Republic of Iraq Ministry of Higher Education And Scientific Research Baghdad University College of Science The effect of colloidal silver concentration and metallic copper against some hospital acquired infections isolates Thesis Submitted to the College of Science/Baghdad University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biotechnology By Alyaa Mohameed Hadi B.Sc., Biotechnology, College of Science, Baghdad University (2007) Supervised By Professor Dr. Alice K. Melconian 2013 1435 ﻦـﻤﺣﺮﻟٱ ﷲِٱ ﻢﺴِﺑ ِ ِ ٰ َ ﱠ ﻢﻴِﺣﺮﻟٱ ِ ﱠ ﺮﻜﺷَﺃ ﻥَﺃ ﻰِﻨﻋﺯﻭَﺃ ﺏﺭ)) ُ ِ َ ﱢَ ٓ ﻰﻠﻋ ﺖﻤﻌﻧَﺃ ﻰِﺘﻟٱ ﻚﺘﻤﻌِﻧ َ َ ﱠ َ َ َ َ َ ﱠ ٓ ﻞﻤﻋَﺃ ﻥَﺃﻭ ﻯﺪﻟَِﺃﻭ ﻰﻠﻋﻭ َ َ َ َ َ ﱠ َ ََ ٰ ﻰِﻨﻠِﺧﺩَﺃﻭ ﻪﺿﺮﺗ ﺎﺤِﻠـﺻ ٰ َ َ َ ُ ً َٰ ﻙِﺩﺎﺒِﻋ ﻰِﻓ ﻚِﺘﻤﺣﺮِﺑ َ َ َ َ ((ﻦﻴِﺤِﻠـﺼﻟٱ َ ٰﱠ ﻢﻴﻈﻌﻟﺍ ﺍ ﻪﺪﺻ ﱢَْ َ ََ (۱۹ ﺔﻳﻵﺍ) ﻞﻤﻨﻟﺍ ﺓﺭﻮﺳ Supervisor Certification We certify that the preparation of this thesis was made by Alyaa M. Hadi under our supervision at Baghdad University/ College of Science, as a partial fulfillment of the requirements for the Master Degree of Science in Biotechnology. Signature Supervisor: Dr. Alice K. Melconian Title: Professor Date: In view of the available recommendation, I forward this thesis debate by the Examining Committee. Signature Name: Dr. Abdul Kareem Al-Kazaz Head of Biotechnology Department Date: Committee Certification We, the examining committee, certify that we have read this thesis and examined the student in its contents and that, according to our opinion, is accepted as a thesis for the degree of Master of science in Biotechnology. Signature Signature Supervisor Name: Dr. Name: Dr. Title: Title: Date: Date: Signature Signature Name: Dr. Name: Dr. Title: Title: Date: Date: : Dedication To my grandfather ... who taught me that even the largest task can be accomplished if it is done one step at a time . To my father .... , who gave me a solid foundation in my life . To my mother .... , who is my rock of support and mentor par excellence To my husband.... , who is my light in darkness , my comfort and laugh in sadness . To my brothers.... , who always supported me with the expression of encouragement . To my son.... , my soul , my angel , who always makes me ambitious to do my best in this life . Acknowledgments In the name of ALLAH , most gracious , most merciful First I would like to express my profound gratitude to my supervisor Professor Dr.Alice K. Melconian for her guidance , scientific support and useful suggestions to solve my problems during the period of this study . I would like to thank the Dean of the college of Science Baghdad university and the Dean of the Medical Technical Institute ,to the Head and chancery of Biotechnology department for providing me with this opportunity to complete my M.Sc study.Grateful acknowledgment is also expressed to the Bacteriology Culture Laboratory of AL-Yarmouk Teaching Hospital and Baghdad Teaching Hospital for helping me get bacterial samples . Many thanks go to Dr.Hanan H. Ghulam for providing me with copper , aluminum and stainless steel materials , without forgetting the great help of my brother Zaid who provided me with different concentrations of colloidal silver solution from the U.K. I also extend my thanks to AL-Nooman Hospital for making it possible for me to do my VITEK tests in their laboratories . Special thanks to Mr. Ammar Khalid who helped me through the period of research and provided me with all important information about VITEK 2 compact system manuals. My acknowledgment is also expressed to M.Sc. student Iman Hatif and Dr. Israa A. Zedan and all the staff of bacteriology laboratory in the Biotechnology Department. I also thank my friends and colleagues during my study for being good groups . My sincere gratitude and love go to my mother , my father , my brothers , my husband and my son , God bless them all , for supporting me through my life . Alyaa M. Hadi August- 2013 Summary I ________________________________________________________________ summary This study is an attempt to prove that metallic copper and colloidal silver solutions have antimicrobial activities against different bacterial species which are considered as a major cause of nosocomial infections . One hundred and twenty samples were collected from different clinical sources ( urine , blood , pus , sputum , stool and wound swabs ) during the period from October /2012 to December /2012 from different hospitals in Baghdad including AL-Yarmouk teaching hospital and Baghdad teaching hospital . According to the collected samples , bacterial isolates were distributed as ( 22 ) Escherichia coli isolates ( 18.3%) , ( 12 ) Pseudomonas aeruginosa isolates( 10%) and ( 14 ) Staphylococcus aureus isolates ( 11.6% ). All these isolates were identified according to morphological and biochemical characteristics by using conventional methods and VITEK2 compact system for confirmation . The susceptibility test of the bacterial isolates was determined using (17) antimicrobials for E.coli isolates , ( 15 ) antimicrobials for P.aeruginosa isolates and ( 19 ) antimicrobials for S.aureus isolates . All these antimicrobials used were detected by using VITEK2 compact system . The results showed variation in resistance to the different antimicrobials tested on bacterial isolates . From each bacterial species three bacterial isolates with the highest resistance level were selected for this study . Since the bacterial species E.coli , P.aeruginosa and S.aureus are considered both as a major cause of nosocomial infections and as the most common species persisted in the clinical samples , these therefore were chosen to(1) evaluate the effectiveness of the metallic copper to reduce the viability of the selected bacterial isolates compared with the effect of both aluminum and stainless steel . (2) to evaluate both the susceptibility of the bacterial isolates to different concentrations of colloidal silver solutions using the agar-well diffusion method and study its effect on the viability reduction of bacterial isolates by using plate-viable count. An inoculum of ( 107 CFU/ml ) from each of the selected bacterial isolates was inoculated onto coupons ( 1cm x 1 cm ) of copper , aluminum and stainless steel Summary II ________________________________________________________________ and incubated at room temperature for various time periods ranging from 30 to180 minutes. Viability was determined using the plate -viable count method. Copper results revealed a complete kill for tested bacterial isolates after 120 minutes of exposure at room temperature indicating a high significant rate of viability reduction(P<0.0001) . In contrast , viable bacterial colonies were detected on aluminum and stainless steel coupons after 180 minutes of exposure . The results showed that the tested isolates were sensitive to colloidal silver solutions at both concentrations. The killing rate studies for various time periods ranging from 30 to 180 minutes at room temperature using plate viable count showed that colloidal silver was highly bactericidal against the tested bacterial isolates at both concentrations . Complete killing was achieved by 10 ppm concentration after( 180 ) minutes of exposure , which indicates high significant (P<0.0001) in viability reduction . Introduction_____________________________________________1___ Introduction Nosocomial infections cause a substantial burden for health and economics worldwide (Von Eiff et al.,2001; Weist et al.,2002). The most common nosocomial infections agents include Staphylococcus aureus , Escherichia coli and Pseudomonas aeruginosa , with additional concern is the emergence and dissemination of organisms with increased resistance to antimicrobial agents (Aavitsland et al.,1992; Lumio,1999). Escherichia coli is an opportunistic pathogen that belongs to a large bacterial family Enterobactereace which are Gram negative bacilli ( rod-shaped), facultative anaerobic , which is normally found as harmless commensalism in human intestine tract . E. coli cause infections in immunocomprimised patients like intestinal infections ,diarrhea and extra intestinal infections such as meningitis , septicemia and urinary tract infections (UTI) (Croxen and Finlay , 2010). P. aeruginosa is a Gram negative , rod shaped , strictly aerobec pathogen (Todar ,2004). It is an opportunistic pathogen that causes severe infections in individuals who are immunocompromised , such as urinary tract infections , respiratory tract infections , skin infections , especially in patients in the Intensive Care Unit (ICU) ( Pollack,2000 ; Stover et al., 2000). The infections caused by Pseudomonas aeruginosa are considered to be dangerous as these bacteria are inherently resistant to many antimicrobials and have the ability to acquire resistance to other effective antimicrobials that are commonly used in hospitals ( Gamal et al .,2007 ). Staphylococcus aureus is a Gram positive cocci , facultative anaerobic and an opportunistic pathogen that colonizes the skin and mucosal surfaces of the human host. It causes wide range of infections ranging ,in severity, from uncomplicated cellulitis , sepsis , impetigo ,to life threatening infections such as endocarditis , osteomylitis , necrotizing pneumonia ( Boucher et al.,2010). One of the most important S.aureus species that cause considerable morbidity and mortality in hospitals is methicillin- resistant S.aureus (MRSA). Owing to expression of an additional penicillin Introduction_____________________________________________2___ binding protein, PBP2a, with decreased affinity to β-lactam agents, MRSA is resistant to all beta-lactam antibiotics, including cephalosporin and staphylococcal penicillins (Berger-Bachi,1999). Many nosocomial infections are caused by these three bacterial species because of a prolonged hospital stay and exposure to contaminated medical devices (Safdar and Maki,2002). Many studies shows that copper surfaces may help diminish surface-related hygiene problems . Dry copper surfaces proved to have a great killing efficiency against a wide range of microbes (Casey et al.,2010 ; Espirito Santo et al.,2010 ).The copper free ions accumulate inside the cell and prevent cell reproduction by damaging the proteins and cell membranes . This will cause cell death within hours (Gant et al.,2007) . Another antimicrobial agent is the colloidal silver solution which consists of ultra fine small silver particles that remain suspended in water. Colloidal silver is different from other conventional antibiotics , microorganisms cannot build resistance to the action of silver (Warren ,2003 ). The silver ions act as a catalyst that effectively disable the metabolism enzymes to take their role ( Hall et al., 1999 ; Iroha et al.,2008 ). The silver antimicrobial properties have been found extensively useful in treating some serious infections caused by different bacterial species ( Wickless and Schwader , 2004). Consequently , the aim of the present study was to succeed in isolation and identification of the methicillin-resistant S. aureus , E. coli and P. aeruginosa and study the effect of the metallic copper in comparison with aluminum and stainless steel and the effect of different concentrations of colloidal silver solution on these isolates. Several steps were carried out to achieve the aims of this study . They are outlined as following : 1-Isolation and identification of methicillin-resistant Staphylococcus aureus , Escherichia coli and Pseudomonas aeruginosa from different clinical sources . 2- Determination of the sensitivity of the identified isolates to different antibiotics to determine the minimum inhibition concentration (MIC) for
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