used to p rep are lead, cadmium, zinc, chromium solutions resp ectively. 4- Acridin orange 1-Samp le collection and identification 25 nasal swabbed samples were obtained from y oung adults t o isolates S. aureus bacteria. 30 samples were obtained from the mid st ream urine from p atients with urinary tract infections t o isolates E. coli bacteria. The samp les were cultured on the blood agar and macconky agar as a differential medium then incubated at 37ċ for 24 hr. Bacterial isolates identification included: microscop ic exam, cultural, morphological, and bio chemical characterist ics of each isolate [4,5]. 2- β-lactamase d etection ( rapid iodometric method) Twenty-four hours bacterial growt h on nutrient agar was p repared for each isolates then 4- 5 colonies were transp orted to an app endrof tubes containing 100 µl of p enicillin G solution and incubated at 37ċ for 30 minutes. Then 50 µl st arch 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 app ear immediately due to starch-iodin interaction. The positive result was recorded if the blue color chan ge to white within one minute [6]. 3- Detection of bacterial isol ates resist ance against some heavy metals The resistance of bacterial isolates against heavy metals was detected by addin g 5 mM of each heavy metals ( Pb(No3 )2, Cdcl, Zncl2, Cro3 ) sep arately to muller hinton medium after cooling to 45-50 ċ. The mixture was mixed immediately after heavy metals adding and then seeded on p lates and kept at 4ċ for 24 hr. after incubation, 5µl of all bacterial isolates were sp read on p lates which contain 5mM of each heavy metals. The p lates were left in room temp erature, to dry , then incubated at 37ċ for 18-24 hr [7]. The b acteria that showing good growt h on the medium with 5mM of the used heavy metals was considered as a heavy metal resistant isolates. 4- Curing of the β-lactamase p lasmid state Small inocu la of β-lactamase p ositive strains for each bacterial isolates were inculate in nutrient broth containing 12.5 µ g/ml of acridin oran ge for 24 hr at 37 ċ [8]. M utants t hat lost the β-lactamase genes were detected by st reaking app rop riate dilutions of the nutrient broth culture on to nutrient agar medium. Aft er incubation for 24 hr the colonies were sufficiently large to be tested by the rap id iodometric method. The isolates that had lost the cap acity to p roduce β-lactamase were tested for resistance to ( Pb(No3)2,Cdcl, Zncl2 and Cro3). ة مجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 Results and Discussion 1-β-lactamase p roduction ( rapid iodometric method ) Table (1) shows t he results of β-lactamase p roduction for staphylococcus aureus and E. coli isolates. The results illustrate that all isolates of two bacterial typ es were p roducing β- lactamase enzy me except two isolates of E. coli d id not show p roduction of β-lactamase, because in some gram-negative bacteria the quantity of β-lactamase enzy me is too litt le to be detected by using rapid iodometric method [6]. 2- Bacterial r esistance against heavy metals Table (2) shows the results of bacterial resistance to some heavy metals. T he results illustrate that most of isolates were resistant to all heavy metals, because the bacteria have evolv ed several ty p es of resistance mechanisms. These mechanisms include the efflu x of metal ions outside the cell, accumulation and comp lexation of the metal ions inside the cell and reduction of the heavy metal ions to a less t oxic st ate [8,9]. 3- β-lactamase p roduction and heavy metals resist ance of staphy lococcus aureus and E. coli in the presence of acridin oran ge The results shows that all the isolates of staphylococcus aureus and E. coli lost the ability of β-lactamase p roduction while the resistance of S. aureus to the heavy metals changed and the bacteria b ecame sensitive t o the used heavy metals. In E. co li isolates the resist ance to t he heavy metals stilled without any change. The growt h in the presence of acridin orange resulted in lossin g of β-lactamase p roduction and heavy metals resistance in S. aureus b ecause the β-lactamase p lasmid in S. aureus also carries genes d etermining resistance to several metal ions. For examp le, in cadmium resist ant S. aureus, two resistant determinants were found on p enicillinase-plasmid p I258, called cadA (drives cad mium ion across the membrane by usin g ener gy from AT P hy drolysis t o confer cadmiu m resist ance) and cadB [10]. In the case of E. coli isolates the growt h of bacteria in the p resence of acrid in oran ge resulted in losin g of the β-lactamase p roduction while the resist ance of E. coli isolates to the heavy metals still without any change, i.e the genes controllin g resistance to t hese heavy metals are not on t he β-lactamase plasmid but e xisting on the other determinants like znt A chromosome which r esp onsible for transp orting Zn+2 and Cd+2 in the p resence of ATP in E. coli . So, the resist ance to t hese heavy metals was not alway s associated with β-lactamase p roduction in E. coli [11] [12]. Conclusion Resistant determinants to the heavy metals are carrying on the β-lactemase p lasmid in S. aureus only . While in E. coli the resistant determinants are carrying on chromosomal genes. Re ferences 1. M ediaros, 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, M C., M orosini, M I., and Blazquez, J. (1998). Ant ibiotics selective environments. Clinical Inf ection Diseases. 27: S5-S11. 3. Perret, C.J. (1999). Nature. Iodometric assay of p enicillinase. 174 :1012. 4. Holt, J.J, Krieg, N.R., Seneath, B.H., Staley, J.T., and Williams, S.T. (1994). Bergy s manual determinative bacteriology 9 th edition. William & Wilken. B altimore. P: 175-248. 5. Jawetz, E.M ., Adelberg, E.A.(1998). M edical M icrobiolo gy , 25 th edition. Ap p leton and L ange. USA. ة مجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 6. WHO. (2002). Techniques for the detection of β-lactamase p roducin g st rains of Neisseria gonorrhoeae.616: 137-143. 7. Nies, D.H. (1999). M icrobial heavy metal resistance. App l. M icrobiol Biotechnol. 51: 730- 750. 8. Jack N. Baldwin, Robert H. Strickland, and M arilyn F. Cox. (2000). β-lactamase genes in Staphy lococcus ep idermidis. Ap p lied microbiolo gy . 18: 628-630. 9. Bouanchaud, D.H., M .R. Scavizz i, and Y.A. Chabbert. (1999). Elimin ation by ethidium bromide of antibiotic resistance in enterobacter and st ap hy lococci. J. Gen. M icrobiol. 54: 417- 425. 10. Butaye, P., Cloeckaert, A., and Schwarz, S. (2003). M obile genes codin g for efflu x mediated antimicrobial resist ance in Gram-p ositive and Gram-negative bacteria. Int J AntimicrobAgents. 22: 205-210. 11. Ot itoloju, A. A.; Rogers, G. B.; Bury , N. R. and Bruce, K. D. (2009). Chromosomal genes conferring tolerance to heavy metal (Ag) to xicity . The Enviromeny. Vol 29: 85-92. 12. Piddock, L. V. (2006). C linically relevant chromosomally encoded mu ltidrug r esistance efflux p ump s in bacteria. Clin. M icrobiol. Rev. 19: 382-402 Table (1): β-lactamase production of staphylococcu s aureu s and E. coli s. aureu s 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 :(+): p ositive 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 growt h), (*) =few growt h ة مجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or 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 growt h), (*) =few growt h Table (4): the results of β-lactamase production and he avy metal resistance of S. aureus in the presenceof acridin orange S. aureus β-lactamas e produ ction Pb(No3)2 Cdcl Zncl2 Cro3 S1 - * * * * S2 - * * * * S3 - * * * * S4 - * * * * S5 - * * * * S6 - * * * * S7 - * * * * S8 - * * * * S9 - * * * * S10 - * * * * Key :( - )= no β-lactamase production , (*) = no growt h of bacterial colon ies Table (5): the results of β-lactamase production and he avy metal resistance of E. coli in the presence of acridin orange E. coli β-lactamas e produ ction 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 growt h of bacterial colonies ة مجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or 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 ة كرام و ) E. coli(الكادمیوم و الكروم في نوعین من البكتریا المرضیة بكتریا موجبة ) S. aureus(بكتریا سالبة لصبغ عشر عزالت من كل نوع بكتیري والمنتجة للبیتاالكتامیز قد تم تنمیتھا . غة كرام، موجود على البیتاالكتامیز بالزمیدلصب acridinبوجود ال e orange . التنمیة بوجود الacridin e orange ج 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 ة بكتریا ال . للمعادن الثقیلة بقیت نفسھا دون أي تغییرE. coliمقاوم 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 ة مجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012