Bacterial Growth and Genetics PDF

Bacterial Growth and Genetics Hongwei Yu, PhD Department of Biomedical Sciences [email protected]  Learning Objectives Describe different types of bacterial stains and morphologies Know bacterial growth curve, growth environments, and major metabolisms and pro...

Bacterial Growth and Genetics Hongwei Yu, PhD Department of Biomedical Sciences [email protected]  Learning Objectives Describe different types of bacterial stains and morphologies Know bacterial growth curve, growth environments, and major metabolisms and products Know the SOD and catalase and their function Know bacterial growth niches inside humans Know the mechanisms and differences of gene transfer in bacteria  Bacterial Stains Simple: Methylene blue, safranin, and crystal violet Methylene blue stain Fix preparation of bacteria on a glass slide Stain with the dye Wash away Stain remain behind to show bacteria Number and Shape Complex: Gram stain, Giemsa, Ziehl- Neelsen, Silver, and India ink Bacterial shape and colony morphology  Gram Stain Staph aureus Purple: Gram positive Retain crystal violet Red: Gram negative Do not retain crystal violet in cell walls Take up safranin counter stain Thick cell wall of peptidoglycan in gram positive make them purple Pseudomonas aeruginosa  Gram Stain Limitations Some bacteria do not have cell walls, do not gram stain well Treponema (syphilis ), too thin to see Mycobacteria (TB), mycolic acids in cell wall Mycoplasma, no cell wall Intracellular bacteria Rickettsia (obligate intracellular) Chlamydia (obligate intracellular, no NAM) Legionella (mostly intracellular)  Giemasa Stain Mixture of methylene blue, eosin, and Azure B Enters cells and stains nucleic acids Used for blood smear, marrow Protozoa Plasmodium Trypanosomes Intracellular bacteria Chlamydia Rickettsia Borrelia (sometimes intracellular) Wright-Giemsa Stain  Ziehl-Neelsen Stain (carbo fuchsin) “Acid fast” stain Contain carbofuschsin Used to detect mycobacterium (especially TB) Also used for Nocardia, stain mycolic acid in cell wall Protozoa (e.g Cryptosproridium oocysts)  Silver Stain Special stain for 3 organisms Pneumocystis pneumonia (HIV/AIDS) Fungal infections Diffuse interstitial pneumonia Legionella Interstitial pneumonia Contaminate water (outbreaks in nursing home) H. pylori Gastric ulcers Direct microscopy of Pneumocystis pneumonia. A. Transbronchial lung biopsy stained with hematoxylin and eosin shows eosinophilic alveolar filling. B. Methenamine silver–stained bronchoalveolar lavage (BAL) fluid. C. Giemsa-stained BAL fluid. D. Immunofluorescent stain of BAL fluid.  India Ink Stain Negative stain Background stained, not bug Unstained organisms stand out in contrast Primarily used for fungus Cryptococcus neoformans Large polysaccharide capsule creates “halos”  Pigments Some bacteria produce special colors Staph aureus Golden, yellow pigment Pseudomonas aeruginosa Blue-green pigment (pyocyanin) Serratia Red pigment Actinomyces Filamentous bacteria Colonies have yellow-orange appearance Known as “sulfur granules” Bacterial Growth Curve  Bacterial Growth Environments Obligate anaerobes, only use fermentation, sugar to acids, make less ATPs Obligate aerobes, only use respiration, require oxygen, makes ATPs Facultative anaerobes, use both respiration and fermentation, E. coli , Strep and Staph Intracellular bacteria  Superoxide Dismutase (SOD) and Catalase Enzymes of aerobic bacteria Superoxide radical (O2-) produced by bacterial metabolism (e- donated by NADPH. NADP converted back to NADPH through HMP shunt) CAT NADPH OX SOD CAT SOD converts O2- to O2 or to H2O2 Catalase converts H2O2 to oxygen and water Needs these enzymes to survive in oxygen environment Reactive oxygen species (ROS) are O2- , H2O2, and ·OH (oxidative stress or Myeloperoxidase (MPO) damage) Neutrophil Anaerobes lack SOD and/or catalase. HOCL Obligate Aerobes Use oxygen to generate ATP Oxygen is final electron acceptor during respiration. Respiration equals to electron transport chain (ETS). Contain SOD and catalase Key Bacteria Pseudomonas aeruginosa (but can use nitrate as electron acceptor via respiration, anaerobe) Mycobacterium tuberculosis Nocardia (opportunistic infections)  Obligate Anaerobes Fermentation Pathways of Different Bacteria Use fermentation (No oxygen) Lack catalase and/or SOD, susceptible to ROS Pyruvate has to re-generate NAD as electron acceptor. Byproducts are gases like CO2 or H2 Produce short chain fatty acids (SCFA), Facultative acetic acids, propionic acid, butyric and isobutyric acids, “foul smell”. Often present in abscesses (dental). Live near mucosal surfaces Key Bacteria: Actinomyces, Bacterioides, Obligate Clostridium, Fusobacterium Facultative  Key Anaerobic Infections n Abdominal abscesses/perorations (abscess pocket) n Contain many gram negative flora of GI tract n Also contain Bacteroides fragilis (anaerobe) n B. fragilis resistant to many antibiotics n Treatment: below diaphragm: Metronidazole (Flagyl) + Gram- agent (Cipro or quinolone) n Aspiration pneumonia n Mouth anaerobe enters lungs n Peptostreptococcus, Fusobacterium, Prevotella n Treatment: above diaphragm, Clindamycin  Facultative Anaerobes n Can live without oxygen but use it if available n Perform respiration and fermentation n Pasteur effect: Oxygen inhibits fermentation n Many common bacteria fall in this category. n Staph n Strep n E. coli  Aerotolerant Anaerobes n Similar to facultative anaerobe n Always use fermentation even in presence of oxygen n Rare n Most aerotolerant anaerobes have superoxide dismutase and (non-catalase) peroxidase but don't have catalase. They can protect themselves from ROS. n Few clinical examples, Cutibacterium acnes.  Obligate intracellular bacteria n Rickettsia Cannot synthesize their own ATP, depend on host for ATP. n Orientia Will not gram stain well (inside other cells) n Ehrlichia Difficult to grow (need cell culture) n Anaplasma Rickettsia Rocky Mountain Spotted Fever n Coxiella Diagnosed clinically or with serology (antibody test) n Chlamydia Chlamydia Diagnosis: Nucleic acid amplification testing n Chlamydophila (DNA testing) Facultative intracellular bacteria n Mycobacterium (macrophages) n Legionella (macrophages) n Salmonella (intestinal cells) n Shigella (intestinal cells) n Neisseria (urethral epithelial cells) n Listeria (monocytes, macrophages) n Brucella (macrophages and neutrophils) n Francisella (macrophages) n Yersinia pestis (macrophages)  Bacterial Gene Transfer Bacteria often transfer genetic material Key for evolution of antibiotic resistance Four major mechanisms Transformation Conjugation Transduction Transposition  Transformation Direct uptake DNA from surrounding environment Allows for evolution of DNA over time Useful technique in micro lab Introduce genes to bacteria Streptococci (all strains) Hemophilus influenzae type b Neisseria gonorrhea Helicobacter pylori Adding DNase degrades naked DNA, preventing transformation.  Conjugation the transfer of DNA from a bacterial cell (donor) to another (recipient) via pilus. Cell to cell contact mediated by F pilus. Cells carrying the plasmid are designated F+. Cells lacking the F factor are the recipients of DNA and are designated F- Require physical contact of two organisms DNA transfer as a single strand via plasmids  High Frequency Strains F plasmid can incorporate into chromosome, termed high frequency recombination (Hfr) strains Used to map genes Process take time Can interrupt at various time intervals See which genetic materials transferred Plasmid site is origin of genetic transfer Initial materials transferred is the closest to the plasmid With multiple experiments can make a map  Transduction Transfer of DNA via bacteriophage that infect bacteria Virus picks up DNA, transfers to another bacteria Lytic vs lysogenic phages. Generalized vs specialized transduction Generalized: virus randomly pick up host DNA when packaging into a virion, then transferred to another bacteria Specialized: Transfer of specific genes, virus always inserts to host DNA at the same site (lysogeny). When excised, packaged into virus with nearby specific host DNA. Phages that replicate only via lytic cycle: virulent Phages that does both lysis and incorporate host DNA: temperate  Lytic vs Lysogenic Phages Transduction happens in two ways: Lytic cycle: nuclear materials enters bacteria, multiplies and lysis bacterial cells, release progeny virtues. Lysogenic: nuclear materials enters bacteria, incorporate into host DNA, may later become excised (enters lytic cycle). Phages that replicate only via lytic cycle: virulent Phages that does both lysis and incorporate host DNA: temperate  Lysogeny Genes for some bacterial toxins are transferred to non- toxic strains via lysogeny Examples: Diphtheria toxin Erythrogenic toxins (S. pyogenes; Scarlet fever) Shiga-like toxin (EHEC) Cholera toxin Botulinum toxin  Transposition Transposons are DNA segments within bacterial DNA Can be excised and re-integrated in new location in DNA Once excised, can also be moved to plasmid Mechanism of transfer of resistance to antibiotics Bacteria #1 is resistant Transposon carries resistance gene Transposon moved to plasmid which then transfer to other bacteria (VRE)  Antibiotic Resistance Transfer between VRE and MRSA Enterococcus faecalis (VRE) Nosocomial UTI infections Vancomycin resistance is common Staphylococcus aureus (MRSA subtype) Boils, SSSS, endocarditis MRSA MRSA+VRE (double resistance)