II. USING ANTIBIOTICS AND CHEMICAL AGENTS TO CONTROL BACTERIA

B. WAYS IN WHICH OUR CONTROL AGENTS AFFECT BACTERIA

Fundamental Statements for this Learning Object:

1. Many antibiotics (penicillins, cephalosporins, vancomycin, bacitracin) inhibit normal synthesis of peptidoglycan by bacteria and cause osmotic lysis. They do this by inactivating the enzymes or the transporters involved in peptidoglycan synthesis.
2. A few antimicrobial chemotherapeutic agents (INH, ethambutol) inhibit normal synthesis of the acid-fast cell wall.
3. A very few antibiotics (polymyxin, colistin, daptomycin) alter the bacterial cytoplasmic membrane causing leakage of molecules and enzymes needed for normal bacterial metabolism.
4. Some antimicrobial chemotherapeutic agents (fluoroquinolones, sulfonamides, trimethoprim) inhibit normal nucleic acid replication in bacteria.
5. Many antibiotics (tetracyclines, macrolides, oxazolidinones, streptogramins) alter bacterial ribosomes, interfering with translation of mRNA into proteins and thereby causing faulty protein synthesis.
6. There are 2 common antimicrobial modes of action for disinfectants, antiseptics, and sanitizers: damaging the lipids and/or proteins of the semipermeable cytoplasmic membrane of microorganisms resulting in leakage of cellular materials; and denaturing microbial enzymes and other proteins.
7. A number of factors which influence the antimicrobial action of disinfectants and antiseptics, including the concentration of the chemical agent, the temperature
at which the agent is being used, the kinds of microorganisms present, the number of microorganisms present, and the nature of the material bearing the microorganisms.
8. Endospore producers such as Bacillus species, Clostridium species, and acid-fast bacteria like Mycobacterium tuberculosis are harder to eliminate.

 

LEARNING OBJECTIVES FOR THIS SECTION


The basis of chemotherapeutic control of bacteria is selective toxicity (def). Selective toxicity means that the chemical being used should inhibit or kill the intended pathogen without seriously harming the host. A broad spectrum agent (def) is one generally effective against a variety of Gram-positive and Gram-negative bacteria; a narrow spectrum agent (def) generally works against just Gram-positives, Gram-negatives, or only a few bacteria. Such agents may be cidal or static in their action. A cidal (def) agent kills the organism while a static (def) agent inhibits the organism's growth long enough for body defenses to remove it. There are two categories of antimicrobial chemotherapeutic agents: antibiotics and synthetic drugs. Antibiotics (def) are metabolic products of one microorganism that inhibit or kill other microorganisms. Synthetic drugs (def) are antimicrobial drugs synthesized by chemical procedures in the laboratory. Many of today's antibiotics are now actually semisynthetic and some are even made synthetically.

We will now look at the two sides of the story with regards to controlling bacteria by means of chemicals:

1. Ways in which our Control Agents Affect Bacteria

2. Ways in which Bacteria May Resist Our Control Agents

We will now look at the various ways in which our control agents affect bacteria altering their structures or interfering with their cellular functions.


USING ANTIBIOTICS AND CHEMICAL AGENTS TO CONTROL BACTERIA

B. Ways in which Our Control Agents Affect Bacteria (see Common Antibiotics)

1. Many antibiotics inhibit normal synthesis of peptidoglycan (def) by bacteria and cause osmotic lysis.

As learned earlier, in order for bacteria to increase their size following binary fission, links in the peptidoglycan must be broken, new peptidoglycan monomers must be inserted into the growing cell wall, and the peptide cross links must be resealed.

New peptidoglycan synthesis occurs at the cell division plane by way of a collection of cell division machinery known as the divisome. The following sequence of events occur at the divisome:

1. Bacterial enzymes called autolysins:

a) Break the glycosidic bonds between the peptidoglycan monomers at the point of growth along the existing peptidoglycan; and

b) Break the peptide cross-bridges that link the rows of sugars together (see Fig. 1, steps 1-3).

2. The bactoprenols help assemble the peptidoglycan monomers, transport those monomers across the cytoplasmic membrane, and insert the monomers into existing peptidoglycan (see Fig. 2, steps 1-6)

3. Transglycosylase enzymes then insert and link new peptidoglycan monomers into the breaks in the peptidoglycan (see Fig. 3, steps 1-2.

4. Finally, transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong (see Fig. 4, steps 1-2).

 

Flash animation showing how penicillins inhibit peptidoglycan synthesis.
© Juliet V. Spencer, Stephanie K.M. Wong, authors, Licensed for use, ASM MicrobeLibrary.

Interference with this process results in the formation of a weak cell wall and osmotic lysis of the bacterium. Agents that inhibit peptidoglycan synthesis include the penicillins (penicillin G, methicillin, oxacillin, ampicillin, amoxicillin, ticarcillin, etc.), the cephalosporins (cephalothin, cefazolin, cefoxitin, cefotaxime, cefaclor, cefoperazone, cefixime, ceftriaxone, cefuroxime, etc.), the carbapenems (imipenem, metropenem), the monobactems (aztreonem), and the carbacephems (loracarbef). Penicillins, monobactams, carbapenems, and cephalosporins are known chemically as beta-lactam antibiotics because they all share a molecular structure called a beta-lactam ring (see Fig. 5). The glycopeptides (vancomycin, teichoplanin) and lipopeptides (daptomycin) also inhibit peptidoglycan synthesis.

 

Flash animation showing how penicillins inhibit peptidoglycan synthesis.
© Juliet V. Spencer, Stephanie K.M. Wong, authors, Licensed for use, ASM MicrobeLibrary.

 

Flash animation showing how vancomycin inhibit peptidoglycan synthesis.
© Juliet V. Spencer, Stephanie K.M. Wong, authors, Licensed for use, ASM MicrobeLibrary.

c. Bacitracin

Bacitracin (see Common Antibiotics) binds to the transport protein bactoprenol after it inserts a peptidoglycan monomer into the growing cell wall. It subsequently prevents the dephosphorylation of the bactoprenol after it releases the monomer it has transported across the membrane. Bactoprenol molecules that have not lost the second phosphate group cannot assemble new monomers and transport them across the cytoplasmic membrane. As a result, no new monomers are inserted into the growing cell wall. As the autolysins continue to break the peptide cross-links and new cross-links fail to form, the bacterium bursts from osmotic lysis (see Fig. 8).

 

Flash animation showing how bacitracin inhibit peptidoglycan synthesis.
© Juliet V. Spencer, Stephanie K.M. Wong, authors, Licensed for use, ASM MicrobeLibrary.

 

2. A few antimicrobial chemotherapeutic agents inhibit normal synthesis of the acid-fast cell wall

A few antimicrobial chemotherapeutic agents inhibit normal synthesis of the acid-fast cell wall of the genus Mycobacterium (see Common Antibiotics).

a. INH (isoniazid) appears to block the synthesis of mycolic acid, a key component of the  acid-fast cell wall of mycobacteria (see Fig. 9).

 b. Ethambutol interferes with the synthesis of the outer membrane of acid-fast cell walls (see Fig. 9).

 

3. A very few antibiotics alter the bacterial cytoplasmic membrane (def) causing leakage of molecules and enzymes needed for normal bacterial metabolism.

A very few antibiotics, such as polymyxins, colistins, and daptomycin (see Common Antibiotics), as well as many disinfectants (def) and antiseptics (def), such as orthophenylphenol, chlorhexidine, hexachlorophene, zephiran, alcohol, and triclosans, alter the bacterial cytoplasmic membrane (def) causing leakage of molecules and enzymes needed for normal bacterial metabolism.

a. Polymyxins and colistins act as detergents and alter membrane permeability in Gram-negative bacteria. They cannot effectively diffuse through the thick peptidoglycan layer in gram-positives.

b. Daptomycin disrupts the bacterial cytoplasmic membrane function by apparently binding to the membrane and causing rapid depolarization. This results on  a loss of membrane potential and leads  to inhibition of protein, DNA and RNA synthesis, resulting in bacterial cell death.

c. Pyrazinamide inhibits fatty acid synthesis in the membranes of Mycobacterium tuberculosis.

 

 

 

4. Some antimicrobial chemotherapeutic agents inhibit normal nucleic acid replication in bacteria (see Common Antibiotics).

a. Fluoroquinolones

Fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, gatifloxacin, etc., (see Common Antibiotics))) work by inhibiting one or more of a group of enzymes called topoisomerase (def), enzymes needed for supercoiling, replication, and separation of circular bacterial DNA (see Fig. 10). For example, DNA gyrase (topoisomerase II) catalyzes the negative supercoiling of the circular DNA found in bacteria. It is critical in bacterial DNA replication, DNA repair, transcription of DNA into RNA, and genetic recombination. Topoisomerase IV, on the other hand, is involved in the relaxation of the supercoiled circular DNA, enabling the separation of the interlinked daughter chromosomes at the end of bacterial DNA replication.

In Gram-negative bacteria, the main target for fluoroquinolones is DNA gyrase (topoisomerase II), an enzyme responsible for supercoiling of bacterial DNA during DNA replication; in Gram-positive bacteria, the primary target is topoisomerase IV, an enzyme responsible for relaxation of supercoiled circular DNA and separation of the inter-linked daughter chromosomes.

 

b. Sulfonamides

Sulfonamides (sulfamethoxazole, sulfanilamide) and diaminopyrimidines (trimethoprim) (see Common Antibiotics) block enzymes in the bacteria pathway required for the synthesis of tetrahydrofolic acid, a cofactor needed for bacteria to make the nucleotide bases thymine, guanine, uracil, and adenine (see Fig. 11).

This is done through a process called competitive antagonism whereby a drug chemically resembles a substrate in a metabolic pathway. Because of their similarity, either the drug or the substrate can bind to the substrate's enzyme. While the enzyme is bound to the drug, it is unable to bind to its natural substrate and that blocks that step in the metabolic pathway (see Fig. 12). Typically, a sulfonamide and a diaminopyrimidine are combined. Co-trimoxazole, for example, is a combination of sulfamethoxazole and trimethoprim.

Sulfonamides such as sulfamethoxazole tie up the first enzyme in the pathway, the conversion of para-aminobenzoic acid to dihydropteroic acid (see Fig. 11). Trimethoprim binds to the third enzyme in the pathway, an enzyme that is responsible for converting dihydrofolic acid to tetrahydrofolic acid (see Fig. 11). Without the tetrahydrofolic acid, the bacteria cannot synthesize DNA or RNA.

c. Metronidazole

Metronidazole (see Common Antibiotics) is a drug that is activated by the microbial proteins flavodoxin and feredoxin found in microaerophilc and anaerobic bacteria and certain protozoans. Once activated, the metronidazole puts nicks in the microbial DNA strands.

 

d. Rifampin

Rifampin (rifamycin) (see Common Antibiotics) blocks transcription (def) by inhibiting bacterial RNA polymerase, the enzyme responsible for transcription of DNA to mRNA.

 

 

5. Many antibiotics alter bacterial ribosomes (def), interfering with translation (def) of mRNA into proteins and thereby causing faulty protein synthesis (see Common Antibiotics).

To learn more detail about the specific steps involved in translation during bacterial protein synthesis, see the animation that follows. Protein synthesis is discussed in greater detail in Unit 6.

a. Aminoglycosides

The aminoglycosides (streptomycin, neomycin, netilmicin, tobramycin, gentamicin, amikacin, etc. (see Common Antibiotics)) bind irreversibly to the 16S rRNA in the 30S subunit of bacterial ribosomes. Although the exact mechanism of action is still uncertain, there is evidence that some prevent the transfer of the peptidyl tRNA from the A-site to the P-site, thus preventing the elongation of the polypeptide chain. Some aminoglycosides also appear to interfere with the proofreading process that helps assure the accuracy of translation (see Fig. 13). Possibly the antibiotics reduce the rejection rate for tRNAs that are near matches for the codon. This leads to misreading of the codons or premature termination of protein synthesis (see Fig. 14). Aminoglycosides may also interfere directly or indirectly with the function of the bacterial cytoplasmic membrane. Because of their toxicity, aminoglycosides are generally used only when other first line antibiotics are not effective.

 

For a more detailed description of any specific antimicrobial agent, see the website of RxList - The Internet Drug Index.

 

6. Modes of action for disinfectants, antiseptics, and sanitizers

 

Concept map for How Antibiotics and Chemical Agents Affect Bacterial Structures and Function:
Agents that Alter Prokaryotic Ribosomal Subunits,
Inhibit RNA Polymerase, and Denature Enzymes

 

TPS Question

 

 

 

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