Microbiology Lecture - The Microbiome - PDF

Dr Jorge Garcia-Lara MICROBIOLOGY Unit: ISCM Module: UM1010 Yr. 2023-2024 Microbiology The Gram-Stain Basic tools & notions by : LECTURE 2...

Dr Jorge Garcia-Lara MICROBIOLOGY Unit: ISCM Module: UM1010 Yr. 2023-2024 Microbiology The Gram-Stain Basic tools & notions by : LECTURE 2 INTERACTIVE Dr. GARCIA-LARA SESSION Bitesize 1 SLIDES Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 1  Dr Jorge Garcia-Lara More fundamental concepts Gram stain Fundamental Concept 1 Gram-positives (G+) Gram-negatives (G-) Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 2  Dr Jorge Garcia-Lara Under the microscope Gram-positive Gram-negative cocci Staphylococcus aureus Neisseria gonorrhoea bacilli Clostridium difficile Escherichia coli 3  Dr Jorge Garcia-Lara The bacterial cell wall : just a polymer of two sugars & small aminoacid chains N-acetyl- N-acetyl- glucosamine muramic acid cell wall cytoplasmic membrane 4  Dr Jorge Garcia-Lara Another unique bacterial feature : Gram stain, plus Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 5  Dr Jorge Garcia-Lara Another unique bacterial feature : Gram stain, plus Safranin Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 6  Dr Jorge Garcia-Lara Another unique bacterial feature : Gram stain, plus Crystal violet Safranin Crystal violet + Iodine + Iodine Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 7  Dr Jorge Garcia-Lara Another unique bacterial feature : Gram stain, plus Ethanol Safranin Crystal violet + Iodine Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 8 Dr Jorge Garcia-Lara 9  Dr Jorge Garcia-Lara Hans Christian Gram Paul Ehrlich Denmark Germany 1853-1938 1854-1915 Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 10  Dr Jorge Garcia-Lara Bacterial pathogens, just like usBacterial growth (they are heterotrophs) Fundamental Concept 2 some water liquid growth medium some sugar some protein + agar some salt some essential supplements 11 solid growth medium  Dr Jorge Garcia-Lara THEORY Conditions of bacterial growth nutrients nutrients, oxygen, pH become compromised and toxic products of growth start accumulating adapted to new environment and optimal conditions for growth, i.e.,, nutrients, pH, nutrients, oxygen, pH oxygen,... depleted and toxic products reach levels leading to cellular death adaptation to new environment 12  Dr Jorge Garcia-Lara HEORY Conditions of bacterial growth growth range for most temperature bacterial pathogens pH extreme alkalophiles alkalophiles neutrophiles acidophiles extreme acidophiles 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 acidic pH basic 13  Dr Jorge Garcia-Lara Conditions of bacterial growth oxygen strict facultative fermenter aero- aerobe anaerobe tolerant (respiration) micro- aerophile strict aero- anaerobe sensitive 14  Dr Jorge Garcia-Lara Conditions of bacterial growth oxygen Mycobacterium Staphylococcus Staphylococcus aero- tuberculosis aureus aureus tolerant (respiration) Helicobacter pilori Clostridium aero- difficile sensitive 15  Dr Jorge Garcia-Lara A vast array of growth media General (non-selective) growth medium Contains the nutrients necessary for general growth for a large variety of bacteria. It can be 'rich' or 'minimal'. Selective medium : Rich or minimal medium that contains the nutrients and supplements necessary for the growth of a selected organism/s. Differential medium : Rich or minimal medium that contains the nutrients and supplements that enable growth and differentiation of various organism/s. 16  Dr Jorge Garcia-Lara THEORY Taxonomy and Diagnosis : Gram, growth & biochemistry Diplococci CLOSTRIDIA LISTERIA HELICOBACTER STREPTOCOCCI PSEUDOMONAS HAEMOPHILUS STAPHYLOCOCCI ENTEROCOCCI ENTEROBACTERIA 17  Dr Jorge Garcia-Lara MICROBIOLOGY Unit: ISCM Module: UM1010 Yr. 2023-2024 histological vaginal smears by : LECTURE 2 CORE Dr. GARCIA-LARA SLIDES Bitesize 2 Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 18  Dr Jorge Garcia-Lara What are we made of ? 19  Dr Jorge Garcia-Lara The body houses 10x more bacteria than cells 20  Dr Jorge Garcia-Lara A few fundamental and modern concepts All concepts below refer to all the microorganisms present in a defined environment. The difference resides on the old Microflora methodology used to determine presence. Environment = new Microbiota ≠ MIcrobiome ≠ Metagenome Taxonomic classification It includes all the The collection of (culture, biochemistry microorganisms, their genomes and genes and 16s rRNA) genomes, biotic and from the members of21 abiotic factors. a microbiota  Dr Jorge Garcia-Lara Just in case it was not clear enough Please, look at the pictures and the legends carefully. Are the terms properly assigned ? Whiteside et al. 2015. The microbiome of the urinary tract—a role beyond infection. Nature Rev. Urology. 12, 81-90. 22  Dr Jorge Garcia-Lara Number of publications with these terms pancreatic cancer cancer bacteria microbiome 4,715,292 129,179 2,605,814 126,902 Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 23  Dr Jorge Garcia-Lara Activity A couple of calculations Write down your body weight in kg (paper/computer) Divide the number by 100 Multiply it by 3 Placed the digits within boxes equivalent to these below. Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 24  Dr Jorge Garcia-Lara A few facts : The microflora/microbiota is 1-3% of our Body Weight The majority appear to be bacteria Most of them are generally "harmless" Human viruses and viruses of our bacterioflora (called bacteriophages) are also there There are also eukaryotic microorganisms (fungi & protozoa) in our microbiota, but less and with less diversity than bacteria Key problems in identification & characterization through culture or molecular technologies : o moderate numbers of many microbes (particularly eukaryotes) o inability to grow o technical difficulties in molecular determinations 25 Fungi growing on agar medium  Dr Jorge Garcia-Lara MICROBIOLOGY Unit: ISCM Module: UM1010 Yr. 2023-2024 The healthy Microflora Lactobacillus spp. by : LECTURE 2 CORE Dr. GARCIA-LARA SLIDES Bitesize 3 Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 26  Dr Jorge Garcia-Lara Beneficial effects of the microbiota : maturation of the immune system, e.g., exposure of T regulatory cells to intestinal microbes decreases: reactions to self-antigens reactions to harmless antigens from nonpathogenic microbes contribute to immune development: Peyer's patches and other gut-associated lymphoid tissue epithelial cell renewal source of essential nutrients (e.g., aminoacids, propionate, short chain fatty acids (SCFAs), vit. K, vit. B12 and folic acid) aids in the extraction of energy and nutrients from food, e.g., Escherichia coli short-chain fatty acids (SCFAs) and amino acids production of antibacterial bacteriocins/colicins e.g., Escherichia coli and fatty acids maintenance of pH (e.g., acidic vagina), e.g., Lactobacillus spp. And we give them food, shelter and transport 27  Dr Jorge Garcia-Lara A sort of "healthy" bacterial flora on our body surfaces skin (physical barrier) antimicrobial peptides (e.g., cathelicidins) acid toxic fats... Staphylococcus epidermidis 28  Dr Jorge Garcia-Lara Carriage is a disease risk factor moist skin areas & higher pH (axilla, perineum, scalp, between toes) 1 in 3 in the population carry Staphylococcus aureus 29  Dr Jorge Garcia-Lara Clinical presentations (disease and system involved) Staphylococcus aureus meningitis sinusitis stye boils furuncules sepsis pneumonia endocarditis nephritis emesis scalded skin CAI syndrome diarrhea cystitis UTI TSS impetigo osteomyelitis 30  Dr Jorge Garcia-Lara The skin "normal flora" Exposed dry areas: have relatively few resident organisms e.g., Staphylococcus epidermidis Moist areas typically have: S. aureus, corynebacteria & Candida spp. Fatty areas (or with limited oxygen) the hair follicles, sebaceous glands and sweat support anaerobic organisms e.g., Propionibacterium acnes and other diphteroids Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 31  Dr Jorge Garcia-Lara MICROBIOLOGY Unit: ISCM Module: UM1010 Yr. 2023-2024 Microbiota Density & Diversity by : LECTURE 2 CORE Dr. GARCIA-LARA SLIDES Bitesize 4 Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 32  Dr Jorge Garcia-Lara Do you recognize this person ? Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 33  Dr Jorge Garcia-Lara Do Stillyou ? recognize this person ? Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 34  Dr Jorge Garcia-Lara Do Stillyou ? recognize this person ? Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 35  Dr Jorge Garcia-Lara Do Stillyou ? recognize this person ? Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 36  Dr Jorge Garcia-Lara Do Stillyou ? recognize this person ? Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 37  Dr Jorge Garcia-Lara Do you recognize this person ? What am I going to say now ? Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 38  Dr Jorge Garcia-Lara The mouth microbiota There are 20,000,000,000 bacteria in your mouth distributed in some 1000 different species which amount to some 10mg The salivary microbiome is affected by diet, genes, and environment it is in turn rather conserved across individuals some examples:Streptococcus spp., Gemella spp., Granulicatella spp., Veillonella spp. image shows an electron micrograph of the microbiota on 39 the human tongue  Dr Jorge Garcia-Lara Who is where ? Staphylococcus aureus "Staph. epidermidis" Scalp Teeth streptococci diphteroids Nose (as skin) Streptococcus mutans Staphylococcus aureus Strep. pneumoniae Pseudomonas aeruginosa Haemophilus influenzae Streptococcus pyogenes Fusobacterium Moraxella catharralis Lactobacilli Escherichia coli Klebsiella Salmonella Streptococcus faecalis Staphylococcus epidermidis Enterococcus faecalis 40  Dr Jorge Garcia-Lara Variability of GI tract microbiota proximal Lactobacillus stomach Helicobacter Veillonella duodenum Bacilli Streptococcaceae jejunum Lactobacilli Actinobacteria Actinomycetaceae ileum Corynebacteriaceae Lachnospiraceae colon Bacteroidetes distal (incl. Enterrobacteria) faeces 41  Dr Jorge Garcia-Lara Activity 30 seconds choose one organism from the preceding slide get your portable computer/mobile telephone determine if it is a gram+/gram- and other aspects of taxonomic classification (Family, Order, Class…) secret award to: – the 3 fastest delivering it – the 3 choosing the most common organisms – the 3 choosing the weirdest organisms Submission : in class Contributors : Individual/group undertaking Output : say it out loud Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 42  Dr Jorge Garcia-Lara http://huttenhowe r.sph.harvard.edu /metaphlan 43  Dr Jorge Garcia-Lara MICROBIOLOGY Unit: ISCM Module: UM1010 Yr. 2023-2024 Where does it come from ? Mother and child Mary Cassatt, 1880 by : LECTURE 2 CORE Dr. GARCIA-LARA SLIDES Bitesize 5 Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 44  Dr Jorge Garcia-Lara Acquiring our microbiota : the birth canal plus Firmicutes ~ Bacteroidetes V Proteobacteria ~ Actinobacteria ~ Fusobacteria ~ Spirochaetes Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 45  Dr Jorge Garcia-Lara Acquiring our microbiota : the birth canal plus Propionibacteriu mother baby m Lactobacillu s Staphylococcus 46  Dr Jorge Garcia-Lara Our microbiome is aging with us or … the other way around, maybe? effect of formula/ more effects with a breast feeding potential nutritional effect 47  Dr Jorge Garcia-Lara GI bacterioflora development Bacteroides Bifidobacteriaceae Clostridium Clostridiaceae Lactobacillaceae Escherichia coli = adult or not (depends in authors) obligate anae. aerobes & facultative anae. G -ve (Bacteroidetes) G +ve (Firmicutes) Bacterial diversity Interindividual variability 48  Dr Jorge Garcia-Lara Is there a geographic distribution of the microbiota ? 49  Dr Jorge Garcia-Lara Geographic microbiota distribution (per individual) G+ G- Rural Africa Europe clusters of individuals with similar microbiota De Filippo et al. 2010. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. PNAS. 107:14691–14696 50  Dr Jorge Garcia-Lara MICROBIOLOGY Unit: ISCM Module: UM1010 Yr. 2023-2024 From health to disease megacolon by : LECTURE 2 CORE Dr. GARCIA-LARA SLIDES Bitesize 6 Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 51  Dr Dr Jorge Jorge Garcia-Lara Garcia-Lara Microbiota CoNS Strep. mutans CoNS Streptococci Corynebacterium Staph. aureus Strep. salivarius Staph. aureus Staphylococci Pseudomonas Strep. viridans Strep. mitis Neisseria Neisseria Enterobacteriaceae Strep. pneumoniae Strep. oralis Strep. viridans Micrococcus Haemophilus Granulicatella Strep. pneumoniae Gemella Actinomyces Lactobacillus Propionibacterium Conynebacterium Bifidobacterium Neisseria Moraxella catharralis Veillonella Haemophilus influenzae. Haemophilus parainfuenzae otitis Prevotella Porphyromonas gingivalis pneumonia Fusobacterium nucleatum Treponema denticola meningitis Candida spp. CMV endocarditis Coxsackie Trichomonas 52 Entoamoeaba gingivalis  Dr Jorge Garcia-Lara The mycobiome (pityriasis) (candidiasis) Candida Malasezzia"it is dry here" lipase, protease callous degrading "it is enzymes wet/moist sulphur producers here for growth (and likely from AA breakdown more easy to get nutrients" Staphylococci Corynebacteri a Micrococcus Aspergillu "it is dry and (aspergillosis) s acid in here" 53  Dr Jorge Garcia-Lara Pityriasis/Tinea versicolor Mlalasezia furfur (basidiomycota, septate hyphae, conidia) typical skin microbiota scaly and discoloured skin patches sometimes itchy (pruritic) chest, abdomen, back & neck Pityriasis versicolor on the back M. furfur Electron micrograph M. furfur lactophenol cotton blue stain 54  Dr Jorge Garcia-Lara Co-presence and exclusion (do not look at it with with an absolute view) most relationships are strongly niche-specific. only a few microorganisms form links across multiple body areas closely related microorganisms co-occur in the same niche most exclusive relationships occur between more distantly related microorganisms. Streptococcus - signature bacterium in the oral cavity. Bacteroidetes – signature bacterium in the gut. Either you have Prevotella or you have Lactobacillus in the vagina. Either you have Propionibacterium or you have Staphylococcus on the skin. Either Streptococcus or Tannerella in the mouth Prevotella and Treponema like to be together in the mouth Faust, Karoline, J. Fah Sathirapongsasuti, Jacques Izard, Nicola Segata, Dirk Gevers, Jeroen Raes, and Curtis Huttenhower. ‘Microbial Co-Occurrence Relationships in the Human Microbiome’. Edited by Christos A. Ouzounis. PLoS Computational Biology 8, no. 7 (12 July 2012): e1002606. https://doi.org/10.1371/journal.pcbi.1002606. 55  Dr Jorge Garcia-Lara MICROBIOLOGY Unit: ISCM Module: UM1010 Yr. 2023-2024 Dysbiosis Clostridium difficile by : LECTURE 2 CORE Dr. GARCIA-LARA SLIDES Bitesize 7 Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 56  Dr Jorge Garcia-Lara EXAMPLE 2 Dysbiosis in disease Implication rather than conclusive causal relation in the majority of cases (ongoing research process) GI tract disorders ❑ Inflammatory Bowel Disease (IBD): ❖ the most prevalent forms are Crohn's Disease (CD) and ulcerative colitis (UC) ❖ patients exhibit decreased microbial population and functional diversity (lower Firmicutes, higher Bacteroidetes; e.g., Enterobacteriacea and Pasteurellaceae) ❑ something similar occurs with irritable bowel syndrome (IBS) and colorectal cancer (CRC) metabolic disorders: obesity (increase in Firmicutes and decrease in Bacteriodetes), type 2 diabetes CNS-related disorders: e.g., early colonisation of the intestinal tract by microbes appears to be important for the post-natal development of the enteric nervous system 57  Dr Jorge Garcia-Lara EXAMPLE 1 Dysbiosis in disease Increased incidence of Clostridium difficile infections (CDI) - enterocolitis Indication of CDIs predomimance as a Hospital Acquired Infection (HAI) also called nosocomial infections, and compared to Community Acquired Infections (CAIs) Seemingly moderate increased incidence translated into: $0.3 billion 3000 deaths 58  Dr Jorge Garcia-Lara EXAMPLE 2 Clostridium difficile Transmission electron microscopy image (TEM) vegetative cell spore differentiating cell 59  Dr Jorge Garcia-Lara The path to Clostridium difficile dysbiosis EXAMPLE 2 pseudomembranous sepsis-like mild diarrhoea death colitis picture Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 60  Dr Jorge Garcia-Lara The path to dysbiosis EXAMPLE 2 changes Intestinal commensal environment microbiota /microbiome alteration anaerobes dominant anaerobes Microbiome Microbiome C. difficile C. difficile vulnerable to leads to pathogens inflammation who are after predisposes to (barrier effect & the new niche pathologies or mucosal available e.g.: C. difficile colonization tissue damage resistance) colonization factors (toxins) CD, IBD, UC 61  Dr Jorge Garcia-Lara A solution to the a “difficile” problem EXAMPLE 2 molecular pathogenesis (top level) Fecal Microbiota Transplant mucus- exposing sialic ac. 62  Dr Jorge Garcia-Lara EXAMPLE 2 Dysbiosis in disease molecular pathogenesis (top level) Fecal Microbiota Transplant mucus- exposing sialic ac. 63  Dr Jorge Garcia-Lara EXAMPLE 2 Dysbiosis in disease molecular pathogenesis (top level) Fecal Microbiota Transplant Sufficient bacteria remain to release Sialic ac. mucus- exposing sialic ac. 64  Dr Jorge Garcia-Lara EXAMPLE 2 Dysbiosis in disease molecular pathogenesis (top level) Fecal Microbiota Transplant Sufficient bacteria remain to release Sialic ac. mucus- exposing sialic ac. 65  Dr Jorge Garcia-Lara EXAMPLE 2 Dysbiosis in disease molecular pathogenesis (top level) Fecal Microbiota Transplant mucus- exposing sialic ac. 66  Dr Jorge Garcia-Lara EXAMPLE 2 Dysbiosis in disease molecular pathogenesis (top level) Fecal Microbiota Transplant mucus- exposing sialic ac. 67  Dr Jorge Garcia-Lara EXAMPLE 3 The onset: patients & antibiotic exposure Enterococci Anti-gram- positive bacteria (piperacillin, tazobactam) lectin 68  Dr Jorge Garcia-Lara EXAMPLE 4 Small Intestinal Bacterial Overgrowth https://www.bsg.org.uk/web-education-articles-list/management-of-difficult-to-treat-small-intestinal- bacterial-overgrowth-dr-andreyev-and-dr-poon-highlight-a-case-study-on-a-difficult-case-of-sibo/ 69  Dr Jorge Garcia-Lara SIBO Diagnosis – not easy !!! EXAMPLE 4 small intestine aspirate and fluid culture (GOLD STANDARD) breath testing (hydrogen and methane) blood testing (vitamin deficiency) stool evaluation (fat malabsorption) imaging tests e.g., X-rays, CT, MRI (intestinal structural abnormalities) https://www.bsg.org.uk/web-education-articles-list/management-of-difficult-to-treat-small-intestinal- bacterial-overgrowth-dr-andreyev-and-dr-poon-highlight-a-case-study-on-a-difficult-case-of-sibo/ 70  Dr Jorge Garcia-Lara EXAMPLE 4 Small Intestinal Bacterial Overgrowth antibiotics (metronidazole and rifaximin) nutritional supplements (e.g., B12) NOT FMT !!! https://www.bsg.org.uk/web-education-articles-list/management-of-difficult-to-treat-small-intestinal- bacterial-overgrowth-dr-andreyev-and-dr-poon-highlight-a-case-study-on-a-difficult-case-of-sibo/ 71  Dr Jorge Garcia-Lara MICROBIOLOGY Unit: ISCM Module: UM1010 Yr. 2023-2024 ”superorganismic Communication” by : LECTURE 2 CORE Dr. GARCIA-LARA SLIDES Bitesize 8 Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 72  Dr Jorge Garcia-Lara Dysbiosis in disease brain gut kidney microbiome axis Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) Physician Associate Studies (MPAS) 73  Dr Jorge Garcia-Lara The axis 74  Dr Jorge Garcia-Lara The axis Environmental stress & cytokines activate HPA - cortisol production & brain function modulation Cortisol alters gut permeability & barrier Hypothalamus function - affect microbiota composition Pituitary gland Adrenal gland Microbiome affects circulating cytokines affecting brain function microbiota SCFAs (Short Chain Fatty Acids) produce: improve glucose metabolism insulin sensitivity are substrates for colonocytes, decrease pro-inflammatory cytokynes secretion modulate brain & behaviour Bacterial components stimulate the efferent vagal/spinal nerves & they can also reach the CNS 75  Dr Jorge Garcia-Lara The future of the microbiota besides progressing from the current rather phenomenological stage to a causal stage the future resides on manipulating the microbiota to maintain health and treat disease from 'blunt-end tools', like bacteriophage treatment or faecal transplantation to biotechnology approaches 76  Dr Jorge Garcia-Lara School of Medicine Questions? [email protected] @GarciaLaraClan #UCLanMicrobiology Medicinae Baccalaureus Baccalaureaus Chirurgiae (MBBS) 77 Physician Associate Studies (MPAS)