Microbiology Past Paper PDF - Ain Shams University

MICROBIOLOGY Microbiology By: Dr.Nagwa Mohammed Faculty of Medicine Ain Shams University Second Year 2019/2018 1 ...

MICROBIOLOGY Microbiology By: Dr.Nagwa Mohammed Faculty of Medicine Ain Shams University Second Year 2019/2018 1  MICROBIOLOGY Acknowledgments This two-year curriculum was developed through a participatory and collaborative approach between the Academic faculty staff affiliated to Egyptian Universities as Alexandria University, Ain Shams University, Cairo University , Mansoura University, Al-Azhar University, Tanta University, Beni Souef University , Port Said University, Suez Canal University and MTI University and the Ministry of Health and Population(General Directorate of Technical Health Education (THE). The design of this course draws on rich discussions through workshops. The outcome of the workshop was course specification with Indented learning outcomes and the course contents, which served as a guide to the initial design. We would like to thank Prof.Sabah Al- Sharkawi the General Coordinator of General Directorate of Technical Health Education, Dr. Azza Dosoky the Head of Central Administration of HR Development, Dr. Seada Farghly the General Director of THE and all share persons working at General Administration of the THE for their time and critical feedback during the development of this course. Special thanks to the Minister of Health and Population Dr. Hala Zayed and Former Minister of Health Dr. Ahmed Emad Edin Rady for their decision to recognize and professionalize health education by issuing a decree to develop and strengthen the technical health education curriculum for pre-service training within the technical health institutes. 2  MICROBIOLOGY ً‫جىصيف مقشس دساس‬ ‫ تياوات المقشس‬-1 : ‫المسحىي‬/ ‫الفشقة‬ : ‫اسم المقشس‬ : ‫الشمض الكىدي‬ ‫الثاويه‬ microbiology ً‫عمل‬2 ‫ وظشي‬3 : ‫عذد الىحذات الذساسية‬ : ‫الحخصص‬ :‫ هذف المقشس‬-2 This is a course equip students with knowledge and basic skills 2-Course Goals related to nutritional microbiology and the relevance of microbes to (overall aim): human disease. : ‫ المسحهذف مه جذسيس المقشس‬-3 3-Intended Learning Outcomes (ILOs): By the end of the course every student will be able to: A.1. Explain how the microbiome is important for maintaining human ‫ المعلىمات‬.‫ا‬ health. : ‫والمفاهيم‬ A.2 Identify the difference between breast milk and formula. A-Knowledge A.3. Recognize interaction among gut microbiota, host and food. and A.4.Describe the interaction between malnutrition and infection. Understanding: A.5. Explain how a deficiency, excess or imbalance of nutrients may affect immunity. A.6.Explain how inflammation is involved in chronic disease: obesity, cardiovascular disease and type 2 diabetes. A.7.Describe the role of probiotics and prebiotics in treating various medical conditions. A.8. Identify the principles of food safety. B.1. Discuss the main dietary goals in the management of IBD. B.2. Discuss the main dietary goals in the management of food ‫ المهاسات‬-‫ب‬ allergy. B.3. Analyze a case study problem about infectious diseases. : ‫الزهىية‬ B.4.Discuss the role of vitamins in immune function: A, B-6, B-12, C, D, E, beta-carotene and folic acid. B.5. Discuss the role of minerals in immune function: zinc, copper, iron, magnesium and selenium. B- Intellectual B.6.Discuss the concepts of epigenetics and nutrigenomics and Skills: how they relate to immunity and diet. C.1. Implement nutritional management of HIV infection, IBD and ‫ المهاسات المهىية‬-‫ج‬ food allergy. :‫الخاصة تالمقشس‬ C.2.Use the appropriate probiotic or prebiotic for various medical conditions. C- Professional C.3.Apply the reliable, genomic information of nutrigenomics with and Practical 3  MICROBIOLOGY the goal of improving health through personalized nutrition. Skills: D.1. Work in collaboration as a member of an interdisciplinary team to solve a case about infectious diseases. ‫ المهاسات‬-‫د‬ D.2. Gather, organize and appraise information including the use of : ‫العامة‬ information technology where applicable in diagnosis of infectious diseases. D-General and D.3. Present the reliable medical information of nutrigenomics, Transferable prebiotics and probiotics in written, oral and electronic forms. Skills: 1. Microbiota in health and disease. 2. Gut microbiota. :‫ مححىي المقشس‬-4 3. The microbiota in inflammatory bowel disease 4-Course Content 4. Food microbiology and food safety 5. Interaction between malnutrition and infection 6. Nutrition and immunity. 7. Food allergy 8. HIV/AIDS 9. Microorganisms in Human Welfare: genetically engineered organisms, probiotics and single cell proteins 10. Prebiotics. 11. Food and water borne diseases. 12. Molecular genetics, epigenetics and nutrigenomics. 5.1. Lecture/ week (for two hours each) ‫ أسااية الحعليم والحعلم‬-5 5.2. Problem solving 5-Teaching and 5.3. Small group discussion Learning Methods: ‫ أسالية الحعليم والحعلم للطالب‬-6 ‫روي القذسات المحذودة‬ 6-Teaching and Learning Methods for students with limited capabilities: : ‫ جقىيم الطالب‬-7 7- Student Assessment Methods: - Written exam. ‫ األسالية المسحخذمة‬-‫أ‬ -MCQs exam. A-Methods used - Quizzes, assignments, and practical exam. Midterm Exam: At the ---------- week ‫ الحىقيث‬-‫ب‬ Final Exam: At the end of the course B- Timing of Assessment: final written examination:60% 80 marks ‫ جىصيع الذسجات‬-‫ج‬ semester work: 5% quiz 3 marks Attendance : 2 marks Midterm ; 10 marks C-Distribution of Assignment: 10 marks Scores: 4  MICROBIOLOGY total:100% : ‫ قائمة الكحة الذساسية والمشاجع‬-8 8-List of Textbooks and References: ‫ مزكشات‬-‫أ‬ A- Course Notes: ‫ كحة ملضمة‬-‫ب‬ B- Essential Books (Textbooks): -Microbiology: An Introduction/Media Update, 7th ‫ كحة مقحشحة‬-‫ج‬ Tortora, Gerard J.; Case, Christine L.; Funke, Berdell R. - Case Files Microbiology, Third Editionby Eugene Toy C- Recommended Books: and Cynthia R. Skinner DeBord -Clinical Case Studies For The Nutrition Care Process by Elizabeth Zorzanello Emery - Nikolaos Katsilambros, Charilaos Dimosthenopoulos, Meropi Kontogianni and Evangelia Manglara(2010) Clinical Nutrition in Practice Google scholar, pubmed search. ‫ الخ‬...... ‫ دوسيات علمية أو وششات‬-‫د‬ Periodicals, websites,…. etc. 5  MICROBIOLOGY Course Contents Course Description ……………………………………………………….…………...v Chapter 1: Micro biota in health and disease............................…..………..........16 Chapter 2: The gut micro biota………………………………………….…............25 Chapter 3: The micro biota in inflammatory bowel disease ……........34 Chapter 4: Food microbiology and food safety ………………....….....38 Chapter 5: Interaction between Malnutrition and Infection……........53 Chapter 6: Nutrition and Immunity ………………………….…….....61 Chapter 7: Food Allergy…………………………………………….....70 Chapter 8: HIV/AIDS disease and nutrition ……………….…………………....…74 Chapter 9: Microorganisms in human welfare: probiotics…………….…………...81 Chapter 10: Prebiotic……………………………………………..…......87 Chapter 11: Food and waterborne diseases………………………..…....97 Chapter 12: Molecular genetics, epigenetics and nutrigenomics…..…..107 References and Recommended Readings……………….............…......118 ‫حقىق الىشش والحأليف لىصاسة الصحة والسكان ويحزس تيعه‬ 6  MICROBIOLOGY Course Description: This is a course through which the students should gain the knowledge and insight into therapeutic Nutrition and must be able to: Know latest knowledge and basic skills in the theoretical and practical aspects of microbiology and immunology and the relevance of microbes to Therapeutic Nutrition. Core Knowledge By the end of the course every student will be able to: A.1 Explain how the microbiome is important for maintaining human health. A.2 Discuss the difference between breast milk and Formula. A.3. Recognize Interaction among gut microbiota, host and food. A.4.Describe the interaction between malnutrition and infection. A.5. Explain how a deficiency, excess or imbalance of nutrients may affect immunity. A.6.Explain how inflammation is involved in chronic disease: obesity, cardiovascular disease and type 2 diabetes. A.7.Describe the role of probiotics and prebiotics in treating various medical conditions. A.8. Understand the principles of food safety. Core Skills By the end of this course, students should be able to: B.1. Discuss the The main dietary goals in the management of IBD. B.2. Discuss the The main dietary goals in the management of food allergy. B.3. Analyse a case study problem about infectious diseases. B.4.Discuss the role of vitamins in immune function: A, B-6, B-12, C, D, E, beta-carotene and folic acid. B.5. Discuss the role of minerals in immune function: zinc, copper, iron, magnesium and selenium. B.6.Discuss the concepts of epigenetics and nutrigenomics and how they relate to immunity and diet. v 7  MICROBIOLOGY Course Overview Methods of Teaching/Training with Number of Total Hours per Topic ID Topics Lecture Assignments Practical 1 Micro biota in health and disease 2 2 Gut micro biota. 2 2 2 The micro biota in inflammatory bowel disease 3 2 2 Food microbiology and food safety 4 2 2 Interaction between malnutrition and 5 infection 2 2 Nutrition and immunity. 6 2 2 Food allergy 7 2 2 HIV/AIDS 8 2 2 Microorganisms in Human Welfare: 9 2 2 genetically engineered organisms, probiotics Prebiotic 10 2 2 11 Food and water borne diseases 2 2 Molecular genetics, epigenetics and 12 2 2 nutrigenomics Total hours (48) 24 24 8  MICROBIOLOGY Terminology Acquired immunodeficiency syndrome (AIDS)is a disorder resulting from the infection with the human immunodeficiency virus (HIV), which leads to a profound immunosuppression and high susceptibility to life-threatening Allergen: A substance (antigen, see below) that provokes an allergic reaction. Antibodies: Substances produced by B cells that react with antigens and prepare them for destruction. Antigen: Asubstance that triggers an immune response. Antioxidant: Any substance that can delay or inhibit oxidation. Atherosclerosis: A degenerative disease of arteries in which there is thickening caused by the accumulation of material (plaque) beneath the inner lining, eventually restricting blood flow. The plaque characteristically contains cholesterol and macrophages. Cloning is the process of creating many identical copies of a sequence of DNA. The target DNA sequence is inserted into a cloning vector Epigenetics is the study of heritable phenotype changes that do not involve alterations in the DNA sequence. Epigenetics most often denotes changes that affect gene activity and expression, but can also be used to describe any heritable phenotypic change. Fermentation is one-way microorganisms can change a food. Yeast, especially Saccharomyces cerevisiae, is used to leaven bread, brew beer and make wine. Food allergy refers to specific reactions that result from an abnormal immunological response to a food and which can be severe and life-threatening and triggered by minute amounts of the allergen. Food microbiology is the study of the microorganisms that inhabit, create, or contaminate food. 9  MICROBIOLOGY Food safety is a major focus of food microbiology. Pathogenic bacteria, viruses and toxins produced by microorganisms are all possible contaminants of food. Gene amplification is a procedure in which a certain gene or DNA sequence is replicated many times in a process called DNA replication. Highly active antiretroviral therapy (HAART) is a term used to describe the use of a combination of antiretroviral drugs for the treatment of HIV infection. Human microbiome refers specifically to the collective genomes of resident microorganisms. Human Microbiome Project took on the project of sequencing the genome of the human microbiota, focusing particularly on the microbiota that normally inhabit the skin, mouth, nose, digestive tract, and vagina. Inflammation is a basic process whereby tissues of the body respond to injury. Malnutrition is a condition that results from eating a diet in which one or more nutrients are either not enough or are too much such that the diet causes health problems. It may involve calories, protein, carbohydrates, vitamins or minerals. Microbiota is a collective term for the micro-organisms that live in or on the human body. Specific clusters of microbiota are found on the skin or in the gastrointestinal tract, mouth, vagina and eyes. Molecular genetics is the field of biology that studies the structure and function of genes at a molecular level and thus employs methods of both molecular biology and genetics. Non-allergic food intolerance refers to reactions to food that can result from a number of causes, none of which is mediated by the immune system (e.g. pharmacological effects, enzyme deficiencies, irritant and toxic effects). 10  MICROBIOLOGY Nutrigenetics: studies the effect of genetic variations on the interaction between diet and health with implications to susceptible subgroups. Nutrigenomics: studies the effect of nutrients on health through altering genome, proteome, metabolome and the resulting changes in physiology. Nutritional genomics is a science studying the relationship between human genome, nutrition and health. opportunistic infections and malignancies Personalized nutrition uses familial, genetic, or metabolomics information to interpret an individual‘s health risk profile. Prebiotics are food ingredients that induce the growth or activity of beneficial microorganisms (bacteria and fungi). The most common example is in the gastrointestinal tract, where prebiotics can alter the composition of organisms in the gut microbiome. Probiotics are microorganisms that are claimed to provide health benefits when consumed. Recommended daily amount (RDA)for a nutrient indicates the average daily dietary intake level considered sufficient to meet the requirements of nearly all (97–98%) healthy individuals. Single-cell protein (SCP) refers to edible unicellular microorganisms. The biomass or protein extract from pure or mixed cultures of algae, yeasts, fungi or bacteria may be used as an ingredient or a substitute for protein-rich foods, and is suitable for human consumption or as animal feeds. Synbiotics are food ingredients or dietary supplements combining probiotics and prebiotics in a form of synergism. 11  MICROBIOLOGY List of abbreviations AIDS acquired immunodeficiency syndrome ASD autistic spectrum disorder B. lactis Bifidobacterium lactis BCFA branched‐chain fatty acids BCM body cell mass BHA butylated hydroxyanisole BHT butylated hydroxytoluene C. botulinum Clostridium botulinum CA Controlled Atmospheric Storage CD Crohn‘s disease cDNA Complementary DNA CO2 carbon dioxide CRP C-reactive proteins DM Diabetes mellitus DNA Deoxy nucleic acid E.coli Escherichia coli EFSA European Food Safety Authority ELISA Enzyme linked immunosorbent assay GIT gastrointestinal tract GOS galactooligosaccharides H2S hydrogen sulfide HAA heterocyclic aromatic amines HAART Highly active antiretroviral therapy HBV hepatitis B virus 12  MICROBIOLOGY HDL high-density lipoprotein HIV human immunodeficiency virus HMOs human milk oligosaccharides IBD inflammatory bowel disease IBS irritable bowel syndrome IgE immunoglobulin E IL-1 interleukin-1 L. rhamnosus Lactobacillus rhamnosus LAB lactic acid bacteria LDL low-density lipoprotein LPS lipopolysaccharides MALT Gastric mucosa-associated lymphoid tissue MetS metabolic syndrome mRNA Messenger RNA N2 Nitrogen gas NAT N-Acetyltransferase nNRTIs non-nucleoside reverse transcriptase inhibitors NRTIs nucleoside reverse transcriptase inhibitors O2 oxygen PCR polymerase chain reaction PEF Pulsed electric field PIs protease inhibitors RA rheumatoid arthritis RAST radioallergosorbent RDA recommended daily amount RNA Ribonucleic acid 13  MICROBIOLOGY RNI reference nutrient intake S. aureus staph aureus SCFA short chain fatty acids SCP Single-cell protein SLE systemic lupus erythematosus Spp species T2D type 2 diabetes TNF alpha tumor necrosis factor alpha UC ulcerative colitis WHO world health organization Y. enterocolitica Yersinia enterocolitica 14  MICROBIOLOGY Chapter 1: Microbiota in health and disease Objectives Define key microbiota -related terms and concepts Explain how the microbiome is important for maintaining human health. Discuss role of microbiota in Disease. Overview of Microbiota and Related Concepts The microbiota is a collective term for the micro-organisms that live in or on the human body. Specific clusters of microbiota are found on the skin or in the gastrointestinal tract, mouth, vagina and eyes. Human microbiome refers specifically to the collective genomes of resident microorganisms. The Human Microbiome Project took on the project of sequencing the genome of the human microbiota, focusing particularly on the microbiota that normally inhabit the skin, mouth, nose, digestive tract, and vagina.  The human microbiota is made up of trillions of cells - including bacteria, viruses and fungi - and they outnumber our own cells tenfold.  The biggest populations of microbe reside in our gut - the gut microbiota. Other habitats include the skin.  The microbial cells - and their genetic material, the microbiome - live with us in an innate relationship that is vital to normal health, although some species are also opportunistic pathogens that can invade us and cause disease.  The microorganisms living inside the gastrointestinal tract - also known as the gut flora - amount to as much as 4 pounds of biomass, with every individual having a unique mix of species.  The microbiota is important in nutrition, immunity and effects on the brain and behavior.  The microbiota is implicated in numerous diseases when the normal individual balance of microbes is disturbed. Such as obesity, inflammatory bowel disease (IBD), diabetes mellitus, metabolic syndrome, atherosclerosis, 15  MICROBIOLOGY alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), cirrhosis, and hepatocellular carcinoma. Types of microbiota: Bacteria Populations of microbes (such as bacteria and yeasts) inhabit the skin and mucosal surfaces in various parts of the body. Their role forms part of normal, healthy human physiology, however if microbe numbers grow beyond their typical ranges (often due to a compromised immune system) or if microbes populate (such as through poor hygiene or injury) areas of the body normally not colonized or sterile (such as the blood, or the lower respiratory tract, or the abdominal cavity), disease can result (causing, respectively, bacteremia/sepsis, pneumonia, and peritonitis). The Human Microbiome Project found that individuals host thousands of bacterial types, different body sites having their own distinctive communities. Skin and vaginal sites showed smaller diversity than the mouth and gut, these showing the greatest richness. The bacterial makeup for a given site on a body varies from person to person, not only in type, but also in abundance. Bacteria of the same species found throughout the mouth are of multiple subtypes, preferring to inhabit distinctly different locations in the mouth. It is estimated that 500 to 1,000 species of bacteria live in the human gut but belong to just a few phyla: Firmicutes and Bacteroidetes dominate but there are also Proteobacteria, Actinobacteria, Fusobacteria and Cyanobacteria. Fungi Fungi, in particular yeasts, are present in the human gut. Candida species due to their ability to become pathogenic in immunocompromised and even in healthy hosts. Yeasts are also present on the skin, such as Malassezia species, where they consume oils secreted from the sebaceous glands. Viruses Viruses, especially bacterial viruses (bacteriophages), colonize various body sites. These colonized sites include the skin, gut, lungs, and oral cavity. Role of microbiota in Nutrition: In addition to helping themselves to obtain energy from the food we eat, gut microbes are essential to the availability of nutrients for ourselves. Gut bacteria 16  MICROBIOLOGY help us break down complex molecules in meats and vegetables, for example. Without the aid of gut bacteria, plant cellulose is indigestible. Gut microbes may also have an influence on our cravings and feelings of being full after eating via their metabolic activities. The diversity of our microbiota is related to the diversity of our diet, and adolescents trying out a wide variety of foods display a more varied gut microbiota than adults who follow a distinct dietary pattern. Role of microbiota in Immunity Without contact with the microorganisms that colonize us from birth, our adaptive immunity would not exist. Adaptive immunity is the part of our immune system that learns how to respond to microbes after first encountering them, enabling a more rapid defense against disease-causing organisms. The microbiota also has relevance to autoimmune conditions and allergies, which can be more likely to develop when early microbial exposures are disturbed. Role of microbiota in Behavior Because of its involvement in digestion, the microbiota can also affect the brain. Some have even called the gut microbiota a "second brain." Small molecules released by the activity of gut bacteria trigger the response of nerves in the gastrointestinal tract. Links have also been observed between the gut microbiome and brain disorders such as depression and autistic spectrum disorder (ASD). 17  MICROBIOLOGY Role of microbiota in Disease: The human microbiota and infectious diseases Infection is one of the most common diseases caused by dysbiosis of the microbiota. Importantly, infectious disease and its treatment have a profound impact on the human microbiota, which in turn determines the outcome of the infectious disease in the human host Disturbance of the microbiota is also associated with the progression of human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Gastrointestinal bacterial populations have provided insights into gut conditions such as the inflammatory bowel diseases (IBD) of Crohn's disease and ulcerative colitis. Low diversity in the gut microbiota has been linked to IBD as well as obesity and type 2 diabetes. The nature of the gut microbiota has been linked to metabolic syndrome, and dietary modification has shown an effect on this cluster of risk factors via prebiotics, probiotics and other supplements. Gut microbes and their genetics affect our energy balance, and brain development and function. As a result, research into the effects of gut microbes on the developing brain and diet-related disorders is ongoing. 18  MICROBIOLOGY Antibiotic disturbance of the microbiota can lead to disease, including the emergence of infections that display antibiotic resistance. The innate microbiota also plays an important role in resisting intestinal overgrowth of externally introduced populations that would otherwise cause disease - the "good" bacteria compete with the "bad," with some even releasing anti-inflammatory compounds. The human microbiota and liver diseases The intestinal microbiota produces ethanol, ammonia, and acetaldehyde; these products may influence liver function through endotoxin release or liver metabolism. Alterations in the intestinal microbiota play an important role in inducing and promoting liver damage progression as well as in direct injury resulting from different causal agents (e.g., viral, toxic, and metabolic agents). through mechanisms such as the activation of liver cells by bacterial endotoxins. The gut microbiota participates in the pathogenesis of liver cirrhosis complications, such as infections, spontaneous bacterial peritonitis, hepatic encephalopathy, and renal failure. Patients with liver cirrhosis have an altered bacterial composition in their gut. Role of microbiota in Cancer Although cancer is generally a disease of host genetics and environmental factors, microorganisms are implicated in some 20% of human cancers. Particularly for 19  MICROBIOLOGY potential factors in colon cancer, bacterial density is one million times higher than in the small intestine, and approximately 12-fold more cancers occur in the colon compared to the small intestine, possibly establishing a pathogenic role for microbiota in colon and rectal cancers. Microbial density may be used as a prognostic tool in assessment of colorectal cancers. The microbiota may affect carcinogenesis in three broad ways: (i) altering the balance of tumor cell proliferation and death. (ii) regulating immune system function. (iii) influencing metabolism of host-produced factors, foods and pharmaceuticals. Tumors arising at boundary surfaces, such as the skin, oropharynx and respiratory, digestive and urogenital tracts, harbor a microbiota. Substantial microbe presence at a tumor site does not establish association or causal links. Instead, microbes may find tumor oxygen tension or nutrient profile supportive. Decreased populations of specific microbes or induced oxidative stress may also increase risks. Microbes may secrete proteins or other factors directly drive cell proliferation in the host, or may up- or down-regulate the host immune system including driving acute or chronic inflammation in ways that contribute to carcinogenesis. Compromised host or microbiota resiliency also reduce resistance to malignancy, possibly inducing inflammation and cancer. Helicobacter pylori appears to increase the risk of gastric cancer, due to its driving a chronic inflammatory response in the stomach. 20  MICROBIOLOGY Practical: Gut microbiota modifications in early life Objectives Recognize the Early life gut microbiota. Discuss the difference between breast milk and Formula. Early life gut microbiota During the first days and months of life, the microbiota of the infant gut and the temporal pattern in which it evolves varies remarkably from individual to individual. At birth, infants used to be considered to be essentially free of bacteria but this was recently challenged in healthy term pregnancies, when bacteria were found in human placental membranes, amniotic fluid and umbilical cords as well as in the meconium of healthy term newborns. Immediately after birth, bacteria from the mother and/or the environment colonizes the sterile gut of a newborn infant and, within a few days, fecal density reaches 108–1010 bacteria/g. For preterm infants, composition of the gut microbiota resembles bacterial communities colonizing hospital surfaces and feeding and intubation tubing, and are enriched in Staphylococcus epidermis, Klebsiella pneumoniae, and Escherichia coli. The early gut microbiota is often dominated by Escherichia, Clostridium, Bacteroides and Bifido bacterium.The early gut microbiome during the first days of life in infants who are born vaginally shares features with the vaginal microbial community, while in infants born by Cesarean section, the early gut microbiome resembles that of the maternal skin. Moreover, it has been proposed that the 21  MICROBIOLOGY composition of the very first human microbiota could have long lasting effects on the intestine in breastfed infants. After birth, the most important determinant of infant gut colonization is breast feeding. Human milk contains up to 109 live bacteria per liter and is a source of staphylococci, streptococci, lactic acid bacteria and, mostly, bifidobacterial. Among breastfed infants, bifidobacterial and particularly B. longum, B. infantis and B. breve can reach up to 60–90% of the total fecal microbiota. Moreover, the gut microbiota of exclusively breastfed infants exhibits significant differences in gut microbiota as compared to that of formula fed infants. Formula fed microbiota is more complex and is characterized by the predominance of facultative anaerobes such as Bacteroides and Clostridium followed by Staphylococcus, Streptococcus and Enterobacteriaceae, and delayed colonization by bifido bacteria. After the first six months of life, when solid food is introduced to the infant, the gut microbiota becomes more diverse and the abundance of Bacteroides, Clostridium and anaerobic bacteria increases rapidly while the proportion of bifido bacteria becomes more stable. Probiotics in early life Probiotics have increasingly been administrated to children. Bifidobacterium lactis (B. lactis) probiotics beyond early infancy are associated with a reduced risk of nonspecific gastrointestinal infections in children. 22  MICROBIOLOGY Probiotics and weight gain The perinatal use of Lactobacillus rhamnosus (L. rhamnosus) suggest that early modulation of the gut microbiota modifies children‘s growth patterns by inhibiting excessive weight gain during the first few years of life. Healthy infants who received Lactobacillus rhamnosus enriched formula during the first six months of life presented significantly increased body length and weight. The administration of Bifidobacterium breve probiotics to very low birth weight infants was associated with improved weight gain as well as a more rapid growth of Lactobacillus as compared to infants who did not receive probiotic Supplements. Moreover, probiotic treatment of very low birth weight infants with Lactobacillus acidophilus and Bifidobacterium infantis has been shown to reduce morbidity as well as to increase daily weight gain and decrease the length of hospital stay. Probiotics have also been used to treat malnutrition in children: ready-to-use therapeutic food has reduced the prevalence of malnutrition and led to weight gain in children. So early modulation of the gut microbiota through the administration of probiotics is likely to result in increased weight gain. Antibiotics in early life Exposure to antibiotics remains very high perinatally and in the first periods of life. A number of neonates, particularly premature infants, receive antibiotics to prevent or treat bacterial infections. Moreover, many mothers receive antibiotics for prophylaxis of vaginal group B streptococcus and Cesarean section delivery. Antibiotic treatment has been associated with disturbances of the gut microbiota in the actively developing infant gut microbiota. Antibiotic treatment during the first two years of life decreases gut flora biodiversity. Early life antibiotic treatment has been linked with an increased risk of obesity and related metabolic sequelae later in life. 23  MICROBIOLOGY Chapter2: The Gut Microbiota Objectives Recognize Interaction among gut microbiota, host and food. Explain The role of gut microbes on the digestion of macronutrients. Discuss impact of diet on GIT microbiota. Overview of gut microbiota and Related Concepts The gut microbiota is an ecological community of symbiotic, commensal and pathogenic microorganisms. This microbial ecosystem includes many bacterial species which permanently colonize the gastrointestinal tract as well as a large number of microorganisms such as Archaea, viruses, parasites and fungi that come from our environment. The number of these microorganisms can reach 10 12–1014 in the colon, making gut microbiota one of the most densely populated communities, far exceeding that of the soil, the subsoil, and the oceans. In a healthy human adult, the gut microbiota The dominant bacterial species in the human gastrointestinal tract are divided into three phyla: The phylum Bacteroidetes (e.g. Porphyromonas, Prevotella etc.), The phylum Firmicutes (e.g. Ruminococcus, Clostridium, Eubacteria etc.) And the phylum Actinobacteria (Bifidobacterium). Other bacteria such as Lactobacilli, Streptococci and Escherichia coli (E.coli) are found in small numbers. The Bacteroidetes and Firmicutes phyla were found to be the dominant bacterial Populations in the gastrointestinal tract (GIT). 24  MICROBIOLOGY Interaction among gut microbiota, host and food The intestinal bacteria reside at the interface of the external environment and the host, allowing for a tightly regulated crosstalk between the three. The bacteria reside within the lumen of the intestinal tract and interact directly with dietary antigens where they aid in digestion of partially digested food, vitamin biosynthesis, and absorption. The bacteria also act as a first line of defense against pathogens in the gut through competition for food and space. 25  MICROBIOLOGY interactions among the intestinal microbiota, host, and the environment regulate intestinal health and by extension the host. Disturbances in any of these factors can have deleterious effects on the host. The role of gut microbes on the digestion of macronutrients To utilize food energy, the host‐microbiome superorganism has evolved to work as a unit customized to the benefit of both. Up to 10% of the dietary energy in humans can be due to the activities of their intestinal microbiota. This may be beneficial or detrimental depending on the needs of the host. For example, in the context of obesity, having microbes that extract more energy is not favorable but in the context of cachexia this would be of benefit. Carbohydrates Mammalian genomes do not encode for most enzymes required for the degradation of insoluble structural polysaccharides. Undigested food particles arriving at the large intestine are typically comprised of insoluble materials derived from plant cell walls and resistant starch. Specialized bacteria species such as Roseburia bromii, belonging to the Firmicutes, act as primary degraders of insoluble polysaccharides, in turn producing substrates for other amylolytic bacteria. The primary result of carbohydrate fermentation under anaerobic conditions in the gut is the production of short-chain fatty acids (SCFAs) such as acetate, lactate, propionate, and butyrate, of which the last plays a particularly important role in gut homeostasis and health. While lactate and acetate become available to the host systemically, butyrate directly becomes the primary food source of the colonocytes. Butyrate is known to possess 26  MICROBIOLOGY anti‐cancer and anti‐inflammatory properties43 and is involved in gut motility, energy expenditure, and appetite control. Butyrate can also be produced through conversion of other available acids such as acetate and lactate by members of the Firmicutes phyla, including Eubacterium hallii and Anaerostipes spp. Proteins Partially digested proteins, mucus secretions, and amino acids from shed IEC are fermented in the large intestine and serve as a source of carbon and nitrogen for intestinal microorganisms. The amount of protein entering the large intestine depends on the total intake as well as the source of the protein. The digestibility of animal‐derived protein is higher (94–99%) than that derived from plant sources (70–90%)50. Protein catabolism by intestinal microorganisms yields a variety of end products, many which are toxic to the host. Degradation of undigested or endogenous protein, referred to as putrefaction, is particularly prominent in the etiology of ulcerative colitis (UC). Bacterial proteases and peptidases can initiate the degradation process by means of hydrolysis to convert proteins into smaller peptides and amino acids. This process is optimal in the distal colon where pH is higher. Fermentation of amino acids by reductive deamination can, albeit to a much lesser extent, produce short chain fatty acids (SCFA) similar to those found during carbohydrate fermentation. They also produce ammonia, amines, thiols, phenols, and indoles, which are exclusive to amino acid fermentation. The accumulation of these by‐products is pathogenic to the host. For example, phenols and indoles are considered carcinogens, ammonia a mutagen, and thiols a cellular toxin. Another product of protein fermentation is branched‐chain fatty acids (BCFA). The physiological significance of BCFA is not well understood; however, they appear to be important in developmental stages during gestation and immediately following birth. Further, sulfate‐reducing bacteria such as those in the genus Desulfovibrio instigate fermentation of dietary and mucinous sulfate and sulfur amino acids, leading to increased production of hydrogen sulfide (H 2S), which has been associated with pathogenesis of various intestinal diseases including ulcerative colitis. As H2S is involved in many normal processes within the colon, it is likely that its association with etiology of intestinal diseases is relevant only in conditions where H2S levels reach higher than subtoxic levels. Lipids Different members of the gut microbiota are thought to regulate fat absorption via distinct mechanisms. Several roles of microbes in the harvest and storage of dietary lipids have been postulated; however, the exact mechanisms in play remain to be elucidated. Once dietary lipids are absorbed into the enterocytes, they are repackaged into lipoprotein particles known as chylomicrons. These chylomicrons transport lipids in the form of triglycerides to adipocytes, where lipoprotein lipases breakdown 27  MICROBIOLOGY triglycerides into free fatty acids that can then be taken into the tissue for storage. the presence of gut microbes reduced serum levels of chylomicrons. This outcome could have been due to decreased lipid absorption in the gut or an increased lipid clearance in the periphery. So the presence of microbiota did not affect the absorbance rate of lipid, but rather affected the clearance of chylomicrons. Impact of diet on GIT microbiota It has been widely reported that the consumption of modern western diets containing less fiber and vegetables tended to result in the loss of some important microbial species in the western (urban) communities compared to rural communities. comparing an individual whose diet is high in fat and low in fiber with an individual on the opposite diet (e.g. high fiber and low fat), the latter tends to have a smaller amount of pathogenic bacteria and a large amount of beneficial microbes, such as Pre-votella and Xylanibacte. The balance of GIT microbial composition can be achieved as a result of symbiosis which regulates the immune system and protects the host from various diseases. The Mediterranean diet, which is based on a balanced intake of fruits, grains, monounsaturated fat, vegetables and polyunsaturated fats, is considered the standard for a healthy life style. It has been found that such diets have anti- inflammatory capabilities and can be used to reduce inflammation in diseases. Individuals fed on the Mediterranean diet have lower numbers of Bacillaceae, 28  MICROBIOLOGY Proteobacteria and acute phase C-reactive proteins (CRP), but higher Clostridium and Bacteroidetes populations. Vegetarian diets are also recognized as healthy and benefic diets because they can protect the host from various chronic, metabolic and inflammatory disorders. Recent investigations showed that vegetarian diets could increase the number of Faecalibacterium praus-nitzii, Clostridium clostridioforme and Bacteroides Prevotella,but decrease the ratio of Clostridium cluster species. Small amounts of dietary fat can be digested and absorbed but some fat components cannot be metabolized and pass to the colon where they affect the microbial composition and are then excreted in feces. Consequently, the consumption of high-fat foods tends to induce substantial changes in the composition of GI tract microbiota. High fat content and increased calorie consumption have the capability to induce changes in the microbial composition of the GI tract. It has been also found that the children feeding on vegetarian diets rich in plant-based polysaccharides, fibers and starches had a significant increase in the number of Firmicutes, Xylanibacter,Bacteroidetes and Prevotella compared with those consuming a carbohydrate rich European diet. Therefore, it was suggested that children should be provided with a plant-based polysaccharide rich diet which confers protection against inflammatory disease Currently, it is clear that the composition of the microbiota differs among individuals living in different geographic regions and also depends on the long- term diet pattern. 29  MICROBIOLOGY Practical: The gut microbiota role in obesity and diabetes Objectives Recognize obesogenic microbiota in humans. Discuss how Obesity‐proneness: mediated by the gut microbiota. Recognize the role of microbiota in diabetes. the metabolic syndrome (MetS) includes increased waist circumference, hyperglycemia, elevated blood pressure and hyperlipidemia. With time, these conditions present a major risk of developing obesity, type 2 diabetes and atherosclerosis. To date, treatment mainly includes symptom management, and effective prevention strategies are largely lacking.With the exciting discovery of an ―obesogenic‖ microbiota in recent years, with attempts to use microbial manipulations to modulate our gut microbiome as a new preventive and therapeutic approach for different aspects of the MetS. 30  MICROBIOLOGY The “obesogenic” microbiota in humans In humans, clear shifts in the gut microbiome can be observed when comparing obese and lean individuals, with increased Firmicutes to Bacteroides ratio in obese individuals. Nutrient content in the diet directly affects the gut microbiome, where high‐energy diets increase the ratio of Firmicutes to Bacteroides in humans. Thus, a diet high in energy will contribute to development of obesity not only due to the energy content in the diet, but also through maintaining an ―obesogenic‖ gut microbiota. It is clear that a high‐fat diet induces an ―obese‖ microbiota independent of body weight state and the altered gut microbiota in obese individuals is not merely a consequence of the obese state. A leaky gut contributing to inflammation and adiposity The monolayer of epithelial cells lining the gut mucosa has the delicate dual function of being an efficient absorptive layer to nutrients, whilst still maintaining a tight barrier to invading pathogens. This is accomplished through several specific features of the intestinal epithelium: first, the intestine hosts 70–90% of the body‘s immune cells, which together with the extensive enteric nervous system closely monitor events in the gut. Second, the junctions between epithelial cells consist of an intricate collection of proteins comprising the so‐called tight junctions, which can be regulated by the cells themselves but are also affected by bacteria. Similarly, in obesity, these junctions appear leaky, as an increased influx of bacterial cell wall lipopolysaccharides (LPS) is observed as compared to lean individuals. LPS is a component of Gram negative bacteria cell walls and is pro‐inflammatory substance.The low‐grade systemic inflammation observed in obese individuals can be ascribed partly to increased LPS levels in the blood, but also to an increased secretion of pro‐inflammatory cytokines from adipose tissue. Interestingly, a high‐fat diet directly increases LPS blood levels, as LPS can form and bind to micelles during fat absorption and are therefore shuttled into the body from the gut. Obesity‐proneness: mediated by the gut microbiota In humans resistance to diet‐induced obesity is occasionally observed, but the reasons remain unclear why the same amount of energy ingested does not result in the same weight increase in all individuals. In obesity‐prone and obesity‐resistant mice fed a high‐fat diet, fecal carbohydrate calories differed, whereas fecal fat or protein calories did not differ. This surprisingly indicates that lean person display decreased carbohydrate absorption from the diet as compared to obese person, despite similar caloric intake. Carbohydrate absorption is greatly affected by gut microbial activities, and there is differences in microbial composition between obesity‐prone and obesity‐resistant persons. 31  MICROBIOLOGY Bacterial metabolites provide a link between bacteria and host metabolism The gut bacteria degrade dietary component sreaching the colon, giving rise to metabolites such as short‐chain fatty acids (SCFA), which are formed during bacterial digestion of indigestible carbohydrates (e.g. dietary fiber). These bacterial metabolites may play an important role for host physiology, as they can be utilized as energy by colon cells, as well as be absorbed into the blood stream. Thus, a diet low in dietary fiber may affect colonic health. The “diabetic” microbiota Diabetes mellitus (DM) presents in two major forms: type I and type II, characterized by vastly different molecular events leading up to malfunction of glucose homeostasis. In type I diabetes, autoimmune reactions to insulin‐producing beta‐cells in the pancreas results in cell death and a gradual loss of insulin‐production capacity, often starting early in life. The disease requires careful monitoring of blood glucose levels and life‐long administration of insulin. Type II diabetes, on the other hand, is usually described as life‐style‐related, as obesity, little exercise and an unhealthy diet are key risk factors. Type II diabetics can often reduce their need for insulin by adopting a more healthy life style. Another important difference between the two forms of diabetes is the phenomenon of ―insulin resistance‖ in type II diabetics. These patients have no lack of insulin; instead, they become insensitive to insulin and require higher doses of insulin to be able to transport glucose from the blood stream into the cells. Type I diabetes and the gut microbiota It is currently unknown what triggers the autoimmune reactions in type I diabetes, Bacteria in the phylum Bacteroidetes (including Prevotella and unclassified Bacteriodales) may protect against development of type I diabetes, while increased levels of members of the Firmicutes phylum, i.e. Ruminococcus, Oscillospira and Lachnospiraceae, promoted the disease. Type I diabetic children were also characterized by increased gut microbial diversity. This is interesting, as obese and type II diabetic adults generally have lower gut microbial diversity, which may point towards a complex age‐related dynamic of the gut microbiota development. Type II diabetes Diabetes Type 2 alters the composition of gut microbiota and microbiota function such as the secondary metabolite bile acid and butyrate products. These functions are crucial for insulin sensitivity. The status of gut microbiota can be used to success-fully distinguish between type 2 diabetes patients and healthy individuals. Type 2 diabetes was linked to higher amounts of Lactobacilli and lower amounts of Roseburia when comparing the two populations. 32  MICROBIOLOGY Chapter 3: The microbiota in inflammatory bowel disease Objectives Explain Dysbiosis and IBD pathogenesis. Recognize Nutritional and dietetic therapies Overview of inflammatory bowel disease and Related Concepts Inflammatory bowel disease (IBD) comprises two distinct conditions, ulcerative colitis (UC) and Crohn‘s disease (CD) that are characterized by chronic relapsing inflammation of the gut in genetically susceptible individuals exposed to defined environmental risk factors. Dysbiosis and IBD pathogenesis The relationship between the gut commensal microbiota and IBD is complex and can be explained by four broad mechanisms: (i) conventional microbiota dysbiosis, (ii) intestinal inflammation induction by pathogens and/or functionally altered commensal bacteria. (iii) genetic defects in the host resulting in the loss of the commensal microbiota. (iv) defective immunoregulation in the host. Causes IBD is a complex disease which arises as a result of the interaction of environmental and genetic factors leading to immunological responses and inflammation in the intestine. Diet Dietary patterns are associated with a risk for ulcerative colitis. In particular, subjects who were in the highest tertile of the healthy dietary pattern had a 79% lower risk of ulcerative colitis. Gluten sensitivity is common in IBD and associated with having flareups. A diet high in protein, particular animal protein, may be associated with increased risk of inflammatory bowel disease and relapses. 33  MICROBIOLOGY Microbiota As a result of microbial symbiosis and immunity, alterations in the gut microbiome may contribute to inflammatory gut diseases. IBD-affected individuals have been found to have 30–50 percent reduced biodiversity of commensal bacteria, such as decreases in Firmicutes (namely Lachnospiraceae) and Bacteroidetes. Further evidence of the role of gut flora in the cause of inflammatory bowel disease is that IBD-affected individuals are more likely to have been prescribed antibiotics in the 2–5 year period before their diagnosis than unaffected individuals. The enteral bacteria can be altered by environmental factors, such as concentrated milk fats (a common ingredient of processed foods and confectionery) or oral medications such as antibiotics and oral iron preparations. Breach of intestinal barrier Loss of integrity of the intestinal epithelium plays a key pathogenic role in IBD. Changes in the composition of the intestinal microbiota are an important environmental factor in the development of IBD. Detrimental changes in the intestinal microbiota induce an inappropriate immune response that results in damage to the intestinal epithelium. Breaches in this critical barrier (the intestinal epithelium) allow further infiltration of microbiota that, in turn, elicit further immune responses. IBD is a multifactorial disease that is nonetheless driven in part by an exaggerated immune response to gut microbiota that causes defects in epithelial barrier function. Nutritional and dietetic therapies Nutritional deficiencies play a prominent role in IBD. Malabsorption, diarrhea, and GI blood loss are common features of IBD. Deficiencies of B vitamins, fat-soluble vitamins, essential fatty acids, and key minerals such as magnesium, zinc, and selenium are extremely common and benefit from replacement therapy. Dietary interventions, including certain exclusion diets, low fiber diets has some benefits. Microbiome There is evidence of an infectious contribution to inflammatory bowel disease in some patients and this subgroup of patients may benefit from antibiotic therapy. Fecal microbiota transplant is a relatively new treatment option for IBD. Patients benefits similar to those in Clostridium difficile infection. 34  MICROBIOLOGY ⃰Around one-third of individuals with IBD experience persistent gastrointestinal symptoms similar to irritable bowel syndrome (IBS) in the absence of objective evidence of disease activity. The cause of these IBS-like symptoms is unclear, but it has been suggested that changes in the gut-brain axis, epithelial barrier dysfunction, and the gut flora may be partially responsible. Patients of IBD do have an increased risk of colorectal cancer. Practical: The main dietary goals in the management of IBD Objectives Discuss main reasons for malnutrition in patients with inflammatory bowel disease. Recognize the main nutritional deficiencies of inflammatory bowel disease. Recognize main dietary goals in the management of Crohn‘s disease. What are the main reasons for malnutrition in patients with inflammatory bowel disease? Several mechanisms contribute to the malnutrition observed in inflammatory bowel disease (IBD) patients. A decrease in the oral intake of nutrients is a common symptom and is often due to abdominal pain, diarrhoea, anorexia, nausea and vomiting and in cases of strictures or abscesses. Moreover, mucosal inflammation and its associated diarrhoea or bleeding lead to a loss of protein, blood, minerals, electrolytes and trace elements. Multiple resections, bacterial overgrowth and fistulas between the small and large intestine may have an adverse nutritional effect (e.g. vitamins and minerals) owing to a decreased absorptive area and subsequent malabsorption. Furthermore, increased energy requirements because of inflammation or fever can lead to weight loss and altered intermediate metabolism, owing to increased tumor necrosis factor alpha (TNF alpha), interleukin-1(IL-1) and 6, which in turn lead to decreased albumin and other protein synthesis. Pharmacological therapies may also lead to malnutrition. For example, sulfasalazine reduces folic acid absorption, whereas corticosteroids decrease calcium absorption and negatively affect protein metabolism. Lastly, restrictive diets that are recommended to patients by family, friends and physicians or prolonged periods of fasting because of exacerbations or diagnostic procedures also contribute to the risk of malnutrition. The consequences of malnutrition are numerous, and include reductions in bone mineral density, as well as growth retardation and delayed sexual maturity in children. Osteoporosis may also be implicated as a result of pro-inflammatory cytokine profiles. What are the main nutritional deficiencies of inflammatory bowel disease? IBD is associated with a number of nutritional deficiencies, including 35  MICROBIOLOGY anaemia, hypoalbuminaemia, hypomagnesaemia, hypocalcaemia and hypophosphataemia, as well as deficiencies in folic acid, niacin, fat-soluble vitamins and B12 in cases of terminal ileum resection and deficiencies of iron, zinc and copper. Plasma antioxidant concentrations are also reduced in many IBD patients, particularly those with active disease. Patients with diarrhoea and vomiting may also experience electrolyte imbalances. What kind of diet should be administered to a patient with ulcerative colitis exacerbation? For nutritional support, enteral feeds are generally preferred to parenteral nutrition, except from cases of toxic megacolon, extended colon haemorrhage, perforation or obstruction, which require bowel rest and parenteral administration of fluids and nutrients. In times of exacerbation, a liquid diet is first administered, followed by a low-residue diet. In patients with strictures, a low-fibre diet is required in order to prevent obstruction. When the patient enters the remission phase, they should be encouraged to consume a variety of foods from all food groups and dietitians should help patients realise their own intolerances and further compose a balanced diet. Which are the main dietary goals in the management of Crohn’s disease? Medical nutritional therapy for patients with Crohn‘s disease (CD) aims to: _ prevent or restore protein/energy malnutrition _ assess and correct micronutrient deficiencies _ maintain bowel rest in periods of exacerbation _ modify the diet regime according to drug treatment and drug–nutrient interactions _ modulate immune response by modulating cytokines expression (e.g. omega-3 polyunsaturated fatty acids), by reducing gut permeability and enhancing gut barrier (e.g. probiotics), by affecting gene expression and by modulating local immunity in the intestine (e.g. butyrate, glutamine). 36  MICROBIOLOGY Chapter4: Food microbiology and food safety Objectives Recognize food safety related concepts. List the commonest causative agents of foodborne illness. Recognize factors affecting microbial growth. Discuss Microbiological Testing of food. Overview of food microbiology and Related Concepts Food microbiology is the study of the microorganisms that inhabit, create, or contaminate food. Of major importance is the study of microorganisms causing food spoilage. "Good" bacteria, however, such as probiotics, are becoming increasingly important in food science. In addition, microorganisms are essential for the production of foods such as cheese, yogurt, and other fermented foods such as bread, beer and wine. Food safety is a major focus of food microbiology. Pathogenic bacteria, viruses and toxins produced by microorganisms are all possible contaminants of food. However, microorganisms and their products can also be used to combat these pathogenic microbes. Probiotic bacteria, including those that produce bacteriocins, can kill and inhibit pathogens. Alternatively, purified bacteriocins such as nisin can be added directly to food products. Finally, bacteriophages, viruses that only infect bacteria, can be used to kill bacterial pathogens. Thorough preparation of food, including proper cooking, eliminates most bacteria and viruses. However, toxins produced by contaminants may not be heat-labile, and some are not eliminated by cooking. Fermentation is one-way microorganisms can change a food. Yeast, especially Saccharomyces cerevisiae, is used to leaven bread, brew beer and make wine. Certain bacteria, including lactic acid bacteria, are used to make yogurt, cheese, hot sauce, pickles, fermented sausages and dishes such as kimchi. A common effect of these fermentations is that the food product is less hospitable to other microorganisms, including pathogens and spoilage-causing microorganisms, thus Extending the food's shelf-life. Food fermentations are ancient technologies that harness microorganisms and their enzymes to improve the human diet. Fermented foods keep better, have enhanced flavours, textures and aromas, and may also possess 37  MICROBIOLOGY certain health benefits, including superior digestibility. For vegetarians, fermented foods serve as palatable, protein-rich meat substitutes. Some cheese varieties also require molds to ripen and develop their characteristic flavors. Microorganisms associated with foods can be categorized as "spoilage," "pathogenic," or "useful.'' Spoilage microorganisms are those that can grow in a food and cause undesirable changes in flavor, consistency (body and texture), color, or appearance. Also bacterial enzymes may effect slow deterioration of frozen or dried foods during long-time storage. These changes diminish the quality characteristics of foods and may render them ultimately unfit for human consumption. For example, refrigerated perishable foods such as milk, fresh meat, poultry, fish, fruits, and vegetables lose some quality characteristics during normal storage and ultimately spoil, due in part to the activity of microorganisms capable of growth at refrigeration temperatures. Usually, extensive microbial growth (millions of organisms per g or cm2) occurs before quality losses are perceptible. These changes, when perceived by the consumer, serve as an alert that extensive microbial activity has taken place. Pathogenic microorganisms can render foods harmful to humans in a variety of ways. Foods may serve as the vehicle for the introduction of infectious microorganisms into the gastrointestinal tract, e.g., Salmonella and Shigella. Multiplication of certain microorganisms in foods prior to consumption may result in production of toxins, e.g., Clostridium botulinum, Staphylococcus aureus, and Bacillus cereus. Foods may also be the vehicle for microorganisms that form toxins in vivo, e.g., Clostridium perfringens and certain pathogenic Escherichia coli. With some foods, conditions are chosen to favor the development of useful microorganisms such as lactic acid bacteria and yeasts, which are either naturally present or added intentionally. Such foods as cheeses, yogurt, breads, pickles, and fermented sausages offer desirable organoleptic properties and shelf-life. Food as a Selective Environment Microbial activities in foods can be viewed from the perspective of the food as a "selective environment," despite the diversity of microorganisms that contaminate the surfaces of the raw materials. The selectivity is imposed by the physical-chemical characteristics of the food, the additives it contains, the processing techniques, the packaging material, and the storage conditions. It is necessary to distinguish between the shelf-life of two broad categories of foods, namely those that are shelf-stable and those that are perishable. shelf-life will be treated as it relates to microbial activity only. Microbiological shelf-stability of many foods is related to storage conditions. For example, dried and frozen foods are microbiologically shelf stable as long as they 38  MICROBIOLOGY remain dry or frozen. Shelf-stable foods are not necessarily sterile; indeed, many do contain microorganisms. Some shelf-stable canned foods may undergo microbiological spoilage if they are exposed to elevated temperatures permitting the growth of surviving thermophilic spore forming bacteria, whereas these organisms are inactive at ambient temperatures and indeed tend to die during normal storage. Shelf- stable food is distinguished from perishable food in that an attribute or attributes of the shelf-stable food prevent the growth of contaminating microorganisms. For example, certain canned products are heat processed to the degree that they are sterile; the attribute assuring stability of such products is elimination of all living forms. With many shelf-stable foods, other attributes prevent microbial growth. Dried beans are shelf-stable because they contain insufficient moisture to permit microbial growth. Mayonnaise is shelf-stable because it contains sufficient quantities of acetic acid in the moisture phase of the product to prevent growth of contaminating organisms. Certain canned cured meats are shelf-stable, not because they are sterile, but because sublethal heat treatment so injures surviving spores that they are incapable of outgrowth in the presence of salt and nitrite. The distinguishing characteristic of shelf-stable foods, then, is their resistance to microbiological spoilage. Microbial growth in such products is an abnormal and unexpected event. Perishable foods, on the other hand, have a finite shelf-life and if not consumed, will spoil at some time during storage. The exact time of spoilage depends upon a great number of variables. Though various processing procedures, additives, packaging methods, and storage conditions may be applied to increase shelf-life, microorganisms capable of growth survive and ultimately grow. When such growth proceeds to the extent that undesirable changes are perceptible to the processor, preparer, or consumer, the food is deemed of inferior quality or spoiled and is rejected. The distinguishing feature of perishable foods, in contrast to shelf-stable foods, is that microbiological spoilage is an expected event. It will ultimately occur even if the food has been prepared from wholesome raw materials and has been properly processed, packaged, and stored. Microflora of Processed Foods Although the microflora of raw materials is usually heterogeneous, processing of foods (except those that are sterile) often imposes a characteristic and highly specific microbiological flora. The normal flora of severely heat processed, but not sterilized, low-acid canned foods is comprised of thermophilic spore forming bacteria, the most heat-resistant microbial components of the raw materials. The predominating flora of shelf-stable canned cured meats consists of mesophilic aerobic and anaerobic spore forming bacteria, the predominant organisms resistant to the heat process applied to these products. The normal flora of mayonnaise and salad dressing is comprised of small numbers of spore forming bacteria, yeasts, and lactic acid bacteria. Aerobic spore forming bacteria 39  MICROBIOLOGY predominate in dry spices and in a number of dry vegetable products. Molds and yeasts predominate in dried fruits. The normal flora in carbonated beverages is comprised of yeasts. In each of the foregoing, the surviving and predominating microflora reflects the nature of the raw materials, processing conditions, packaging, and storage of the shelf-stable product. However, spoilage is still possible. If the severely heat-processed canned foods were exposed to high temperatures during storage, spoilage due to the germination and outgrowth of thermophilic spore forming bacteria might occur. If shelf-stable canned cured meat were to contain excessive numbers of aerobic spore forming bacteria, growth of these organisms might result in spoilage, despite an adequate heat process and normal levels of salt and nitrite. Excessive levels of yeasts or lactic acid bacteria might result in their growth and subsequent spoilage of the mayonnaise, despite levels of acetic acid that would assure the stability of a product containing "normal" levels of the same organisms. Time/temperature abuse of an ingredient of a carbonated beverage (for example, a flavor) may lead to the development of large numbers of yeasts that could overcome the effect of carbonic acid, which would normally render the same beverage stable. The normal flora in microbiologically shelf-stable products is, therefore, rather specific. If the stabilizing nature of the system should be overcome, this microflora may multiply and cause spoilage—an unexpected event. With perishable products, the microflora that survives processing may be heterogeneous, but that portion of it developing during storage and causing spoilage is usually quite specific. For example, a heterogeneous flora exists on raw red meats, poultry, and fish as a result of contamination from the animal and/or the processing environment. Yet, during refrigerated storage of such products, spoilage is caused predominantly by a highly specific group of microorganisms, namely Pseudomonas and closely related aerobic, psychrotrophic gram-negative bacteria. If the same products are vacuum-packed in oxygen- impermeable films, a different microflora becomes predominant, namely, lactic acid bacteria that grow under both aerobic and anaerobic conditions. In both examples, despite the heterogeneous flora of the finished product, a rather restricted group of microorganisms may develop and ultimately cause sensory changes in the product. Similar relationships exist for many other perishable foods. It follows that since most classes of perishable foods constitute selective environments for rather restricted groups of microorganisms, the spoilage caused by the growth of these microorganisms manifests itself in a characteristic manner, i.e., normal spoilage pattern. For example, when pseudomonads and other closely related gram-negative psychrotrophic aerobic bacteria grow to large numbers on the surface of refrigerated fresh meat, poultry, and fish, sensory changes occur. The first manifestation of spoilage is development of off-odor. As growth proceeds, slime may develop and the 40  MICROBIOLOGY off-odor may intensify. The normal spoilage pattern of a perishable food can be a safeguard, since under certain situations it warns the processor, preparer, or consumer that the food is no longer edible. Changes in processing of perishable foods must take into account the effect these changes may have on the spoilage flora, and thus on the normal spoilage pattern of the food involved. If such changes tend to alter the normal patterns of spoilage the public health aspects must be taken into account. A classic example of this relates to the merchandising of smoked whitefish. For generations this product was merchandised under conditions where the fish was exposed to air. Spoilage was evidenced by the development of bacteria which produced off odors and slime that were readily recognized by the consumer and caused rejection of the product. Then it was discovered that the shelf-life of smoked fish could be significantly increased if the product was packed in an oxygen impermeable film. With extended storage of the product under these conditions, Clostridium botulinum (C. botulinum type E) was able to grow and produce toxin, just as it would have been able to do in the conventionally packaged product. However, under these storage conditions the aerobic bacteria producing off-odor and slime could not develop and the normal spoilage flora was now comprised of lactic acid bacteria that did not produce off-odors. This change in the normal spoilage pattern of the product reduced the probability that the consumer would reject a product that had been held in storage out of refrigeration for an extended period of time. This led to a multistate outbreak of type E botulism. Approaches to microbiological control in Three principal means have been used by regulatory agencies and food processors to control microorganisms in foods. These are (1) education and training. (2) inspection of facilities and operations. (3) microbiological testing. Although food handlers have the potential for contaminating foods with disease- producing microorganisms, i.e., staphylococci, salmonellae, and hepatitis virus, health examination of food handlers is a nonproductive approach to the control of foodborne illness. Specimens from food handlers have traditionally been examined only for a few microorganisms, and such tests do not always detect carriers. Screening tests cannot be made with sufficient frequency to be effective in detecting the carrier status in persons who are continually exposed to the risk of acquiring foodborne pathogens. Negative tests convey to food handlers, managers, and public health personnel the erroneous concept that the workers are free of infections and therefore incapable of transmitting foodborne pathogens to the foods they handle. Although direct transfer of pathogens from food handlers to food is a hazard, far more frequently improper food- handling practices create a hazard that is not circumvented by health examinations. 41  MICROBIOLOGY Education and Training Programs These programs are directed primarily toward developing an understanding of the causes and consequences of microbial contamination and of measures to prevent contamination and subsequent growth. The extent of training required of personnel within processing plants and food service establishments depends upon the technical complexity of the food operation and the level of responsibility of the individuals being trained. In-depth training may be necessary for supervisory personnel, while for others training may relate only to specific aspects of a food operation. Although education and training are necessary parts of any food control program, standing alone they have certain limitations and shortcomings. Personnel turnover in the food industry is both constant and rapid, and thus education of workers must be a continuing rather than a sporadic exercise. It is essential that supervisory personnel be properly trained with respect to the hazards associated with the operations for which they have responsibility. Inspection of Facilities and Operations Inspections of facilities and operations are commonly used to evaluate adherence to good handling practices. Resident inspectors observe all phases of processing from the live animal to the finished product. Microbiological Testing Samples of ingredients, materials obtained from selected points during the course of processing or handling, and finished products may be examined microbiologically to determine adherence to Good Manufacturing Practices. In some instances, foods are examined for a specific pathogen or its toxins, but more often examinations are made to detect organisms that are indicative of the possible presence of pathogens or spoilage or to detect presence of the specific spoilage organisms or their products. Microbiological testing is absolutely essential to the control of certain products, e.g., to assure that dried milk and eggs and confectionery products are free of a Salmonella hazard. Testing is essential to assure that critical raw materials are satisfactory for their intended use, e.g., to assure that the sugar used in canning meets established standards and to assure that critical products used in dried blends are free of Salmonella. Microbiological testing has severe limitations as a control option. The most serious shortcoming is the constraint of time. Most microbiological test results are not available until several days after testing. Therefore, if finished product acceptability must be measured by microbiological testing, the product is held pending results. With perishable foods, this is generally not possible; with shelf stable foods, the warehousing of finished product increases costs. If in-line samples are collected and analyzed, the results are of retrospective value since the finished product has already been produced. 42  MICROBIOLOGY Practical: Food preservation Objectives Recognize food preservation related concepts. List the different techniques of food preservation Understand the principals of food preservation techniques Food preservation prevents the growth of microorganisms (such as yeasts), or other microorganisms (although some methods work by introducing benign bacteria or fungi to the food), as well as slowing the oxidation of fats that cause rancidity. Food preservation may also include processes that inhibit visual deterioration, such as the enzymatic browning reaction in apples after they are cut during food preparation. Traditional techniques Curing The earliest form of curing was dehydration or drying, used as early as 12,000 BC. Smoking and salting techniques improve on the drying process and add antimicrobial agents that aid in preservation. Smoke deposits a number of pyrolysis products onto the food, including the phenols syringol, guaiacol and catechol. Salt accelerates the drying process using osmosis and also inhibits the growth of several common strains of bacteria. More recently nitrites have been used to cure meat, contributing a characteristic pink color. Cooling preserves food by slowing down the growth and reproduction of microorganisms and the action of enzymes that causes the food to rot. The introduction of commercial and domestic refrigerators drastically improved the diets of many in the Western world by allowing food such as fresh fruit, salads and dairy products to be stored safely for longer periods, particularly during warm weather. Before the era of mechanical refrigeration, cooling for food storage occurred in the forms of root cellars and iceboxes. Rural people often did their own ice cutting, 43  MICROBIOLOGY whereas town and city dwellers often relied on the ice trade. Today, root cellaring remains popular among people who value various goals, including local food, heirloom crops, traditional home cooking techniques, family farming, frugality, self- sufficiency, organic farming, and others. Freezing is also one of the most commonly used processes, both commercially and domestically, for preserving a very wide range of foods, including prepared foods that would not have required freezing in their unprepared state. For example, potato waffles are stored in the freezer, but potatoes themselves require only a cool dark place to ensure many months' storage. Cold stores provide large-volume, long-term storage for strategic food stocks held in case of national emergency in many countries. Boiling Boiling liquid food items can kill any existing microbes. Milk and water are often boiled to kill any harmful microbes that may be present in them. Heating Heating to temperatures which are sufficient to kill microorganisms inside the food is a method used with perpetual stews. Milk is also boiled before storing to kill many microorganisms. Sugaring The earliest cultures have used sugar as a preservative, and it was commonplace to store fruit in honey. Sugar tends to draw water from the microbes (plasmolysis). This process leaves the microbial cells dehydrated, thus killing them. In this way, the food will remain safe from microbial spoilage." Sugar is used to preserve fruits, either in an antimicrobial syrup with fruit such as apples, pears, peaches, apricots, and plums, or in crystallized form where the preserved material is cooked in sugar to the point of crystallization and the resultant product is then stored dry. This method is used for the skins of citrus fruit (candied peel), angelica, and ginger. Also, sugaring can be used in the production of jam and jelly. Pickling Pickling is a method of preserving food in an edible, antimicrobial liquid. Pickling can be broadly classified into two categories: chemical pickling and fermentation pickling. 44  MICROBIOLOGY In chemical pickling, the food is placed in an edible liquid that inhibits or kills bacteria and other microorganisms. Typical pickling agents include brine (high in salt), vinegar, alcohol, and vegetable oil. Many chemical pickling processes also involve heating or boiling so that the food being preserved becomes saturated with the pickling agent. Common chemically pickled foods include cucumbers, peppers, corned beef, herring, and eggs, as well as mixed vegetables such as piccalilli. In fermentation pickling, bacteria in the liquid produce organic acids as preservation agents, typically by a process that produces lactic acid through the presence of lactobacillales. Fermented pickles include sauerkraut, nukazuke, kimchi, and surströmming. Lye Sodium hydroxide (lye) makes food too alkaline for bacterial growth. Lye will saponify fats in the food, which will change its flavor and texture. Lutefisk uses lye in its preparation, as do some olive recipes. Modern recipes for century eggs also call for lye. Canning Canning involves cooking food, sealing it in sterilized cans or jars, and boiling the containers to kill or weaken any remaining bacteria as a form of sterilization. It was invented by the French confectioner Nicolas Appert. By 1806, this process was used by the French Navy to preserve meat, fruit, vegetables, and even milk. Although Appert had discovered a new way of preservation, it wasn't understood until 1864 when Louis Pasteur found the relationship between microorganisms, food spoilage, and illness. Foods have varying degrees of natural protection against spoilage and may require that the final step occur in a pressure cooker. High-acid fruits like strawberries require no preservatives to can and only a short boiling cycle, whereas marginal vegetables such as carrots require longer boiling and addition of other acidic elements. Low-acid 45  MICROBIOLOGY foods, such as vegetables and meats, require pressure canning. Food preserved by canning or bottling is at immediate risk of spoilage once the can or bottle has been opened. Lack of quality control in the canning process may allow ingress of water or micro- organisms. Most such failures are rapidly detected as decomposition within the can causes gas production and the can will swell or burst. However, there have been examples of poor manufacture (under processing) and poor hygiene allowing contamination of canned food by the obligate anaerobe Clostridium botulinum, which produces an acute toxin within the food, leading to severe illness or death. This organism produces no gas or obvious taste and remains undetected by taste or smell. Its toxin is denatured by cooking, however. Cooked mushrooms, handled poorly and then canned, can support the growth of Staphylococcus aureus, which produces a toxin that is not destroyed by canning or subsequent reheating. Jellying Food may be preserved by cooking in a material that solidifies to form a gel. Such materials include gelatin, agar, maize flour, and arrowroot flour. Some foods naturally form a protein gel when cooked, such as eels and elvers, and sipunculid worms, which are a delicacy in Xiamen, in the Fujian province of the People's Republic of China. Jellied eels are a delicacy in the East End of London, where they are eaten with mashed potatoes. Potted meats in aspic (a gel made from gelatin and clarified meat broth) were a common way of serving meat off-cuts in the UK until the 1950s. Many jugged meats are also jellied. A traditional British way of preserving meat (particularly shrimp) is by setting it in a pot and sealing it with a layer of fat. Also common is potted chicken liver; jellying is one of the steps in producing traditional pâtés. Jugging Meat can be preserved by jugging. Jugging is the process of stewing the meat (commonly game or fish) in a covered earthenware jug or casserole. The animal to be jugged is usually cut into pieces, placed into a tightly-sealed jug with brine or gravy, and stewed. Red wine and/or the animal's own blood is sometimes added to the cooking liquid. Jugging was a popular method of preserving meat up until the middle of the 20th century. 46  MICROBIOLOGY Burial Burial of food can preserve it due to a variety of factors: lack of light, lack of oxygen, cool temperatures, pH level, or desiccants in the soil. Burial may be combined with other methods such as salting or fermentation. Most foods can be preserved in soil that is very dry and salty (thus a desiccant) such as sand, or soil that is frozen. Many root vegetables are very resistant to spoilage and require no other preservation than storage in cool dark conditions, for example by burial in the ground, such as in a storage clamp. Century eggs are traditionally created by placing eggs in alkaline mud (or other alkaline substance), resulting in their "inorganic" fermentation through raised pH instead of spoiling. The fermentation preserves them and breaks down some of the complex, less flavorful proteins and fats into simpler, more flavorful ones. Cabbage was traditionally buried during Autumn in northern US farms for preservation. Some methods keep it crispy while other methods produce sauerkraut. A similar process is used in the traditional production of kimchi. Sometimes meat is buried under conditions that cause preservation. If buried on hot coals or ashes, the heat can kill pathogens, the dry ash can desiccate, and the earth can block oxygen and further contamination. If buried where the earth is very cold, the earth acts like a refrigerator. Before burial, meat (pig/boar) can be fatted. The tallow of the animal is heated and poured over meat in a barrel. Once the fat hardens the barrel is sealed and buried in a cold cellar or ground. Fermentation Some foods, such as many cheeses, wines, and beers, use specific micro-organisms that combat spoilage from other less-benign organisms. These micro-organisms keep pathogens in check by creating an environment toxic for themselves and other micro- organisms by producing acid or alcohol. Methods of fermentation include, but are not limited to, starter micro-organisms, salt, hops, controlled (usually cool) temperatures and controlled (usually low) levels of oxygen. These methods are used to create the specific controlled conditions that will support the desirable organisms that produce food fit for human consumption. Fermentation is the microbial conversion of starch and sugars into alcohol. Not only can fermentation produce alcohol, but it can also be a valuable preservation technique. Fermentation can also make foods more nutritious and palatable. For example, drinking water in the Middle Ages was dangerous because it often contained pathogens that could spread disease. When the water is made into beer, the boiling during the brewing process kills any bacteria in the water that could make people sick. 47  MICROBIOLOGY Additionally, the water now has the nutrients from the barley and other ingredients, and the microorganisms can also produce vitamins as they ferment. Modern industrial techniques Techniques of food preservation were developed in research laboratories for commercial applications. Pasteurization Pasteurization is a process for preservation of liquid food. It was originally applied to combat the souring of young local wines. Today, the process is mainly applied to dairy products. In this method, milk is heated at about 70 °C (158 °F) for 15–30 seconds to kill the bacteria present in it and cooling it quickly to 10 °C (50 °F) to prevent the remaining bacteria from growing. The milk is then stored in sterilized bottles or pouches in cold places. Vacuum packing Vacuum-packing stores food in a vacuum environment, usually in an air-tight bag or bottle. The vacuum environment strips bacteria of oxygen needed for survival. Vacuum-packing is commonly used for storing nuts to reduce loss of flavor from oxidization. A major drawback to vacuum packaging, at the consumer level, is that vacuum sealing can deform contents and rob certain foods, such as cheese, of its flavor. Artificial food additives Preservative food additives can be antimicrobial—which inhibit the growth of bacteria or fungi, including mold—or antioxidant, such as oxygen absorbers, which inhibit the oxidation of food constituents. Common antimicrobial preservatives include calcium propionate, sodium nitrate, sodium nitrite, sulfites (sulfur dioxide, sodium bisulfite, potassium hydrogen sulfite, etc.). Antioxidants include butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Other preservatives include formaldehyde (usually in solution), glutaraldehyde (insecticide), ethanol, and methylchloroisothiazolinone. Irradiation Irradiation of foodis the exposure of food to ionizing radiation. Multiple types of ionizing radiation can be used, including beta particles (high-energy electrons) and 48  MICROBIOLOGY gamma rays (emitted from radioactive sources such as cobalt-60 or cesium-137). Irradiation can kill bacteria, molds, and insect pests, reduce the ripening and spoiling of fruits, and at higher doses induce sterility. The technology may be compared to pasteurization; it is sometimes called "cold pasteurization", as the product is not heated. Irradiation may allow lower-quality or contaminated foods to be rendered marketable. National and international expert bodies have declared food irradiation as "wholesome"; organizations of the United Nations, such as the World Health Organization and Food and Agriculture Organization, endorse food irradiation. Consumers may have a negative view of irradiated food based on the misconception that such food is radioactive; in fact, irradiated food does not and cannot become radioactive. Activists have also opposed food irradiation for other reasons, for example, arguing that irradiation can be used to sterilize contaminated food without resolving the underlying cause of the contamination. Pulsed electric field electroporation Pulsed electric field (PEF) electroporation is a method for processing cells by means of brief pulses of a strong electric field. PEF holds potential as a type of low- temperature alternative pasteurization process for sterilizing food products. In PEF processing, a substance is placed between two electrodes, then the pulsed electric field is applied. The electric field enlarges the pores of the cell membranes, which kills the cells and releases their contents. PEF for food processing is a developing technology still being researched. There have been limited industrial applications of PEF processing for the pasteurization of fruit juices. For cell disintegration purposes especially potato processors show great interest in PEF technology as an efficient alternative for their preheaters. Modified atmosphere Modifying atmosphere is a way to preserve food by operating on the atmosphere around it. Salad crops that are notoriously difficult to preserve are now being packaged in sealed bags with an atmosphere modified to reduce the oxygen (O 2) concentration and increase the carbon dioxide (CO2) concentration. There is concern that, although salad vegetables retain their appearance and texture in such conditions, this method of preservation may not retain nutrients, especially vitamins. There are two methods for preserving grains with carbon dioxide. One method is placing a block of dry ice in the bottom and filling the can with the grain. Another method is purging 49  MICROBIOLOGY the container from the bottom by gaseous carbon dioxide from a cylinder or bulk supply vessel. Carbon dioxide prevents insects and, depending on concentrati