5.4: Antibiotics

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Antibiotics

substances produced by one microorganism which inhibits or kills other microorganisms

Bacterial structures targeted by antibiotics: source: Christopher Walsh and Michael Fischbauch. Scientific American July 2009

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Additional targets of antibiotics/antimicrobials used to inhibit bacteria

(source: Bauman’s Microbiology)

Cell membrane: polymyxin

RNA polymerase/cell transcription: rifamycin

Gram stain and different types of bacterial cell walls

History of the Gram stain: Hans Christian Gram was a Danish pathologist in the 1800’s. A pathologist tries to determine why humans die by examining the body, tissues, cells of the dead person. Gram was aware bacterial infections could kill people however since bacteria are so small and are usually colorless, Gram found it very difficult to identify bacteria in tissues from the dead. To aid his ability to visualize bacteria from pathology samples, he attempted to develop a staining technique which would stain bacteria one color and human cells a different contrasting color. He developed an early version of the Gram Stain as described below

cell samples were spread over and attached to a glass microscope slide, usually by gentle, quick heating (heat fixing)
The cells were covered by a few drops of crystal violet, then a few drops of Gram’s iodine. The Gram’s iodine acts as a mordant, it complexes with the crystal violet forming large purple aggregates
The cells were then destained/decolorized by rinsing with a lipid/membrane solvent e.g. acetone-alcohol. Only cells with thick cell walls could trap the large crystal violet-iodine complexes inside. Cells which lacked thick cell walls could not hold the purple aggregates inside , thus were “decolorized” (appeared colorless once more)

Gram’s observations. When Gram used different bacteria in his staining procedure, he noted ther were 2 possible outcomes. Some bacteria would stain purple (he called these “positive” hence “Gram-postive cells”), some bacteria lost the purple stain, were colorless, thus “negative” (hence “Gram-negative”). Because the negative cells were so hard to see, a second staining step was added using a stain of contrasting, red color (safrainin). Results of this modified Gram stain thus were:

Gram-positive cells=purple

Gram-negative cells= pink/red

It wasn’t until the 20^th century and development of electron microscopes that the structural basis for differences in Gram staining were understood. Differences in staining are related to differences in bacterial cell wall structures.

Today we describe 3 types of bacterial cell walls based on their Gram- staining behaviors:

some bacteria are stained purple=Gram-positive bacteria
some bacteria are stained pink/red=Gram-negative bacteria
some bacteria are poorly or not stained= Acid-fast bacteria
Reason for differences in staining is because of differences in cell wall structure

Gram stains an bacterial cell walls, continued

1.Gram positive cell walls are characterized by a thick layer of peptidoglycan (the thick layers trap the Gram stain crystal violet: iodine complex which causes the bacteria to appear purple following gram staining). They lack a water repellant outer layer and hence have no protection against toxic chemicals. Examples of bacteria with Gram-positive cell walls: Staphylococcus, Streptococcus, Bacillus, Clostridium, Listeria

2.Gram-negative cell walls have a thin layer of peptidoglycan and a “water-repellant” outer membrane. Because of the thin layer of peptidoglycan, gram-negative bacteria are easily decolorized with lipid-solvents (alcohol/acetone) during the Gram stain procedure and lose the purple crystal violet:iodine complex. Subsequently they are counter-stained with safranin, appearing pink/red . ex E.coli, Salmonella, Yersinia, Vibrio

Endotoxin.: The outer membrane is lipid-rich and protects the bacterium against many toxic substances (e.g. some antibiotics such as penicillin). The outer membrane also contains toxic lipopolysaccharide or LPS, also called “endotoxin”

-when LPS is released as gram-negative bacteria die, the LPS/endotoxin causes

massive cytokine release from host leukocytes.

-This massive cytokine release causes:

- a drop in blood pressure /hypotension

- disseminated blood clotting called “DIC” for “Disseminated Intravascular Coagulation”

-fever.

-As blood pressure drops, blood flow to organs is decreased which may lead to “multiple organ system failure” and endotoxic shock . Death may follow.

3.Acid-fast cell walls: moderate layer of peptidoglycan covered by water-repellant “waxy” layer. Bacteria with acid0fast cell walls are often called “AFB” acid-fast bacteria.

AFB’s are very difficult to stain (hence special acid-fast stain), very slow growing, resistant to many antibiotics/antiseptics/disinfectants, requires months of antimicrobial therapy, resistant to drying in environment, difficult for host phagocytic cells to destroy.

Ex Mycobacterium tuberculosis, M. leprae

Footnote: Comparison of cell walls: cell walls are strong layers outside the cytoplasmic membrane which help protect cells from bursting (a process called “osmotic lysis”) and which provide specific shapes to cells. Cell walls are made of different components.

organism	important component of cell wall	notes
bacteria	peptidoglycan	-target of bet-lactam antibiotics ex penicillin, ampicillin -target of cephalosporins -target of vancomycin
fungi	chitin
plants	cellulose

It is important to note that animal cells (such as human cells) lack cell walls.

Contributors

Karen Carberry-Goh, D.V.M., Ph.D. (Sacramento City College)