MLAB 2434 – CLINICAL MICROBIOLOGY KERI BROPHY-MARTINEZ

1. MLAB 2434 – CLINICAL MICROBIOLOGY KERI BROPHY-MARTINEZ

2. Bacterial Cell Structure, Physiology, Metabolism, & Genetics • Taxonomy • Defined as the orderly classification & grouping of organisms into categories • Based on genotype and phenotype • Kingdom, Division, Class, Order, Family, Tribe, Genus and Species ( these are the formal levels of classification) • Family = “Clan”; has “–aceae” ending • Genus = “Human last name” • Species = “Human first name”

3. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Nomenclature • Family names: Capitalized with “aceae” endings • Genus names: Capitalized • When in print, genus and species are italicized. (Staphylococcus aureus) • When written, genus and species are underlined. (Staphylococcus aureus) • Species names • Staphylococcus sp. is used when referring to the genus as a whole when the species is not identified. • Can be referred to as singular or plural • “sp.” – singular (Staphylococcus sp.) • “spp.” – plural (Staphylococcus spp.) • Abbreviations • First letter of the genus, followed by a period and the species epithet • Ex: Staphylococcus aureus changes to S. aureus

4. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Bacteria Identification – test each bacterial culture for a variety of metabolic characteristics and compare the results with known results. • All organisms are either “prokaryotes”, “eukaryotes”, or “archaeobacteria”

5. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • PROKARYOTES - bacteria • Do not have a membrane-bound nucleus • DNA is a single circular chromosome • Have both cell (plasma) membrane AND cell wall. F= flagellum C=capsule P= pili N= nuclear info R=ribosome CM= cytoplasmic membrane CW= cytoplasmic wall

6. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • EUKARYOTES - fungi, algae, protozoa, animal cells, and plant cells • Cells have nuclei that contains DNA and are complex • Most cells do NOT have a cell wall V=Vesicle M=Mitochondria G= Golgi NM= nuclear membrane N= nucleus NC= nucleolus RER= rough endoplasmic reticulum PM= plasma membrane

7. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Archaeobacteria • Resembles eukaryotes • Found in microorganisms that grow under extreme environmental conditions • Cell wall lacks peptidoglycan

8. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Bacterial Cell Wall

10. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Gram Positive (GP) Cell Wall • Very thick protective peptidoglycan layer • Many GP antibiotics act by preventing synthesis of peptidoglycan • Consists of cross-linked chains of glycan • Also contain teichoic acid and lipoteichoic acid these unique structures makes these bacteria GP

11. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Gram Negative (GN) Cell Wall • Two layers; outer is much thinner than GP cell walls • Outer wall contains several molecules, including Lipid A which is responsible for producing fever and shock in infections with GN bacteria

12. The Cell Wall Gram Stain Gram Positive Gram Negative

13. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • GP cocci in clusters → • GN bacilli (rods) →

14. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Acid Fast Cell Wall – mainly Mycobacteria and Nocardia • Have a GP cell wall structure but also a waxy layer of glycolipids and fatty acids (mycolic acid) • Waxy layer makes them difficult to gram stain • Cannot be decolorized by acid-alcohol, hence the name “acid fast”

15. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Absence of Cell Wall – mainly Mycoplasma and Ureaplasma • Lack of cell wall results in a variety of shapes microscopically • Contain sterols in cell membrane

16. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Some bacteria produce a capsule • Protect the bacteria from phagocytosis • Capsule usually does not stain, but can appear as a clear area (halo-like)

17. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Some bacteria produce slime layers • Made of polysaccharides • Inhibit phagocytosis • Aid in adherence to host tissue or implants

18. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Cell Appendages • Flagella – exterior protein filaments that rotate and cause bacteria to be motile • Polar • Extend from one end • Can occur singly or in multiple tufts • Peritrichous • Flagella found on all sides of bacteria • Pili (fimbriae) – hairlike projections that aid in attachment to surfaces

19. Examples of Flagella

20. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Microscopic Shapes • Cocci (spherical) • Bacilli (rod-shaped) • Spirochetes (helical) • Groupings • Singly • Pairs • Clusters • Chains • Palisading

21. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)

22. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Size and length • Short • Long • Filamentous • Fusiform • Curved • Pleomorphic

23. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Common Bacterial Stains • Gram Stain: • cell wall structure determines the staining characteristics • Procedure to be covered in lab • Acid-fast • stains bacteria with high lipid and wax content in their cell walls

24. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Acridine Orange • stains nucleic acid of both G+ and G- bacteria, either living or dead • used to locate bacteria in blood cultures and other specimens where background material obscures gram stains

25. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Methylene Blue • stain for Corynebacterium diphtheriae to show metachromatic granules and as counter-stain in acid-fast stain procedures • Lactophenol Cotton Blue • fungal stain • Calcofluor White • fungal stain

26. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • India Ink – negative stain for capsules, surrounds certain yeasts

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28. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Microbial Growth and Nutrition Needs • Source of carbon for making cellular constituents • Source of nitrogen for making proteins • Source of energy (ATP) for cellular functions • Smaller amounts of other molecules

29. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Nutritional Requirements for Growth • Autotrophs (lithotrophs) • Able to grow simply, using only CO2, water and inorganic salts • Obtain energy via photosynthesis or oxidation of inorganic compounds • Occur in nature and do not normally cause disease

30. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Heterotrophic • Require more complex substances for growth • Require an organic source of carbon and obtain energy by oxidizing or fermenting organic substances • All human bacteria fall in this category • Within this group, nutritional needs vary greatly

31. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Types of Growth/Culture Media • Minimal medium – simple; not usually used in diagnostic clinical microbiology • Nutrient medium – made of extracts of meat or soy beans • Enriched medium – nutrient medium with extra growth factors, such as blood which encourages small numbers of organisms to flourish • Broths- used to detect small numbers of aerobes, anaerobes and microaerophiles

32. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Nonselective medium- supports growth of most nonfastidious microbes. • Selective medium – contains additives that inhibit the growth of some bacteria while allowing others to grow • Differential medium – contains additives that allow visualization of metabolic differences in bacteria • Transport medium – holding medium to preserve those bacteria present but does not allow multiplication

33. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Selection of primary culture media will vary from lab to lab • Selection of primary media will depend on anatomical site

34. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Environmental Factors Influencing Growth • pH – most media is between 7.0 and 7.5 • Temperature – most pathogens grow at body temperature; grown at 35° C in the lab • Psychrophiles: cold temperatures • 10-20o C • Mesophiles moderate temperatures • 20-40 o C • Thermophiles: high temperatures • 50-60 o C

35. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Gaseous composition • Obligate aerobes – require oxygen • Obligate anaerobes – cannot grow in the presence of oxygen • Facultative anaerobes – can grow with or without oxygen • Microaerophilic- grow better in low oxygen environments ( about 20%) • Aerotolerant anaerobes- grows better in the absence of oxygen • Capnophilic – grow better with extra CO2 (5-10%)

36. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Bacterial Growth • Reproduce by binary fission • Can be fast (as little as 20 minutes for E. coli or slow as 24 hours for M. tuberculosis

37. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Determination of Numbers • Direct counting under microscope • Estimates number of live and dead cells • Direct plate count • Determines the number of CFU (colony-forming units) in broth cultures and urine cultures • Density measurement • Useful to prepare inoculums for antimicrobial susceptibility testing

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39. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Bacterial Biochemistry and Metabolism • Metabolic reactions cause production of energy in form of ATP • Identification systems analyze unknown specimens for: • Utilization of variety of substances as a source of carbon • Production of specific end products from various substrates • Production of acid or alkaline pH in the test medium

40. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Fermentation and Respiration (Oxidation) • Fermentation • Anaerobic process in obligate and facultative anaerobes • The electron acceptor is an organic compound • Does NOT require oxygen

41. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Respiration (Oxidation) • More efficient energy-generating process • Molecular oxygen is the final electron acceptor • Aerobic process in obligate aerobes and facultative anaerobes

42. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Metabolic Pathways • Embden-Meyerhoff-Parnas • Primary cycle for bacteria • Convert glucose to pyruvic acid, a key intermediate • Generates energy in the form of ATP • Pentose Phosphate pathway • Entner-Doudoroff pathway

43. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • From pyruvic acid: • Alcoholic fermentation (ethanol) • Homolactic fermentation (lactic acid) • Heterolactic fermentation (lactic acid, CO2, alcohols, formic and acetic acids • Propionic acid (propionic acid) • Mixed acid fermentation (lactic, acetic, succinic, and formic) • Butanediol fermentation(acetoin and 2,3 butanediol) • Butyric acid fermentation (butyric)

44. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Main oxidative pathway is the Krebs Cycle, resulting in acid and CO2 • Carbohydrate Utilization & Lactose Fermentation • “Sugars” = carbohydrates • Lactose fermentation – key component in identification schemes • Lactose is converted to glucose, so ALL lactose fermenters also ferment glucose

45. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Genetic Elements and Alterations • Plasmid • Extra piece of DNA • Code for antibiotic resistance and other virulence factors are often found on plasmids • Sometimes passed from one bacterial species to another. This is how resistance is acquired.

46. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Plasmid replication • Three methods • Transformation • Transduction • Conjugation

47. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) • Mutations • “They don’t always read the book” • Changes that occur in the DNA code • Results in changes in the coded protein or in the prevention of its synthesis

48. References • Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams and Wilkins. • http://andyannie.pbworks.com/w/page/5454436/Reproduction • http://animals.howstuffworks.com/fish/eels-slippery1.htm • http://fanaticstars.wordpress.com/2009/04/01/on-break/ • http://www.istockphoto.com/stock-illustration-82213-coffee-break.php • http://pathmicro.med.sc.edu/infectious%20disease/infectious%20disease%20introduction.htm • http://realneo.us/content/yellowstone-national-park-hit-swarm-earthquakes • Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.