Incidence of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Various Fermented Foods
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Incidence of Antibiotic Resistance in Lactic Acid Bacteria Isolated From Various Fermented Foods By Rania Mohamed Saeed B.Sc (agric) honour Faculty of Agriculture - University of Khartoum November 2004 A Dissertation Submitted to the University of Khartoum in partial fulfillment of the requirement of the degree of M.Sc. in Food Science and Technology Supervisor Dr. Nuha Mohammed Elkhatim Yousif Dept. of Food Science and Technology Faculty of Agriculture - University of Khartoum December - 2007 i DEDICATION TO MY FAMILY... WITH LOVE ii Acknowledgment I would like to express my deepest thanks to my supervisor Dr. Nuha Mohammed Elkhatim for her close supervision, supporting, advising and encouraging me throughout this study, and thanks for her personal guidance and fruitful criticism from which I benefit much. My thanks are also to all staff of the Department of Plant and Agric. Biotechnology and the Department of Food Science and Technology, Faculty of Agriculture, University of Khartoum. My deep gratitude goes to all staff of the Central Laboratory-Shambat Campus for unlimited help and friendly attitude during the practical work. I am pleased to express my appreciation to Mr. Abed Elazeez Mustafa, Mr. Mohammed Osman, my Friends and to my colleagues Hunda and Sara for their unlimited and continuous help. Thanks are first and last to Allah who enabled me to conduct this study by the grace of Him who gave me strength and patience. iii Abstract In this study, 25 isolates of lactic acid bacteria (LAB) were isolated from different types of fermented foods consumed in Sudan. Phenotypic tests revealed that, all isolates were homofermentative LAB. Among these, 40% were identified as lactobacilli, 44% as enterococci, and 16% of them were identified as pediococci. Standard disk diffusion method was used to determine the resistances of these isolates to 8 different antibiotics ; amoxicillin, ceftriaxone, ciprofloxacin, chloramphenicol, erythromycin, tetracycline, tobramycin, and vancomycin. All isolates showed multiple resistances to the antibiotics used, specifically, tobramycin, ceftriaxone and ciprofloxacin. 50% of Lactobacillus isolates and 27% of Enterococcus isolates were resistant to vancomycin, whereas all Pediococcus isolates were resistant to vancomycin. 36% of Enterococcus isolates were resistant to amoxicillin and erythromycin, whereas, Lactobacillus and Pediococcus isolates were susceptible amoxicillin and erythromycin. All isolates were susceptible to chloramphenicol and tetracycline. iv ﻣﻠﺨﺺ اﻷﻃﺮوﺣﺔ ﺧﻼل هﺬﻩ اﻟﺪراﺳﺔ ﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻰ 25 ﻋﺰل ﻣﻦ ﺑﻜﺘﻴﺮﻳﺎ ﺣﻤﺾ اﻟﻼآﺘﻴﻚ ﻣﻦ أﻧﻮاع ﻣﺨﺘﻠﻔﺔ ﻣﻦ اﻷﻏﺬﻳﺔ اﻟﻤﺨﻤﺮة اﻟﻤﺴﺘﻬﻠﻜﺔ ﻓﻲ اﻟﺴﻮدان . أآﺪت اﻻﺧﺘﺒﺎرات اﻟﻤﻈﻬﺮﻳﺔ أن ﺟﻤﻴﻊ اﻟﻌﺰﻻت ﻋﺒﺎرة ﻋﻦ ﺑﻜﺘﻴﺮﻳﺎ ﺣﻤﺾ اﻟﻼآﺘﻴﻚ ﻣﺘﺠﺎﻧﺴﺔ اﻟﺘﺨﻤﻴﺮ، و ﻣﻨﻬﺎ ﺗﻢ اﻟﺘﻌﺮف ﻋﻠﻰ ﺛﻼث ﻣﺠﻤﻮﻋﺎت أﺳﺎﺳﻴﺔ و هﻲ : pediococci . و(lactobacilli%44) ، enterococci ((%16(%40) ﺗﻢ اﺳﺘﺨﺪام ﻃﺮﻳﻘﺔ ﻧﺸﺮ اﻷﻗﺮاص اﻟﻘﻴﺎﺳﻴﺔ ﻟﺘﺤﺪﻳﺪ ﻣﻘﺎوﻣﺔ هﺬﻩ اﻟﻌﺰﻻت ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ اﻟﺘﺎﻟﻴﺔ : أﻣﻮآﺴﻴﺴﻠﻴﻦ، ﺳﻔﺘﺮﻳﺎآﺴﻮن، آﻠﻮراﻣﻔﻴﻨﻜﻮل، ﺳﻴﺒﺮوﻓﻠﻜﺴﺎﺳﻴﻦ، إرﻳﺜﺮوﻣﻴﺴﻴﻦ، ﺗﻴﺘﺮاﺳﻴﻜﻠﻴﻦ، ﺗﺒﺮاﻣﻴﺴﻴﻦ وﭬﺎﻧﻜﻮﻣﻴﺴﻴﻦ. وﺟﺪ أن ﺟﻤﻴﻊ ﻋﺰﻻت ﺑﻜﺘﻴﺮﻳﺎ ﺣﻤﺾ اﻟﻼآﺘﻴﻚ ﻣﻘﺎوﻣﺔ ﻟﻠﺴﻔﺘﺮﻳﺎآﺴﻮن ، ﺳﻴﺒﺮوﻓﻠﻮآﺴﺎﺳﻴﻦ و واﻟﺘﺒﺮاﻣﻴﺴﻴﻦ. آﺎﻧﺖ ﻣﻘﺎوﻣﺔ Lactobacillus و 27% ﻣﻦ ﻋﺰﻻت 50Enterococcus % ﻣﻦ ﻋﺰﻻت ﻣﻘﺎوﻣﺔ ﻟﻠﭭﺎﻧﻜﻮﻣﻴﺴﻴﻦ .Pediococcusﻟﻠﭭﺎﻧﻜﻮﻣﻴﺴﻴﻦ ، ﺑﻴﻨﻤﺎ آﺎﻧﺖ ﺟﻤﻴﻊ ﻋﺰﻻت آﺎﻧﺖ ﻣﻘﺎوﻣﺔ ﻟﻸﻣﻮآﺴﻴﺴﻴﻠﻴﻦ و 36 % أﺧﺮى ﻣﻨﻬﺎ آﺎﻧﺖ 36Enterococcus% ﻣﻦ ﻋﺰﻻت ﺣﺴﺎﺳﺔ Pediococcus و Lactobacillusﻣﻘﺎوﻣﺔ ﻟﻺرﻳﺜﺮوﻣﻴﺴﻴﻦ ، ﺑﻴﻨﻤﺎ آﺎﻧﺖ ﻋﺰﻻت ﻟﻸﻣﻮآﺴﻴﺴﻠﻴﻦ و اﻹرﻳﺜﺮوﻣﻴﺴﻴﻦ . ﺟﻤﻴﻊ ﻋﺰﻻت ﺑﻜﺘﺮﻳﺎ ﺣﻤﺾ اﻟﻼآﺘﻴﻚ آﺎﻧﺖ ﺣﺴﺎﺳﺔ ﻟﻠﻜﻠﻮراﻣﻔﻴﻨﻜﻮل و اﻟﺘﻴﺘﺮاﺳﻴﻜﻠﻴﻦ. v Table of Contents page Dedication ……………………………………………………… i Acknowledgement ……………………………………………. ii Abstract ……………………………………………………….. iii Arabic abstract………………………………………………… iv Table of contents ……………………………………………… v List of tables …………………………………………………... vii List of figures …………………………………………………. viii Chapter 1: Introduction…………………………………………. 1.1. Introduction ………………………………………… 1 Chapter 2: Literature Review…………………………………... 2.1. Food fermentation …………………………………………. 4 2.1.1. Lactic acid fermentation …………………………………. 5 2.1.2. Types of fermented foods ……………………………….. 6 2.1.2.1. Fermented milk products………………………………… 6 2.1.2.2. Fermented meat products ……………………………….. 6 2.1.2.3. Fermented vegetable products ………………………… 7 2.1.2.4. Cereal -based (European breads) …………… 7 2.1.2.5. African fermented cereal-based foods…………………… 8 2.2. Lactic acid bacteria 10 2.3. Antibiotic gents……………………………………………… 12 2.3.1. Mechanism of action of antibiotics. 13 2.3.2. Types of antibiotics……………………………………….. 14 2.4. Antibiotic resistance……………………………………….. 18 2.4.1. Types of bacterial resistance ……………………………. 18 2.4.1.1. Intrinsic resistance…………………………………….. 18 2.4.1.2. Acquired resistance …………………………………… 18 2.4.2. Transmission of resistance gene………………………… 19 2.4.3. Mechanisms of resistance ……………………………… 20 2.5. Antibiotic resistance of LAB 21 Chapter 3: Materials and Methods …………………………….. 3.1. Microbiological sampling and isolation …………………… 24 vi 3.2. Phenotypic characterizations………………………………. 25 3.2.1. Cell morphology ………………………………………… 25 3.2.2. Catalase activity…………………………………………. 25 3.2.3. Gram reaction …………………………………………… 25 3.2.4. Endospore stain …………………………………………. 26 3.2.5. Growth in different temperatures ………………………… 26 3.2.6. Growth in NaCl concentrations…………………………... 26 3.2.7. Growth at pH 9.6 …………………………………………. 27 3.2.8. Production of acid and/or gas (CO2) from glucose ……….. 27 3.3. Antibiotic susceptibility testing ……………………………. 27 Chapter 4: Results and Discussion………................................... 4.1. Phenotypic characterization of LAB isolates………………... 30 4.2. Antibiotic susceptibility testing……………………………. 33 4.2.1. Antibiotic resistance of Lactobacillus sp…………………. 36 4.2.2. Antibiotic resistance of Enterococcus sp………………. 36 4.2.3. Antibiotic resistance of Pediococcus sp……………... 41 Chapter 5: Conclusions and recommendations………………... 5.1. Conclusions.............................................................................. 44 5.2. Recomendations....................................................................... 45 REFERENCES............................................................................... 46 APPENDIX...................................................................................... 63 vii List of Tables Table 1 Antibiotic discs used, their abbreviation, concentration 29 and action Table 2 Characteristics Used To Differentiate Isolated LAB 31 Table 3 The genera of LAB isolated from different sources 34 35 Table 4 Antibiotic Resistance of LAB Isolated From Different Fermented Foods viii List of Figures Figure 1 Reaction of Lactobacillus to Different Antibiotics 37 Figure 2 Inhibition Zone of Lactobacillus Isolates Towards the Different 38 Antibiotics Tested Figure 3 Reaction of Enterococcus to Different Antibiotics 39 Figure 4 Inhibition Zone of Enterococcus Isolates Towards the Different 40 Antibiotics Tested Figure 5 Reaction of Pediococcus to Different Antibiotics 42 Figure 6 Inhibition Zone of Pediococcus Isolates Towards the Different 43 Antibiotics Tested ix CHAPTER 1: INTRODUCTION 1.1. Introduction Lactic acid bacteria (LAB) are constituted of a heterogeneous group of Gram-positive bacteria united by a constellation of morphological, metabolic and physiological characteristics (Axelsson, 1998). Lactococcus, Leuconostoc, Pediococcus, Enterococcus, Lactobacillus and Streptococcus are example of bacterial genera that are members of the LAB group (Adams & Moss, 2000). They can be found in different nutrient rich habitats like mucosal membranes of humans and animals, on plants and in many food and feed systems (Holzapfel et al., 2001). LAB have strictly fermentative metabolism from which lactic acid is the main metabolite during the fermentation of carbohydrates (Axelsson, 1998; Holzapfel et al., 2001). The fermentation pattern of LAB can be described as homo- or heterofermentative depending on how they ferment carbohydrates under non-limited conditions. Homofermentative LAB use the glycolysis pathway with lactic acid as the main product. Heterofermentative LAB use the 6P-gluconate pathway or phosphoketolase pathway and the main end products are equal amounts of lactic acid, carbon dioxide and ethanol (Adams & Moss, 2000). LAB have a long and safe history of use in the production and consumption of fermented foods and beverages (Wood and Holzapfel, 1995; Caplice and Fitzgerald, 1999; Leroy and Vuyst, 2004). Therefore, lactic acid bacteria are widely used as probiotics or in starter cultures in food fermentation 1 process (Schleifer and Ludwig, 1995). Lactic acid fermentation as a means of food preservation is probably one of the oldest ‘biotechnological processes’ rooted in the cultural history of mankind (Geisen and Holzapfel, 1996). About 50 years ago, antibiotics were introduced for the treatment of microbial diseases. Since then, the greatest threat to the use of antibiotics for therapy of bacterial infections has been the development of antibiotic resistance in pathogenic bacteria that consequently cannot be treated by previously successful regimens. Extensive recent reviews of the application of antibiotics in human and veterinary medicine (Levy, 1997; WHO, 1997), agriculture (Natural Research Council and Institute of Medicine US, 1998; Falkiner, 1998) and aquaculture (Reilly and Kaferstein, 1997) have documented the evolution and enrichment of antibiotic resistant bacteria. Antibiotic resistance has been shown to have occurred rarely in bacteria collected before the antibiotic era (Hughes and Datta, 1983). Shortly after the introduction of each new antibiotic compound, emergence of antimicrobial