II. USING ANTIBIOTICS AND CHEMICAL AGENTS TO CONTROL BACTERIA
C. WAYS IN WHICH BACTERIA MAY RESIST OUR CONTROL AGENTS
The overall purpose of this Learning Object is:
1) to learn mechanisms bacteria use to resist our antibacterial control agents; and
2) to introduce a variety of bacteria that are frequently resistant to our antibacterial control agents.
LEARNING OBJECTIVES FOR THIS SECTION
We will now look at the two sides of the story with regards to controlling bacteria by means of chemicals:
1. Ways in which Control Agents Affect Bacterial Structures or Function
2. Ways in which Bacteria May Resist Our Control Agents
We will now look at the various ways in which bacteria become resistant to our control agents.
USING ANTIBIOTICS AND CHEMICAL AGENTS TO CONTROL BACTERIA
C. Ways in which Bacteria May Resist Our Control Agents
Some opportunistic pathogens, such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Enterococcus species, have low-permeability membrane barriers and are thereby intrinsically resistant to many antibiotics. Most bacteria, however, become resistant to antibiotics by way of one or more of the following mechanisms coded for by genes in the nucleoid (def) or in plasmids (def):
1. Producing an enzyme capable of destroying or inactivating the antibiotic;
2. Altering the target site receptor for the antibiotic to reduce or block its binding; and
3. Preventing the entry of the antibiotic into the bacterium and/or actively transporting the antibiotic out of the bacterium.
4. Modulating gene expression to produce more of the bacterial enzyme that is being tied up or altered by the antibiotic.
We will now look at each of these mechanisms of resistance.
a. Producing enzymes that destroy or inactivate the antibiotic (see Fig. 2).
As an example, penicillins, monobactams, carbapenems, and cephalosporins are known chemically as beta-lactam antibiotics (see Fig. 2A) and many bacteria become resistant to these antibiotics by producing various beta-lactamases that are able to inactivate some forms of these drugs. Beta-lactamases break the beta-lactam ring of the antibiotic, thus destroying the drug. (Penicillinase is a beta-lactamase that inactivates certain penicillins.)
To overcome this mechanism of resistance, sometimes beta-lactam antibiotics such as amoxicillin, ticarcillin, imipenem, or ampicillin are combined with beta-lactamase inhibitors such as clavulanate, tazobactam, or sulbactam (see Fig. 1) - chemicals that resemble beta-lactam antibiotic (see Fig. 2A). These agents bind to the bacterial beta-lactamases and neutralize them . Bacteria may become resistant to aminoglycosides (streptomycin, neomycin, netilmicin, tobramycin, gentamicin, amikacin, etc.) by enzymatically adding new chemical groups to these antibiotics, thus inactivating the drug.
b. Altering the target site receptor for the antibiotic in the bacterium to reduce or block its binding.
For example, bacteria may become resistant to to macrolides (erythromycin, azithromycin, clarithromycin, dirithromycin, troleandomycin, etc.) by producing a slightly altered 50S ribosomal subunit that still functions but to which the antibiotic can no longer bind (see Fig. 3A). Bacteria may become resistant to beta-lactam antibiotics (penicillins, monobactams, carbapenems, and cephalosporins) by producing altered transpeptidases (penicillin-binding proteins) with greatly reduced affinity for the binding of beta-lactam antibiotics. Bacteria may become resistant to vancomycin by producing altered cross-linking peptides in the peptidoglycan to which the antibiotic no longer bonds. Bacteria may become resistant to fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, etc.) by producing altered DNA gyrase or topoisomerases (see Fig. 3B).
c. Altering the membranes and transport systems to prevent the entry of the antibiotic into the bacterium and/or actively transport the antibiotic out of the bacterium.
By altering porins (def) (see Fig. 4) in the outer membrane of a gram-negative bacterium or by altering carrier (transport) proteins (def) (see Fig. 5) used to transport the drug through a bacterium's cytoplasmic membrane, the bacterium may block entry of the drug. In addition, the bacterium may produce transporter molecules in the cytoplasmic membrane capable of an energy-driven efflux that pumps the antibiotic back out of the bacterium (see Fig. 6).
d. Modulating gene expression to produce more of the bacterial enzyme that is being tied up or altered by the antibiotic.
Genetic control of enzyme activity refers to controlling transcription of the mRNA needed for an enzyme's synthesis. In prokaryotic cells, this involves the induction or repression of enzyme synthesis by regulatory proteins that can bind to DNA and either block or enhance the function of RNA polymerase, the enzyme required for transcription.
Bacteria also use translational control of enzyme synthesis. In this case, the bacteria produce antisense RNA that is complementary to the mRNA coding for the enzyme. When the antisense RNA binds to the mRNA by complementary base pairing, the mRNA cannot be translated into protein and the enzyme is not made. (See Fig. 11).
Mutations or genetic recomination may result in a modulation of gene expression or translational events that favors increased production of the enzyme being tied up or altered by the antimicrobial agent (see Fig. 12). Since enzymes are normally produced in limited amounts, production of excessive amounts of enzyme may allow for the metabolic activity being blocked by the agent to still occur.
Exposure to antibiotics doesn't cause bacteria to become drug resistant. The above changes in the bacterium that enable it to resist the antibiotic occur naturally as a result of mutation (def) or as a result of genetic recombination (def).
For example, when under stress from antibiotics, some bacteria switch on genes whose protein products can increase the mutation rate within the bacterium 10,000 times as fast as the mutation rate that occurs during normal binary fission. This causes a sort of hyperevolution where mutation acts as a self defense mechanism for the bacterium by increasing the chance of forming an antibiotic-resistant mutant.
In addition, genetic recombination as a result of transformation, transduction, and conjugation can transfer antibiotic resistance from one bacterium to another.
Exposure to the antibiotic selects for strains of the organism that have become resistant through these natural processes. Misuse of antibiotics, such as prescribing them for nonbacterial infections (colds, influenza, most upper respiratory infections, etc.) or prescribing the "newest" antibiotic on the market when older brands may still be as effective simply inceases the rate at which this natural selection for resistance occurs. According to the Centers for Disease Control and Prevention, as many as one-third (50 million out of 150 million) of antibiotic prescriptions given on an outpatient basis are unneeded. Patient noncompliance with antimicrobial therapy, namely, not taking the prescribed amount of the antibiotic at the proper intervals for the appropriate length of time, also plays a role in selecting for resistant strains of bacteria.
The spread of antibiotic resistance in pathogenic bacteria is due to both direct selection and indirect selection. Direct selection refers to the selection of antibiotic resistant pathogens at the site of infection. Indirect selection is the selection of antibiotic-resistant normal floras within an individual anytime an antibiotic is given. At a later date, these resistant normal floras may transfer resistance genes to pathogens that enter the body. In addition, these resistant normal flora may be transmitted from person to person through such means as the fecal-oral route or through respiratory secretions.
As an example, many gram-negative bacteria possess R (Resistance) plasmids (def) that have genes (def) coding for multiple antibiotic resistance through the mechanisms stated above, as well as transfer genes coding for a conjugation (sex) pilus (see Figs. 7-10). It is possible for R-plasmids to accumulate transposons (def) to increase bacterial resistance. Such an organism can conjugate with other bacteria and transfer to them an R plasmid. E. coli, Proteus, Serratia, Enterobacter, Salmonella, Shigella, and Pseudomonas are bacteria that frequently have R-factor plasmids.
As another example, increasing numbers of strains of Neisseria gonorrhoeae have penicillinase plasmids and are known as PPNG (penicillinase-producing Neisseria gonorrhoeae). As a result, penicillin is no longer the drug of choice for gonorrhea. Other antibiotic resistant strains of bacteria that are increasingly becoming a medical problem include MRSA (methicillin-resistant Staphylococcus aureus), MDRSA (multiple drug resistant Staphylococcus aureus), VRE (vancomycin-resistant Enterococcus), MDRTB (multiple drug resistant Mycobacterium tuberculosis), and PRP (penicillin-resistant pneumococci).
In addition to plasmids, conjugative transposons also frequently transmit antibiotic resistance from one bacterium to another. Conjugative transposons are transposons normally found within the bacterial nucleoid, but they can excise and transfer themselves from the donor bacterium's nucleoid to the nucleoid of a recipient bacterium.
Another mechanism that protects some bacteria from antibiotics is antibiotic tolerance. In the case of antibiotic tolerance, the tolerant bacterium is not killed but simply stops growing when the antibiotic is present. It then is able to recover once the antibiotic is no longer in the host. For example, Streptococcus pneumoniae tolerant to vancomycin appear to repress their autolysins in the presence of the drug and don't undergo osmotic lysis.
Bacteria such as E. coli, Proteus, Enterobacter, Serratia, Pseudomonas, Staphylococcus aureus, and Enterococcus mentioned above, are the leading cause of the over two million nosocomial infections (def) seen each year in the U.S. and 50%-60% of these infections are caused by antibiotic resistant bacteria.
For further information on bacterial pathogenesis, see the online Microbiology Web Textbook at the University of Wisconsin-Madison.
Bacterial endospores (def), such as those produced by Bacillus and Clostridium, are also resistant to antibiotics, most disinfectants, and physical agents such as boiling and drying. Although harmless themselves, they are involved in the transmission of some diseases to humans, (e.g., anthrax (Bacillus anthracis), tetanus (Clostridium tetani), botulism (Clostridium botulinum), and gas gangrene (Clostridium perfringens).
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