MLAB 2434 – CLINICAL MICROBIOLOGY KERI BROPHY-MARTINEZ

MLAB 2434 – CLINICAL MICROBIOLOGY KERI BROPHY-MARTINEZ

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”

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

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”

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= flagellumC=capsuleP= piliN= nuclear infoR=ribosomeCM= cytoplasmic membraneCW= cytoplasmic wall

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=MitochondriaG= GolgiNM= nuclear membraneN= nucleusNC= nucleolusRER= rough endoplasmic reticulumPM= plasma membrane

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

Archaeobacteria Resembles

eukaryotes Found in

microorganisms that grow under extreme environmental conditions

Cell wall lacks peptidoglycan

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

Bacterial Cell Wall

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

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

Gram Negative (GN) Cell WallTwo layers; outer is much thinner

than GP cell wallsOuter wall contains several

molecules, including Lipid A which is responsible for producing fever and shock in infections with GN bacteria

The Cell Wall

Gram Positive Gram Negative

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

GP cocci in clusters →

GN bacilli (rods) →

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

Acid Fast Cell Wall – mainly Mycobacteria and NocardiaHave 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”

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

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)

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

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

Examples of Flagella

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

Microscopic Shapes Cocci (spherical) Bacilli (rod-shaped) Spirochetes (helical)

Groupings Singly Pairs Clusters Chains Palisading

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

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

Size and lengthShortLongFilamentousFusiformCurvedPleomorphic

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

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

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

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

India Ink – negative stain for capsules, surrounds certain yeasts

I NEED A BREAK!!!!!

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

Microbial Growth and Nutrition NeedsSource of carbon for making

cellular constituentsSource of nitrogen for making

proteinsSource of energy (ATP) for cellular

functionsSmaller amounts of other

molecules

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

Nutritional Requirements for GrowthAutotrophs (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

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

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

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

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

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

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%)

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

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

Is It Time For a Break?

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

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

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

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 pathwayEntner-Doudoroff pathway

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)

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

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

Genetic Elements and AlterationsPlasmid

• 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.

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

Plasmid replication Three

methods• Transformatio

n• Transduction• Conjugation

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

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%20diseas

e%20introduction.htm http://realneo.us/content/yellowstone-national-park-hit-swarm-earthqua

kes Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of

Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.