The Impact Of Microbes On Health, Industry & The Environment Flashcards

Question

What is the main method by which antibiotics can be produced?

Answer 1

Antibiotic production; fed batch culture NOTION 1.4

Answer 2

1. Penicillins 2. Carbapenems 3. Cephalosporins 4. Monobactams

Answer 3

• Beta-lactams are the most important class of antibiotics targeting cell wall biosynthesis. • Beta-lactam group is the pharmacophore of all Beta-lactam antibiotics. • Beta-lactam rings were unknown to chemists before the discovery of penicillin

Answer 4

NOTION 1.5

Answer 5

NOTION 1.6

Answer 6

NOTION 1.7

Answer 7

EU statistics: - Infections by antibiotic-resistant bacteria = 400,000, - Deaths = 25,000 - Hospital days = 2.5 million

Answer 8

- Vertical Transfer - Horizontal Transfer NOTION 1.8

Answer 9

• Host enzymes modify and inactivate the drug (e.g. beta-lactam antibiotics) • Modification of the drug target (mutations of the target enzyme e.g. peptidoglycan transpeptidase) • Reducing the drug’s intracellular concentration: drug-specific transporters that pump the antibiotic out of the bacterium NOTION 1.9

Answer 10

• Beta-lactamases are bacterial enzymes that have evolved as defence mechanisms against the penicillins • A bacterium carrying the beta-lactamase gene will be penicillin resistant • Beta-lactamases cleave the beta-lactam ring in penicillin

Answer 11

They could be used to produce antibiotics or chemotherapeutics: - In this case, the microorganism would be given a transformed foreign gene - And therefore an enzyme would be expressed - And a novel metabolite synthesised - This novel metabolite could then be used as a therapy In addition, they could also be used to manufacture vaccines, or hormones (insulin): - In This case, the microorganism would be given a transformed foreign gene - A protein would then be expressed - This encoded protein could then be used as a therapy

Answer 12

- Diagnosed Type 2 Diabetes: 10.3 million - Diagnosed Type 1 Diabetes: 0.5-1.0 million - Undiagnosed Diabetes: 5.4 million

Answer 13

NOTION 2.1

Answer 14

- Human insulin can be expressed in E Coli. - Avoids use of cadavers or animal insulin - Insulin gene can be modified through mutation to engineer the properties of insulin (long acting, short acting) - Requirement to express two insulin polypeptides A and B: production in large quantities in two separate fermentations

Answer 15

NOTION 2.2

Answer 16

NOTION 2.3

Answer 17

1. Hepatitis B virus (HBV) affects the liver 2. Sickness; vomiting, jaundice, fatigue, dark urine and abdominal pain 3. Cirrhosis and liver cancer may eventually develop; death of 15 to 25% of those with chronic disease 4. Transmission by exposure to infectious blood or body fluids. Routes; intravenous drug use and sexual intercourse. 5. A vaccine was needed; vaccine would be first anti-viral vaccine that is also an anti-cancer vaccine.

Answer 18

Vaccine administered to: Children and Adolescents. Babies normally get 3 doses of hepatitis B vaccine. At risk adults, including: • Sex partners of people infected with hepatitis B • Intravenous drug users • People with multiple sex partners • People with chronic liver or kidney disease • People with jobs that expose them to human blood • People who travel to countries where hepatitis B is common

Answer 19

Anyone with a life-threatening allergy to yeast should not get hepatitis B vaccine.

Answer 20

NOTION 2.4

Answer 21

NOTION 2.5

Answer 22

Hepatitis B vaccine; • viral envelope proteins, hepatitis B surface antigen (HBsAg). • produced by yeast cells, into which the genetic code for HBsAg has been inserted

Answer 23

• Recombivax HB (Merck), • Engerix-B (GSK), • Elovac • Genevac B (Serum Institute)

Answer 24

NOTION 2.6

Answer 25

500 million people in the tropics and subtropics become infected with malaria each year.

Answer 26

Suffer burning fever and severe pain.

Answer 27

Three million—mostly children—die

Answer 28

Artemisinin: • Chinese herbal medicine, discovered in the 4th century • Source is leaves of Artemisia annua (annual wormwood) • 25,000 hectares of wormwood + $300M per annum to treat all sufferers

Answer 29

• Artemisinin synthesis pathway; each arrow is a separate enzyme reaction • Each enzyme is encoded by a separate Artemisia plant gene NOTION 2.7

Answer 30

Synthetic biology is the use of synthetic gene circuits to endow a cell with new functions. Genes are assembled into new pathways with new regulatory properties using engineering principles.

Answer 31

• Aim: clone all 16 plant genes into baker’s yeast • express kg quantities cheaply in fermenters • Artemesinin successfully produced for $0.25 / dose (was $2.5)

Answer 32

NOTION 2.8

Answer 33

Strain improvements: for example - : Overexpression of an enzyme in the sterol synthesis pathway to ‘steal’ more metabolic flux from Acetyl CoA. NOTION 2.9

Answer 34

Advantages: 1. SAFETY: Cadaver material, Hepatitis B Cell Culture 2. EASE: Hard to purify insulin from a pancreas 3. COST: Expensive to purify artemisinin, insulin 4. FLEXIBILITY: Whole new pathways engineered (Artemisinin), novel variants created (Insulin)

Answer 35

Glucose -> 2CO2 + 2Ethanol

Answer 36

1. Saccharomyces cerevisiae 2. Fermentation 3. It is an aerobic process

Answer 37

NOTION 3.1

Answer 38

NOTION 3.2

Answer 39

Fermentation • Produces 2 moles ATP / mole glucose • Is redox balanced; - oxidation during glycolysis - reduction during fermentation • Pyruvate dehydrogenase and alcohol dehydrogenase overexpressed in yeast

Answer 40

Global production of fuel ethanol tripled between 2000 and 2007 from 17 billion to more than 52 billion liters.

Answer 41

Feedstock = sugar-rich crops: sugar cane, sugar beet, potato, cassava, sweet corn/maize.

Answer 42

Ethanol is only partially a renewable energy source: sunlight energy used to fix CO2 into sugars, but energy input required for distillation of 10% ethanol into 95% ethanol.

Answer 43

Vitamins = Organic compound required by an animal in limited amounts.

Answer 44

Humans are ‘auxotrophic’ for these compounds (no synthesis e.g. Vitamin B12). Disease, and often death result from diets deficient in these vitamins.

Answer 45

Only bacteria are metabolically capable of making vitamin B12.

Answer 46

1. Vitamin A (beta-carotene, retinol) 2. Vitamin B1 (thiamin) 3. Vitamin B2 (riboflavin, vitamin G) 4. Vitamin B3 (niacin, vitamin P, vitamin PP) 5. Vitamin B5 (pantothenic acid) 6. Vitamin B6 (pyridoxine, pyridoxamine, or pyridoxal) 7. Vitamin B7 (biotin, vitamin H) 8. Vitamin B9 (folic acid, folate, vitamin M) 9. Vitamin B12 (cobalamin) 10. Vitamin C (ascorbic acid) 11. Vitamin D (ergocalciferol D2, or cholecalciferol D3) 12. Vitamin E (tocopherol) 13. Vitamin K (naphthoquinoids)

Answer 47

Vitamin B12 deficiency caused by mal-absorption, due to; • leaky gut and/or gut inflammation • pernicious anemia (autoimmune condition)

Answer 48

Symptoms linked to a lack of vitamin B12 include: • Yellowing of the skin • Changes or loss of some sense of touch • Feeling less pain • Walking problems • Vision problems

Answer 49

Vitamin B12 required in human for; • metabolism of propionyl-CoA which is derived from the degradation of some amino acids • Methionine synthase requires vitamin B12. This enzyme methylates homocysteine to form methionine in the methionine salvage pathway

Answer 50

• A multi-gene, multi-enzyme biosynthetic pathway to produce vitamin B12 • A cobalt-containing enzyme cofactor NOTION 3.3

Answer 51

Vitamin B12 is produced in microorganisms starting from glycine and succinyl CoA.

Answer 52

Fermentative

Answer 53

Pseudomonas and Nocardia species used

Answer 54

B12 yields improved by: • (i) random mutagenesis • (ii) And by genetic engineering; multigene expression systems improve the vitamin B12 production levels in propionibacteria

Answer 55

Essential amino acids are not made by human (and many other animal species)

Answer 56

Humans and other mammals are auxotrophic for the following amino acids: - Isoleucine - Lysine - Leucine - Methionine - Phenylalanine - Threonine - Tryptophan - Valine - Histidine - Arginine

Answer 57

Amino Acid’s can help the growth of animals e.g cows & chicken.

Answer 58

• Amino acids are used as additives to animal feed. • Needed since normal animal feed protein e.g. soya protein has low content of the essential amino acids: Lysine, methionine, threonine, and tryptophan

Answer 59

DL-Methionine, produced by chemical synthesis, used in poultry feed.

Answer 60

L-Lysine production by fermentation started in Japan during the 1960s; main limiting amino acid for pigs and the second for poultry.

Answer 61

MSG: Mono-sodium glutamate MSG = Flavour enhancer Intensifies the meaty, savory flavour of food

Answer 62

Corynebacteria—cultured with ammonia and carbohydrates from sugar beets, sugar cane, tapioca or molasses—excrete amino acids into a culture broth from which L-glutamate is isolated.

Answer 63

C. glutamicum is: • Gram positive, facultatively anaerobic, heterotrophic bacterium • irregular rod shape in a V-formation • Non-pathogenic, soil living. • Wild-type strains produce about 10 g/L glutamic acid • Genetic engineering yields are now upwards of 100g/L

Answer 64

Corynebacterium glutamicum produces glutamic acid as a normal amino acid producing reaction from TCA cycle intermediates…but very efficiently NOTION 3.4

Answer 65

• Clostridium acetobutylicum produces acetone by fermentation of starch • Uses glycolysis intermediate acetyl CoA • ABE process, (Acetone Butanol Ethanol fermentation process)

Answer 66

Chaim Weizmann • Lectured in chemistry at the University of Manchester during 1910s • Discovered how to use microbial fermentation to produce acetone (propan-2-one) • Acetone required to produce cordite – propellant used in shells and bullets in First World War • Became first President of Israel in 1949

Answer 67

• Pilot plant production of acetone gin factory in London, 1915 • Distilleries requisitioned for the war effort • 30,000 tonnes of acetone produced during the war • National collection of horsechestnuts initiated during first world war to provide starch source for acetone fermentation

Answer 68

Microorganism advantages for SCP: • High rate of multiplication • Genetic modification to vary amino acid composition • Flexible ability to utilise vast range of carbon and energy sources, including waste products

Answer 69

Microorganism disadvantages for SCP: • In all cells, nitrogen is found in protein, and nucleic acid • Meat has high ratio of protein: nucleic acid • SCP has much lower ratio of protein: nucleic acid • If SCP is consumed, nucleic acid breakdown produces uric acid, which can cause gout and kidney stones

Answer 70

Utilization of collagenous by-products from the meat packing industry: production of single-cell protein by the continuous cultivation of Bacillus megaterium.

Answer 71

Alfalfa residual juice can be used for production of single-cell protein.

Answer 72

Growth of Aureobasidium pullulans on straw hydrolysate.

Answer 73

Quorn is made up of Textured mycoprotein i.e a Single Cell Protein Fusarium venenatum is a filamentous fungus, used in the production of Quorn mycoprotein. Continuous culture process is used: glucose and ammonium sulphate are used as medium, plus vitamins and minerals = high value.

Answer 74

Animals harbour a complex microbial community (=microbiota) in their intestines.

Answer 75

Particularly important for herbivores (plant eaters), as microbes help digesting the plant material, but omnivores (incl. humans) and carnivores (meat eaters) also have a microbiota.

Answer 76

➢Ruminants, large microbial fermentation chamber (“rumen”) in the stomach ➢Often re-gurgitate food for further chewing

Answer 77

➢Monogastric animals ➢Microbial fermentation takes place in the large intestine ➢Often perform coprophagy (consumption of faeces) to extract further nutrients

Answer 78

Hundreds of different microbes are found in different body sites – bacteria, archaea, Eukaryotes and viruses.

Answer 79

Few dominant bacterial phyla in the gut: - Actinobacteria - Bacteroidetes (20-30% of faecal bacteria) - Firmicutes (60-70% of faecal bacteria) - Proteobacteria NOTION 4.1

Answer 80

Yes, high diversity at the levels of strains, species and genera present, particularly within Firmicutes and Bacteroidetes.

Answer 81

Oral cavity: 1 billion/ml Skin: 1 million/ cm2 Stomach & duodenum: 10-1000/ ml Jejunum/ ileum: 0.01- 100 million/ ml Colon: 10-100 billion/ ml

Answer 82

Oral cavity: - pH = 5-7 - Transit = sec - min - Digestive enzymes, immune interactions Stomach & duodenum: - pH = 1-3 - Transit = 1-3 hours - hydrochloric acid, digestive enzymes (proteases) Jejunum/ ileum: - pH = 6-7.5 - Transit = 1-5 hours - digestive enzymes, bile, main site for immune interactions Colon: - pH = 5-7 - Transit = 10 hours - several days - strictly anaerobic, immune interactions

Answer 83

➢ Colon is most densely colonised due to favourable pH and slow transit time ➢ Facultative anaerobes use up oxygen → anaerobic

Answer 84

Development of microbiota from birth can be seen in the following graph: NOTION 4.2 Birth: - Natural delivery = Vaginal & faecal bacteria - Caesarean = Skin bacteria Feeding regime: - Breast feeding: high no of bifidobacteria - Formula-feeding: more complex microbiota Weaning: - Introduction of solid foods → major changes in microbiota towards adult composition Environmental exposure: - Cleanliness, pets, older siblings etc. - Antibiotics, medication, vaccination - Dietary habits Genetic make up & immune system of the individual also impacts on the gut microbiota.

Answer 85

- Variation between individuals and between different geographic regions/cultures - Different microbial profiles in Westernised societies compared to developing world

Answer 86

Early childhood is crucial for proper maturation of the immune system, to enable it to distinguish: - Resident microbiota & harmless environmental agents (i.e should be tolerated) - Pathogens (specific bacterial, viruses, fungi, worms etc) (i.e should be attacked)

Answer 87

Development of tolerance to environmental agents requires the presence of the microbiota, with certain species likely to play specific roles and/or overall microbial diversity being important → Hygiene hypothesis: exposure to microbes protects against inflammatory diseases (allergies etc.) NOTION 4.3

Answer 88

Continuous cross-talk between gut microbes and the immune system throughout life → immune homeostasis.

Answer 89

Microbiota provides colonisation resistance against pathogens by: 1. Direct inhibition → Competitive exclusion: competition for nutrients, occupation of gut wall binding sites → Production of bacteriocins, acids, etc 2. Immune-mediated colonisation resistance → Microbial cells and metabolites stimulate the immune system to elicit an immune response 3. Modulation of gut physiology → Energy supply to gut epithelium → Gut barrier

Answer 90

1. Gastrointestinal infections 2. Irritable bowel syndrome 3. Inflammatory bowel disease 4. Colorectal cancer 5. Obesity, Diabetes 6. Heart Disease 7. Autoimmune disorders 8. Depression, Autism ➢ Relative importance of microbiota to other factors (eg. direct diet effects, host genetics)? ➢ Specific microbes or microbial community effects? ➢ Microbiota changes associated with disease do not necessarily have a causative relationship

Answer 91

Beneficial effects of the microbiota: 1. Barrier function against pathogens 2. Immune maturation and stimulation 3. Release & transformation of dietary phytochemicals and host metabolites with health-promoting properties 4. Production of metabolites - Energy supply - Regulation of host metabolism (satiety, lipid metabolism, behaviour etc.) - Anti-inflammatory & anti-carcinogenic effects - Lowering of pH → pathogen inhibition, Ca2+-availability increases

Answer 92

Detrimental effects of the microbiota: 1. May harbour opportunistic pathogens; e.g. antibiotic-associated C. difficile infection) 2. May contribute to inflammatory diseases in predisposed individuals (e.g. Crohn’s disease) 3. Release & transformation of toxic compounds from the diet or environment 4. Production of metabolites - Energy supply (obesity) - Regulation of host metabolism (satiety, lipid metabolism, behaviour etc.) - Potentially toxic metabolites, e.g. ethanol, acetaldehyde (liver disease)

Answer 93

NOTION 4.4

Answer 94

NOTION 4.5

Answer 95

High fibre diets help provide: ➢ Supply of short chain fatty acids ➢ Release of phytochemicals ➢ Bulking, shorter transit time ➢ Increased resistance to pathogens (barrier function)

Answer 96

A low fibre diet can lead to: ➢ Production of toxins, carcinogens ➢ Lower bacterial activity - lower supply of protective metabolites, e.g. butyrate - weaker barrier function

Answer 97

Carbohydrate variation occurs due to: - Different monosaccharide constituents - Different linkages - Linear or branched - Non-sugar constituents

Answer 98

Usage as energy source requires: → carbohydrate binding and cleavage (different enzymes required) → transport → fermentation of monosaccharides

Answer 99

NOTION 4.6

Answer 100

NOTION 4.7

Answer 101

NOTION 4.8

Answer 102

NOTION 4.9

Answer 103

90-95% of bacterially produced SCFA are estimated to be absorbed by the host.

Answer 104

Local effects of SCFA in the gut ➢ Butyrate is the main fuel for the colonic wall ➢ Anti-cancerous and anti-inflammatory effects, in particular butyrate ➢ Influence on gut physiology → gut motility & transit time, barrier function ➢ Physical effects: lowering of luminal pH → protection against pathogens → increased absorption of calcium

Answer 105

Systemic effects of SCFA in host tissues ➢ Contributes to energy supply ➢ Regulation of host physiology: lipid and glucose metabolism etc ➢ Influence of behaviour: satiety, hunger

Answer 106

They can aid in the conversion of: ➢ Other dietary constituents proteins, fats, phytochemicals ➢ Xenobiotics compounds not normally found in an organism, e.g. drugs, environmental pollutants ➢ Host-derived compounds, e.g. bile acids

Answer 107

The biotransformation of these compounds, into other compounds may affect: - Absorption status (whether it resides in the gut or is taken up by the host) - Host physiology, e.g. anti-inflammatory effects

Answer 108

They can help produce: ➢ Vitamins ➢ Neurotransmitters, e.g. gamma-aminobutyric acid

Answer 109

Many microbially derived metabolites are taken up and further metabolised by the host. NOTION 4.10

Answer 110

- Acetate = Metabolised mainly in muscle & organs - Propionate = Metabolised mainly by the liver - Butyrate = Metabolised mainly by the liver

Answer 111

1. Isolate & characterise gut bacteria in pure culture - Growth characteristics & traits - Genome sequence, transcriptomes, proteomes 2. In vitro microbial community models - Faecal microbiota or synthetic communities from cultured strains - Batch or continuous culture 3. In vivo trials: Microbiota analysis in faecal or gut/ biopsy samples - Healthy vs diseased, effect of diet etc 4. Ex vivo studies (cell culture, biopsies, organs sections)

Answer 112

Molecular & analytical techniques: - Microbiota changes in compositional, activity, metabolite production etc - Analysis of host measures: blood & urine metabolites, health parameters etc

Answer 113

Level of culturability partially dependent on the natural environment. For example: - Soil: nutrients may be scarce, frequent environmental changes => Microbes often dormant or slow growing - Gut: Relatively stable environment, nutrient rich, constant turnover (growth at certain rate to persist, majority of gut microbes likely culturable)

Answer 114

Complex culture media => Growth of many bacteria => Difficult and laborious to identify individual bacteria => Difficult to obtain rare bacteria Selective culture media => Growth only of target bacteria => Many different media/ conditions required for whole microbiota, limits to selectivity => Suitable for rare bacteria

Answer 115

High throughput methods (‘culturomics’), use of genome information and application of molecular techniques to target specific bacteria (e.g. molecular probes).

Answer 116

Anaerobic cabinet: - Maintained at 37*C (body temperature) - Anaerobic atmosphere (mainly nitrogen; no oxygen) Hungate technique R.E: - Strong glass tubes of medium filled with CO2, inoculated and sealed with rubber screw cap - Works because CO2 is heavier than air

Answer 117

Batch Cultures: - All nutrients and microbes added at the start => closed system - Different growth phases (Lag, Exponential/ Log, Stationary, Death) - Can only study high substrate concentrations, which are exhausted during growth - Products accumulate as growth proceeds => can influence metabolism, change pH etc NOTION 5.1

Answer 118

Continuous culture: - Parameters can be kept constant: substrate concentration, pH etc - Products are constantly diluted/ removed - Steady state: level of microbial growth dependent on substrate concentration in feed - Self regulating

Answer 119

Continuous culture: - Maintained at 37*C, specific pH, anaerobic - Constant nutrient inflow - Single stage: Stimulates a specific region of the gut - Multi stage: Stimulates passage through whole gut Example of a 5 stage fermentor step: NOTION 5.2

Answer 120

16S rRNA gene is often used as marker for microbial diversity: - Present in all living organisms (18S rRNA in eukaryotes) - Codes for structural component of ribosomes - Level of sequence similarity can be used to estimate relatedness of different organisms - More difference between the 16S rRNA genes of two organisms -> less closely related

Answer 121

Phylogenetics - Evolutionary relationship between organisms

Answer 122

NOTION 5.3

Answer 123

Design primers or probes to utilise 16S rRNA gene as a phylogenetic marker.

Answer 124

Primer: short oligonucleotide required for amplification of DNA; for techniques involving the polymerase chain reaction (PCR).

Answer 125

Probe: short oligonucleotide containing a marker (normally fluorophore) that can bind to DNA; for techniques such as fluorescence in situ hybridisation (FISH).

Answer 126

NOTION 5.4

Answer 127

Amplify 16S rRNA gene for further analysis 1. Primers against conserved regions → amplify 16S rRNA genes from all bacteria 2. Primers against variable regions → amplify only specific bacterial groups or species 3. Probes against variable regions → label specific bacterial groups or species

Answer 128

Non-targeted methods: investigate whole community e.g. changes within community in response to a particular substrate.

Answer 129

Targeted methods: investigate specific microbes/groups e.g. determine whether a probiotic bacterium can be detected in faeces.

Answer 130

Methods that lead to identification or provides specific information of the microbes e.g. sequencing, qPCR, FISH, phylogenetic microarrays.

Answer 131

Profiling methods: fingerprint of microbial community to investigate changes over time, differences between individuals, etc.; identity of microbes remains unknown without further downstream analysis, e.g. DGGE

Answer 132

NOTION 5.5

Answer 133

- Amplification of gene from whole community with universal primers (using conserved regions) - Grouping of different sequences into phylotypes or Amplicon Sequence Variants (ASVs) - Allows assessment of relative changes within microbial community & level of microbial diversity NOTION 5.6

Answer 134

Determination of changes over time in a continuous culture fermentor with faecal samples from 3 human donors (D2, D7, D19) in the absence (top) and presence (bottom) of 10 mM lactate. NOTION 5.7

Answer 135

Total number of bacteria can vary between different indidvuals & over time!

Answer 136

A change in relative abundance does not necessarily mean a change in absolute numbers.

Answer 137

Quantitative real time PCR (qPCR): - PCR amplification in presence of fluorescent dye that measures total amount of DNA - Detection of fluorescence signal in real time (while the PCR is running) - The more target DNA present at the start of the PCR, the earlier a fluorescence signal is detected NOTION 5.8

Answer 138

- Use conserved 16S rRNA gene primers for amplification of total bacteria - Use primers for specific bacterial groups to determine their abundance

Answer 139

Fluorescence in situ hybridisation (FISH) ➢ PCR-independent technique ➢ Hybridise probe specific to bacterial species, genus, family etc. with 16S rRNA in bacterial cells ➢ Count under microscope, or use flow cytometry for quantification

Answer 140

NOTION 5.9

Answer 141

Metagenomics ➢ Sequencing of total DNA from sample, without amplification of specific genes → investigation of all genes, not just phylogenetic markers genes → functions, metabolism, etc. ➢ Generates massive datasets, assignment of correct function to genes can be difficult (many genes remain uncharacterised, annotation purely based on sequence similarity) ➢ Does not take into consideration expression level of genes

Answer 142

Metatranscriptomics, metaproteomics - Investigation of RNA or proteins from sample

Answer 143

Metabolomics - Investigation of metabolites in faeces, urine, blood

Answer 144

Functional metagenomics ➢ Expression of cloned sample DNA in heterologous host (e.g. E. coli) ➢ Screening for clones with the desired properties, e.g. Specific enzyme function for degradation of non-digestible carbohydrates present in bacteria present in original sample

Answer 145

NOTION 5.10

Answer 146

- Dental caries & periodontal disease - Infectious diseases - Intensive care - Haematology and Oncology patients - Children - General Practice - Hospital Acquired Infection (HAI)

Answer 147

NOTION 6.1

Answer 148

Aberdeen Royal Infirmary – serves 530 000 people – 700,000 specimens per year

Answer 149

Employs – Biomedical Scientists – Secretaries – Medical Microbiologists and Virologists

Answer 150

- Advice on the diagnosis of infection - Supervision and review of lab tests - Advice on interpretation of test results - Advice on treatment/management (antibiotics) - Infection control/prevention - Antibiotic policy

Answer 151

Bacteria, viruses, fungi, parasites & prions

Answer 152

Within sterile sites there shouldn’t be any organisms found, meaning they are easier to identify: - Blood, CSF, Lung, Bladder Within non sterile sites a selective process is required, due to the commensal flora. At some points the commensal causes the disease: - Skin, Nasopharynx, Urethra, Gut

Answer 153

- Urinary tract infection = Mid stream urine - Chest infection = Sputum - Tonsilitis/ pharyngitis = Throat swab - Wound or site of infection = Swab or pus - Diarrhoea = Faeces - Bacteraemia = Blood culture - Meningitis = Cerebro-spinal fluid

Answer 154

- Light microscopy (x1000 magnification) - Unstained - to see pus cells (e.g., urine, CSF) - to see parasites (faeces) - Gram stain - to see bacteria and yeasts/fungi - ZN stain or auramine stain - to see Mycobacteria - Viruses not visible in light microscope

Answer 155

- Rapid - telephone urgent information - Not sensitive - Can’t usually identify particular species

Answer 156

Slow but sensitive Type of media: - Non selective e.g Blood (strep), Chocolate (Hemophilus) - Selective e.g MacConkey - enteric gram -ve microbes - Specialised e.g Mycobacteria Things to consider: - Atmosphere - Temperature - Duration of incubation

Answer 157

Identifying strains (variants) is important: ˗ Evolution ˗ Natural history ˗ Pathogenesis ˗ Diagnosis ˗ Treatment ˗ Prevention of disease spread - Outbreak investigations - Surveillance - Transmission routes

Answer 158

T = Transferable R = Reliable (sensitivity, specificity) A = Able to discriminate different strains C = Cheap E = Effort required (labour) S = Standardised laboratory methods & interpretation (Kits)

Answer 159

NOTION 6.2

Answer 160

NOTION 6.3

Answer 161

Chromogenic substrate is used: - Galateridase: coliforms => pink - Glucaranidase: E.coli => purple

Answer 162

1. Testing serum with antibodies. You target antigenic variations in cell surface. 2. Expensive, time consuming, low discrimination 3. Campylobacter, Salmonella 4. Conventional method for E. coli • >700 serotypes • O lipopolysaccharide • H flagella • K capsule – O157 (O26, O111, O103 often less severe illness)

Answer 163

Phage typing – Inoculate phage onto lawn of bacteria – Area of lysis at site phage inoculation – Susceptibility / resistance to each phage – Used for further strain discrimination Used for – E. coli O157 – Salmonella enterica serovars

Answer 164

1. Knowledge of normal flora at site 2. Knowledge of likely pathogens at site 3. Clinical significance in light of clinical picture – if YES, antibiotic sensitivity tests performed

Answer 165

Molecular methods most common now – Real time PCR (RNA/DNA viruses) Other methods – Antigen detection – Serology to determine immunity – Cell or tissue culture - inoculate a “cell line” – Electron microscopy little used now

Answer 166

Microscopy of different stages – parasites, cysts and ova in faeces – blood films for malaria Culture rarely possible Serology sometimes useful

Answer 167

NOTION 6.4

Answer 168

- Methicillin Resistant Staph aureus (MRSA) - Clostridium difficile - Organisms with Extended Spectrum β-lactamases (ESBLs) - Noroviruses

Answer 169

Characterised by signs and symptoms – inflammation – pain – pyrexia – tachycardia – rigors – increased white cell count – Increased C reactive protein (CRP) Note that infection can also be latent or sub clinical.

Answer 170

A pathogen is an organism which can cause disease.

Answer 171

A commensal is an organism which is part of normal flora e.g. E. coli in the gut, Staph aureus in the nose, axilla

Answer 172

Koch’s Postulates – Organism must be found in all cases of the disease – Able to be cultured outside the body for several generations – Should reproduce the disease on inoculation

Answer 173

Knowledge of: – Normal flora for site – Organism’s pathogenicity – Clinical context

Answer 174

Pathogenicity = The capacity of a micro-organism to cause an infection. Requirements: – Infectivity • Ability to become established – Virulence • Ability to cause harmful effects once established

Answer 175

Attachment • E. coli – P-fimbriae – Receptor on uroepithelial cells Acid resistance • Helicobacter pylori – Urease – Makes ammonia from urea

Answer 176

Conferred by virulence factors: Genetically determined microbial components: • Invasiveness • Toxin production • Evasion of immune system Specific to strains, not species.

Answer 177

Streptococcus pyogenes (Group A streptococci): – necrotising fasciitis – cellulitis – Connective tissue breakdown • Hyaluronidase • Collagenase – Fibrinolysis • Streptokinase

Answer 178

• Exotoxins are released extracellularly by the micro-organism • Enterotoxins are exotoxins which act on the GI tract • Endotoxin is structurally part of the Gram negative cell wall

Answer 179

Enterotoxin: Cholera • Vibrio cholerae – Colonises small intestine • Enterotoxin production – Increases cAMP levels • Inhibits uptake of Na+ and Cl- ions • Stimulates secretion of Cl- and HCO3- ions – Passive (massive) outflow of H2O • Causes death by dehydration • Treated by rehydration

Answer 180

• Certain exotoxins of Strep pyogenes and Staph aureus • Able to stimulate division of T cells in the absence of specific antigen • overwhelming cytokine production causes “toxic shock”

Answer 181

• Component of the Gram-negative bacterial cell wall – Lipopolysaccharide - Lipid A - Oligosaccharide core - Specific polysaccharide chain • E. coli and other Gram-negative bacilli • Neisseria meningitidis • Induces severe uncontrolled host response – Cytokine production – Fever, rigors, hypotension, tachycardia, collapse

Answer 182

• Simple framework to classify clinically important organisms • Different species have varying capacity to cause disease (pathogenicity) • Different classes of antibiotics are effective against Gram positive and Gram negative bacteria • Gram stain can give an early indication of the type of bacteria which may be causing infection

Answer 183

Staphylococci (clusters) Streptococci (chains)

Answer 184

Coagulate Test: - If coagulate positive = Staphylococcus aureus (either MSSA or MRSA) - Or can be coagulase negative! If coagulase negative: - Can be many different species - Skin commensals - Can be pathogenic in presence of foreign bodies/ prostheses - Or in immunocompromised individuals

Answer 185

• Commonly penicillin resistant due to βlactamase • By a different mechanism, some strains are “methicillin resistant”. Methicillin resistant Staph aureus (MRSA) poses major problems for infection control in hospitals.

Answer 186

NOTION 7.1

Answer 187

NOTION 7.2/ 7.3

Answer 188

• Alpha- haemolytic (partial haemolysis) turn blood agar green • Beta-haemolytic (complete haemolysis) turn blood agar clear • Non haemolytic

Answer 189

Streptococcus pneumoniae (pneumococcus): pneumonia, meningitis, septicaemia Viridans streptococci • many different species • normal oral flora • cause of infective endocarditis (infection of heart valves)

Answer 190

• Further identified by Lancefield groupings (based on surface antigens) • Groups A-G • A, B and D clinically most important

Answer 191

Sore throats, cellulitis, necrotising fasciitis (“flesh eating bug”)

Answer 192

• “Difficult” to culture • Can be carried asymptomatically in the gut of healthy people – up to 3% of adults, 66% of babies • But also an important cause of diarrhoea, associated with toxin production – potentially fatal • Increased risk with antibiotic use, and anything else that disrupts the normal gut flora • Pseudomembranous colitis • Spreads in hospital via spores • Detect antigen & toxin in stool sample by ELISA

Answer 193

• Toxins cause tetanus • Uncontrolled muscle spasm due to loss of inhibition at neuro-muscular junction • antigenically modified toxin (toxoid) used for immunisation

Answer 194

Source of ‘Botox’

Answer 195

Bacillus anthracis: Cause of anthrax It is a human infection associated with exposure to infected animals or contaminated animal products. Bacillus anthracis: Cutaneous: - Easily rupture: dark brown/ black eshar at base of shallow ulcer (surrounded by induration/ oedema) - As ulcer matures base becomes black - In uncomplicated cases, lesions will heal over 1-3 weeks - Untreated cutaneous diseases associated with fatality rate 10-20%. But if treated, rarely fatal.

Answer 196

• Staphylococcus aureus • Streptococcus pyogenes • Streptococcus pneumoniae • Clostridium difficile

Answer 197

• Neisseria spp • Moraxella catarrhalis

Answer 198

• Neisseria meningitidis (meningococcus) • Neisseria gonorrhoeae

Answer 199

• Causes meningitis – inflammation of the meninges and septicaemia • Important when isolated form a sterile site e.g. blood cultures or CSF • PCR available on EDTA blood for N. menigitidis • Can be life threatening

Answer 200

• Causes urethritis in men, pelvic inflammatory disease in females • Spread by sexual contact

Answer 201

• Coliforms – organisms which are in the family Enterobacteriaceae • Mostly inhabitants of the human gut

Answer 202

Lactose fermentation is a useful preliminary test in identifying Gram negative bacilli.

Answer 203

• Several virulence mechanisms: pili, capsule, endotoxin and exotoxins produced • Ferments lactose • Over 160 serotypes based on O antigen (LPS) • Strains vary considerably in disease potential • Important cause of urinary tract infection (UTI) and bacteraemia

Answer 204

Enterotoxogenic E.coli - Commonest cause of traveller’s diarrhoea Enterohaemorrhagic E.coli - Bloody diarrhoea - Haemolytic uraemic syndrome (HUS) associated with E.coli O157

Answer 205

• Does not ferment lactose • Salmonella enterica >1500 serotypes • Self-limiting enterocolitis with or without bloody diarrhoea (incubation period 12-48 hrs) • Second commonest cause of bacterial diarrhoea in UK

Answer 206

• Cause of typhoid fever • Fever, constipation early stages • Organism can be isolated from blood cultures as well as faeces • Risk during foreign travel but vaccine available

Answer 207

• Shigella spp – Cause of diarrhoea, dysentery – in UK usually travel related • Klebsiella spp – Occasional cause of UTI and pneumonia • Proteus spp – Causes UTI – often associated with stones

Answer 208

• Camplyobacter spp = Gram negative bacilli • Microaerophilic – likes low O2 content • Source is domestic animals, chicken • Faecal-oral route • Foul smelling then bloody diarrhoea (incubation period 2-5 days) • Commonest cause of bacterial diarrhoea in UK

Answer 209

• Natural habitat is human stomach • Damages mucosa and causes ulcers

Answer 210

• Cocco-bacilli (mixed appearance) • Causes respiratory tract infection (second to Strep pneumoniae) • Capsulate form (type “b”) was formerly an important cause of meningitis (Hib conjugate vaccine)

Answer 211

• Water and soil organism – drains, sinks, mops • Can contaminate medical equipment • Hospital acquired cause of sepsis: pneumonia, UTI • Respiratory infection in cystic fibrosis • Sensitive to limited range of antibiotics

Answer 212

• E. coli • Salmonella spp • Campylobacter spp • Pseudomonas spp • Helicobacter pylori • Haemophilus influenzae • Bacteroides and other anaerobes

Answer 213

– Mycobacterium species – Spirochaetes – Chlamydia / Chlamydophila

Answer 214

Mycobacterium species are seen using the Ziehl-Neelsen (ZN) Stain. Resistant to decolourisation by acid or alcohol following staining with carbol fuchsin → ACID AND ALCOHOL FAST BACILLI (AAFB)

Answer 215

WHO figures for 2013: – 9 million new cases worldwide – India accounted for 24% of new diagnoses – 3.5% of these new cases were Multi-Drug Resistant (MDR-TB) – 1.5 million deaths

Answer 216

1.1 million/9 million HIV co-infected

Answer 217

• Mycobacterium leprae – cannot be cultured • Mainly India, Nepal, Brazil, parts of Africa • Around 250,000 new cases per year • Attacks peripheral nerves

Answer 218

• Long, spiral-shaped bacteria • Not easily visualised by light microscopy • Not easily culturable • Dark ground microscopy or immunofluorescence • Often diagnosed by serology

Answer 219

Treponema pallidum = Syphilis Borrelia burgdorferi = Lyme disease Leptospira interrogans = Leptospirosis

Answer 220

• Primary syphilis: non-painful skin lesion (chancre) at the site of infection (skin or mucous membranes) • Secondary syphilis (6-8 weeks after primary symptoms): generalised systemic illness and rash • Latent phase: symptomatic episodes may occur • Tertiary syphilis (years after primary symptoms): central nervous system • Congenital syphilis: stillbirth, neonatal death or disease

Answer 221

• Lyme disease = Transmitted by ticks • Stage 1: Skin rash (erythema chronicum migrans) appears at the site of the tick bite • Stage 2: Systemic illness occurs in some patients weeks or months later when patients suffer cardiac or neurological and musculoskeletal symptoms • Stage 3: Chronic disease, occurring years later when patients present with chronic skin, nervous system or joint abnormalities • Diagnosed by clinical assessment and serology (antibody detection)

Answer 222

NOTION 7.4

Answer 223

• Virus infects cells of the respiratory tract • Destruction of respiratory epithelium – Secondary bacterial infections • Altered cytokine expression leading to fever – e.g interleukin-1 and interferon – Avian influenza virus (eg H5N1) causes a “cytokine storm”

Answer 224

- Antigenic DRIFT: Minor changes (natural mutations) in the genes of flu viruses, occurs gradually over time to generate antigenic variants - Antigenic SHIFT: Abrupt major changes in virus antigenic structure

Answer 225

- Poliomyelitis (poliovirus) - Aseptic meningitis (many enteroviruses) - Myocarditis (coxsackie B viruses) - Pancreatitis (coxsackie B viruses) - Respiratory infections (many enteroviruses)

Answer 226

NOTION 7.5

Answer 227

Herpes simplex virus – cold sores (type 1) and genital lesions (type 2)

Answer 228

Papillomaviruses – cervical carcinoma Retroviruses – lymphomas and leukaemias

Answer 229

Human disease and sickness, after eating contaminated food with either infectious or toxic agents (pathogenic microorganisms) that enter the body through the ingestion of food. May be organism or toxin it produces that causes disease.

Answer 230

Incidence of 2+ human cases of same disease linked to same source.

Answer 231

Pathogen – An agent that causes disease to its host – Most are infectious microbes • bacteria ; viruses; (fungi); (protozoans)

Answer 232

NOTION 8.1

Answer 233

1. Camplyobacter = 6000 2. Norovirus = 3000 3. Rotavirus = 1300 4. Salmonella = 1000 5. Cryptosporidium = 600 6. E Coli O157 = 250 7. Giardia = 200 8. Shigella = 100 9. Hepatitis A = 50 10. Listeria = 15 11. Yersinia enterocolitica = 5 NOTION 8.2

Answer 234

Infectious agent (living organism) in food – ability to grow and replicate in the gastrointestinal tract • may release toxins in vivo • May invade mucus and cells lining the intestine, causing inflammation – Escherichia coli O157, Salmonella, Campylobacte

Answer 235

Toxin in food – Toxin already present in food – Causative organism may grow and release toxin – Bacillus cereus, Clostridium botulinum, C. perfringens

Answer 236

Different types and severity of symptoms depend on ➢type of pathogen/toxin in food (or environment: water) ➢amount ingested (mass or volume) ➢the concentration of toxin/pathogen (infectious dose) ➢the individual’s health status Symptoms: Include one or more of: • nausea and vomiting • diarrhoea and dehydration • abdominal pain, headache • fever • paralysis

Answer 237

Main food poisoning species = S. enterica • >2000 serotypes – Enteritidis, Typhimurium • Often named after place of isolation

Answer 238

Causes gastro-enteritis – 12 to 72 hours after consumption – 4 –7 days duration – Symptoms - headaches, vomiting, diarrhoea, fever – normally resolves without intervention – death in severe cases – Control now high in UK (poultry vaccinated) – Most cases from abroad – Reduction in 2020 due to COVID travel/socialising restrictions

Answer 239

• Hosts: mammals, birds, reptiles (asymptomatic) • Transmission: contaminated food - Raw meat - Poultry (numbers decreasing due to vaccination strategy) - Eggs - Dairy products, chocolate • No human vaccines – Apart from S. typhi (vaccination available if going abroad) – Good hygienic practice advised

Answer 240

Listeria monocytogenes (Gram +ve) • Environmental bacterium: survives well in harsh conditions • Very few cases in the UK – ~10-20 in Scotland per year – increasing, esp in elderly • Low infective dose & long incubation time • Severe illness – Flu-like symptoms →septicemia, meningitis – abortion and death occurring in 20% of severe cases

Answer 241

• Association with ‘ready to eat foods’ – smoked salmon, paté, cooked meats • More common in US and Europe – different diet? e.g. pate, cold cured meats, soft cheeses • Foods often contaminated during processing from equipment – Difficult to sterilise during routine cleaning – Tolerance to and growth during refrigeration makes “Use by” dates important on refrigerated items – Bacterium killed by thorough cooking

Answer 242

• Spore forming, soil living bacteria • Found in dried foods: rice, spices, beans • Illness - self limiting – < 24 hr duration – Two toxins → different disease • Emetic form (heat stable toxin) - 1-6 hr onset - abdominal cramps, nausea, vomiting • Diarrhoeal form (heat labile toxin) - 8-16 hr onset - abdominal cramps, diarrhoea

Answer 243

• Spore forming anaerobic bacteria (Gram +ve) • Common in environment (eg soil) • Needs low salt, neutral pH and anaerobic conditions - Home canning, native meat ageing, oils

Answer 244

• Produce potent neurotoxins – Heat labile - destroyed by cooking – “The most poisonous poison”, writer Carl Lamanna (1959) – 1g of toxin could theoretically kill >1million people – Botox is prepared from botulinum toxin type A ! • Botulism – Food, infant formula, wounds – Severe symptoms of descending paralysis (face → limbs). Patient remains alert

Answer 245

• Potatoes AND C. botulinum grow in soil, • Proper cooking and storage kills spores and inhibits toxin formation. • Baking in aluminium foil creates anaerobic environment • Spores germinate during subsequent storage to produce toxin • April 1994, large outbreak in the USA, Greek restaurant • Thirty people affected; 4 required mechanical ventilation

Answer 246

• Additional Bacillus spp. - B. subtilis, subtilisin toxin, low toxicity but can be allergenic • Staphylococcus aureus - Causes mastitis so can infect dairy products - Cytolytic toxins infecting red/white blood cells • Clostridium perfringens - Spore former, four different toxins, - Meat/poultry contaminant • Vibrio spp. - Raw, undercooked shellfish

Answer 247

• Protozoan parasite – Cryptosporidium parvum • Infects many animals – diarrhoea in young livestock • Human disease – From drinking infected water (private water supplies, reservoirs) • Chlorination ineffective, install filtration, UV or ozone

Answer 248

Lyme Disease is caused by bacterium Borrelia burgdorferi. Transmitted by ticks ➢ Commonly carried by deer ➢ Parasites that feed on the blood of humans/animals. If animal infected with bacterium tick picks it up and passes it onto the next animal/human the tick bites ➢ Bacteria move through the skin, into the bloodstream and the lymphatic system

Answer 249

➢ Stage 1: Skin rash (erythema chronicum migrans) appears at the site of the tick bite (3-30 days) ➢ Most cases cured with 2- 4 weeks of oral antibiotics ➢ Stage 2: Systemic illness occurs in some patients weeks or months later – cardiac, neurological and musculoskeletal symptoms ➢ Stage 3: Chronic disease, occurring years later when patients present with chronic skin, nervous system or joint abnormalities ➢ Diagnosis: clinical assessment and (antibody detection)

Answer 250

Enteropathogenic E. coli (EPEC) = pathogenic E. coli causing disease in intestinal tract

Answer 251

➢Enterotoxigenic (ETEC) (VTEC) (STEC) Produce (vero)(shiga) toxins that stimulate the lining of the intestines causing them to secrete excessive fluid, thus producing diarrhea. ➢Enterohaemorrhagic (EHEC) Produce toxin that damages intestinal wall causing bleeding ➢Enteroinvasive (EIEC) Invade intestinal epithelial cells, produce adhesin proteins ➢Enteroaggrevative (EAEC) Cells aggregate and adhere to mucosal surface cells producing characteristic biofilms ➢Diffusely adherent (DAEC) Produce specific adhesins and aggregate

Answer 252

• Also called – STEC Shiga toxin producing E. coli – VTEC Verocytotoxin producing E. coli • ~100 VTEC strains associated with human disease • Reservoirs mainly ruminants – Transmitted through contaminated food (red meat) and direct contact with animals / faeces – Environmental cycling route to humans

Answer 253

• One or more verocytotoxin genes – vt1 & vt2 (stx1 & stx2 ) • Attachment & effacement gene (eae) – Helps organism adhere to gut cell wall - Persists and produces toxins (shiga toxins = stx) - Increased fluid into colon lumen → bloody diarrhoea - Toxin into blood stream, receptors in kidney → haemolytic uremic syndrome → kidney failure, patients need dialysis. At most severe can cause multiple organ failure and death • Distinguished by genotyping (PCR)

Answer 254

• Main EHEC serotype = E. coli O157:H7 • ~250 cases per year in Scotland • Symptoms – severe bloody diarrhoea – HUS (haemolytic uraemic syndrome = kidney failure) – Possible death • Low infective dose

Answer 255

- Exposure - After 3 days get onset of diarrhoea, abdominal cramping and tenderness - After further 3 days of exposure to shiga toxin get bloody diarrhoea - Risk of dehydration In 85% of patients haemorrhagic diarrheal symptoms resolve after seven days. Remaining 15% develop systemic infections – often haemolytic uraemic syndrome (HUS).

Answer 256

Key treatment ➢ fluid replacement, maintenance of hydration ➢ Severe HUS may require hemodialysis ➢ Avoid antibiotics as subsequent cell lysis releases more shiga toxin into circulation exacerbating risk of HUS

Answer 257

• Scout camp, May 2000 • 18 of 226 scouts infected • Where did infection come from: food, water, faeces? • Food & water = negative • Sheep, 300, grazing until day before camp • Wet weather • Mud, sheep faeces = positive

Answer 258

Testing farmer’s sheep – 12/15 ewes positive (10^1-10^4 cfu/g) - 2/13 lambs positive (10^4/10^6 cfu/g) • Estimated 10^10 E.coli O157 cells shed onto field per day → 80 E.coli O157 cells /cm2 soil - Soil survival study: viable after 15 weeks • 30-200 mg soil/day ingested for children camping • 4-24 E.coli O157 cells ingested by the scouts → disease in 18 scouts As few as 10 viable bacteria can cause disease

Answer 259

Required to link specific isolates together • Describe evolutionary relationships • Distinguish highly pathogenic and less dangerous strains • Investigate epidemiology of outbreaks

Answer 260

NOTION 8.3

Answer 261

NOTION 8.4

Answer 262

- Slow & sensitive - Type of media: - Non selective e.g Blood, chocolate - Selective e.g MacConkey - Specialised e.g Mycobacteria - Atmosphere, temperature, duration of incubation - Blood cultures used to detect bacteremia

Answer 263

Immunocapture techniques • Immunomagnetic separation (IMS) • Selects out other ‘contaminants’ in sample • Concentrates and purifies target organism either with/without prior culturing If sample has – matrix (food, soil, ..) which interferes with isolation – inhibitory substances (affect subsequent typing steps) – low concentration (affect sensitivity of subsequent typing steps) NOTION 8.5

Answer 264

Phenotyping – identifying bacterial strains based on observable characteristics. Gram stains, shape, growth, also on disease symptoms (EHEC, EIEC etc).

Answer 265

Genotyping – identifying bacterial strains based on DNA sequence set of genes within organism (molecular typing)

Answer 266

Serotyping – distinguishing related bacterial strain based on the presence of characteristic surface antigens.

Answer 267

Ultimately the most accurate. Examples of use = • Presence of virulence (vir) genes • MLVA: Multiple-locus variable number of tandem repeats analysis • MLST: Multi locus sequence typing • WGS: Whole genome sequencing

Answer 268

• Often PCR-based typing methods • Used for E. coli O157 and other toxin producing E. coli • Test for presence of specific virulence genes: - stx1, stx2 (Verotoxin/ shigatoxin) - eaeA (adhesion genes: attaching & effacing) - rfbE (O157 - Lipopolysaccharide synthesis gene)

Answer 269

• Used for a range of bacteria - E. coli O157 - Salmonella - Listeria • Genomic regions of tandem repeated motifs are sequenced: nnnnn ATTGCA ATTGCA ATTGCA nnnnn • Involves PCR amplification across repeat region • Different product lengths are produced depending on number of repeats within amplification region. Therefore you can then work out how closely related two different strains are.

Answer 270

NOTION 9.1

Answer 271

NOTION 9.2

Answer 272

Advantages: 1. Rapid 2. Direct testing of samples (soil/ food) 3. Detection of non culturable organisms 4. Multiplex many assays Disadvantages: 1. Cost (equipments & reagents) 2. Technical expertise 3. Might have inhibitors 4. May require prior enrichment to increase organism numbers 5. May identify dead cells

Answer 273

To help identify different strains of pathogens, cell surface structures that are capable of eliciting an immune responses are used = antigens.

Answer 274

Pathogenic E. coli can be distinguished based on the presence of specific lipopolysaccharide antigens (O) and flagella antigens (H). For example E. coli O157:H7 has cell surface lipopolysaccharide antigens classed as O157 and flagella antigen type H7.

Answer 275

Phage typing • Viruses that infect bacteria are called bacteriophages or phages and some of these can only infect a single strain of bacteria. • Phage specificity can thus identify different strains of bacteria within a single species. • Differentiate bacteria into a number of phage types by their pattern of infection • Identify specific bacteriophage sequences (obtained as part of bacterial whole genome sequence information)

Answer 276

In general terms • Epidemiology is the scientific study of factors affecting the health and illness of individuals and populations In microbiological terms • it involves the isolation and identification of the causative organism from both the human case(s) and the source

Answer 277

Based on assumption that strains of the causal organism isolated from different sources are related • Clonally related • Similar or identical in their phenotypic, serotypic and genotypic characteristics

Answer 278

Need to use techniques capable of discriminating closely related strains (often genotyping) • To confirm source of an outbreak • To establish routes of transmission • To determine and monitor reservoirs

Answer 279

• Outbreak cases – Isolates from the different sources under investigation are similar or identical in their phenotypic and genotypic characteristics – There is a common source of the organism for all cases – Two or more cases from separate households • Sporadic cases – Many cases are unresolved – Cases without links to other cases – But may be part of an unrecognised outbreak

Answer 280

• PCR typing of specific virulence genes - stx1- stx2+ • Phage typing - presence of specific bacteriophage - phage type 21/28 • Clinical and ruminant isolates indistinguishable by pulsed field gel electrophoresis (PFGE) genotyping

Answer 281

– 3911 cases – 22% of cases developed Haemolytic uraemic syndrome (HUS) = 70x normal rate – 800 people suffered HUS and bloody diarrhoea (patients can require kidney dialysis) – Total number of deaths = 53 – Older patients, median age 42 years – 68% women – Delay in recognising ‘outbreak’ because cases spanned different regions and healthcare documentation records in Germany – Also picked up in 17 other countries

Answer 282

• “High probability source is tomatoes, cucumbers or green salads.” Based on survey of people and commonly consumed foods/ places visited • Established that outbreak started in northern Germany • Finally linked to eating at a single restaurant - Patient memory unreliable - Partly time lapse - Partly neurological symptoms linked to EHEC/HUS • Restaurant bills and orders scrutinised However • no epidemiological chain of exposure • no microbiological serotyping • Failure to proclaim their uncertainty Further typing showed • different EHEC strain on cucumbers than in clinical cases • E. coli O8:H19 = not source of outbreak Identification of clinical isolates → E. coli O104:H4 (cell surface antigen O104, flagellin H4) • fenugreek seeds most likely link

Answer 283

A new super virulent strain • mix of EHEC and EAEC types - Enterohaemorrhagic, enteroaggrevative • Toxin positive like EHEC - stx2+ • Adherence typical of EAEC - aggregative adherence genes: aggA, aggR, aap - (plasmid encoded)

Answer 284

Profound implications for – disease detection → new detection methods – Reporting → global problems – food safety → global co-ordination – understanding of microbial pathogenesis – understanding of microbial evolution

Answer 285

NOTION 9.3

Answer 286

1. Epidemiological studies: identify the cause(s) of outbreaks 2. Case-control studies: determine the importance of particular risk factors (e.g. eating burgers) for sporadic cases 3. Spatial disease mapping: demonstrate the importance of environmental risk factors (e.g. cattle and human population densities) 4. Quantitative Microbial Risk Assessment (QMRA): determine risk of disease from a particular infection pathway

Answer 287

NOTION 9.4 - Red = linked to food - Blue = linked to environment Therefore largest numbers of cases within food outbreaks

Answer 288

Linear Regression Models of E. coli O157 incidence in rural Grampian • Predict the relationship between two variables or factors • R-squared is a statistical measure of how close the data are to the fitted regression line • R-squared is always between 0 and 1 (0 and 100%): - 0 indicates that the model explains none of the variability of the data - 1 indicates that the model explains all the variability of the data The results found that: • Cattle density/ human population density = 0.48 (R^2) • Sheep density/ human population density = 0.33 (R^2) • % human pop. with private water supply = 0.17 (R^2) NOTION 9.5

Answer 289

• Risk of disease from a particular infection pathway • Probability distributions of different events • The risk of illness = not an average but a distribution • Example: the risk of E. coli O157 infection from: – a single beef burger meal = 10-6 – a day visit to fields grazed by cattle = 10-4 • Therefore 100x more likely to be infected visiting a field recently grazed by cattle compared to eating a beef burger.

Answer 290

• Risk of cattle being colonised • Level of colonisation • Likelihood that abattoir eliminates contamination • Level in meat leaving abbatoir • Carriage of organism in butcher • Die off rate of bacterium • Probability of meat being processed into a burger • Probability of burger being under-cooked • Probability of person not having any immunity

Answer 291

• Typical human infection lasts about five days: self-limiting severe diarrhoea, abdominal cramps, fever • Commonest known cause of bacterial infectious intestinal disease in developed world - WHO estimates around 1% of population affected in developed countries every year. - Campylobacter infection causes almost half of all diarrhoea cases in UK - 10% of reported cases are hospitalised • Economic cost c.£900M in the UK

Answer 292

• The biggest cause of gastrointestinal illness - >50,000 recognized cases annually in UK - (6000 annually in Scotland) • Reservoirs - poultry - cattle and sheep - Wild birds and animals • Transmission - mainly foodborne - rarely water or environment • Gram-negative bacterium: mainly spiral-shaped, “S”-shaped, or curved, rodshaped

Answer 293

• Epidemiology study in NE Scotland Study (2005-2016) • Collect Campylobacter isolates from - Clinical cases - Foods: Chicken, turkey - Abbatoirs - Reservoirs: Cattle, sheep, pigs, wild birds • Culture purified campylobacter isolates, • Perform subsequent “genotyping” analysis • Chicken is principal source of human infection – 80% retail chicken contaminated – eating undercooked chicken – cross-contamination of other food NOTION 9.6/9.7/9.8

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