II. USING ANTIBIOTICS AND CHEMICAL AGENTS TO CONTROL BACTERIA

C. WAYS IN WHICH BACTERIA MAY RESIST CHEMICAL CONTROL AGENTS

Fundamental Statements for this Learning Object:

1. Most bacteria become resistant to antibiotics by way of one or more mechanisms that are coded for by genes in the bacterial chromosome and/or in bacterial plasmids.
2. Bacterial genes may code for production of an enzyme that inactivates the antibiotic.
3. Bacterial genes may code for an altered target site receptor (ribosomal subunit, enzyme, etc.) for the antibiotic to reduce or block its binding.
4. Bacterial genes may code for altered membrane components that prevent the entry of the antibiotic into the bacterium and/or using an efflux pump to transport the antibiotic out of the bacterium.
5. Bacterial genes may code for modulated gene expression to produce more of the bacterial enzyme that is being tied up or altered by the antibiotic.
6.
When under stress from antibiotics, some bacteria switch on genes whose protein products can increase the mutation rate within the bacterium causing a hyperevolution to increase the chance of forming an antibiotic-resistant mutant that is able to survive.
7. Horizontal gene transfer as a result of transformation, transduction, and conjugation
can transfer antibiotic resistance from one bacterium to another. Horizontal gene transfer enables bacteria to respond and adapt to their environment much more rapidly than mutation by acquiring large DNA sequences from another bacterium in a single transfer.
8.
Another mechanism that protects some bacteria from antibiotics is antibiotic tolerance whereby the tolerant bacterium, called a dormant persister, is not killed but simply stops growing when the antibiotic is present.
9. CDC estimates that “more than two million people in the United States get infections that are resistant to antibiotics and at least 23,000 people die as a result.”

 

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 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 Chemical 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 as a result of mutation or horizontal gene transfer. Mutation (def) in bacterial DNA can alter the order of nucleotide bases in a gene and alter that gene product. Horizontal gene transfer (def) can alter or add bacterial genes, again altering the bacterium's gene products. See function of DNA.

Most bacteria, become resistant to antibiotics by way of one or more of the following mechanisms that are coded for by genes in the bacterial chromosome or in plasmids (def):

1. Producing an enzyme capable of inactivating the antibiotic;
2. Altering the target site receptor for the antibiotic to reduce or block its binding;
3. Preventing the entry of the antibiotic into the bacterium and/or using an efflux pump (def) to transport the antibiotic out of the bacterium; and/or

4. Modulating gene expression to produce more of the bacterial enzyme that is being tied up or altered by the antibiotic.

 

Nice 2013 summary of antibiotic resistant cases and associated deaths; from the CDC.
Improving antibiotic use among hospitalized patients; a 2014 report from CDC.

Estimates of Healthcare-Associated Infections (HCIs) 2011; from CDC.

Getting Smart About Antibiotics; a 2015 report from CDC.
Preventing Antibiotic-Resistant Infections in Hospitals - United States, 2014; a 2016 report from CDC.

 

We will now look at each of these mechanisms of resistance.

1. Producing enzymes that inactivate the antibiotic (see Fig. 1).

Bacteria may acquire new genes that code for an enzyme that inactivates a particular antibiotic or group of antibiotics. For example:

a. Bacteria typically become resistant to penicillins, monobactams, carbapenems, and cephalosporins are known chemically as beta-lactam antibiotics (see Fig. 2) 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 inactivating 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 Common Antibiotics) - chemicals that resemble beta-lactam antibiotic (see Fig. 2). These agents bind to the bacterial beta-lactamases and neutralize them.

b. Bacteria may become resistant to aminoglycosides (streptomycin, neomycin, netilmicin, tobramycin, gentamicin, amikacin, etc.) and streptogramins by enzymatically adding new chemical groups to these antibiotics, thus inactivating the drug.

2. Altering the target site receptor for the antibiotic in the bacterium to reduce or block its binding.

Antibiotics work by binding to some bacterial target site, such as a 50S ribosomal subunit, a 30S ribosomal subunit, or a particular bacterial enzyme such as a transpeptidases or a DNA topoisomerase. Bacteria may acquire new genes that alter the molecular shape of the portion of the ribosomal subunit or the enzyme to which the drug normally binds. For example:

a. 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. 3).

b. 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.

c. Bacteria may become resistant to vancomycin by producing altered cross-linking peptides in the peptidoglycan to which the antibiotic no longer bonds.

d. Bacteria may become resistant to fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, etc.) by producing altered DNA gyrase or other topoisomerases to which the drug no longer binds (see Fig. 4).

 

 

3. Altering the membranes and transport systems to prevent the entry of the antibiotic into the bacterium and/or using an efflux pump (def) to transport the antibiotic out of the bacterium.

Antibiotics that target ribosomes or enzymes within the bacterium must first pass through the porins in the outer membrane of gram-negative and acid-fast bacterial cell walls, and then the cytoplasmic membrane in the case of all bacteria. Subsequently, the antibiotic has to accumulate to a high enough concentration within the bacterium to inhibit or kill the organism.

a. A Gram-negative or an acid-fast bacterium may block the entry of an antmicrobial drug by acquiring genes that alter the porins (def) in the cell wall's outer membrane (see Fig. 5).

b. A bacterium may block the entry of an antmicrobial drug by acquiring genes that alter the carrier (transport) proteins (def) used to transport the drug through the bacterium's cytoplasmic membrane (see Fig. 6). This is generally not a common mechanism of antibiotic resistance.

c. A bacterium may acquire genes coding for an energy-driven efflux pump (def) in its the cytoplasmic membrane that is able to to pump the antibiotic out of the bacterium and preventing it from accumulating to a high enough concentration to inhibit or kill the organism (see Fig. 7). This is the most common method bacteria use to prevent toxic levels of antimicrobial drugs from accumulating within the cytoplasm.

 

4. Modulating gene expression to produce more of the bacterial enzyme that is being tied up or altered by the antibiotic.

Remember that enzymes function as catalysts and are present in cells in small amounts because they are not altered as they carry out their specific biochemical reactions. As mentioned in the previous section, numerous antimicrobial drugs work by inactivating bacterial enzymes and blocking metabolic reactions. Making a particular enzyme and the amount of enzyme that is made is under genetic control.

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 noncoding RNAs (ncRNAs) or antisense RNAa such as microRNAs (miRNAs) that are complementary to an early portion of the mRNA coding for the enzyme. When the noncoding RNA binds to the mRNA by complementary base pairing, ribosomes cannot attach, the mRNA cannot be translated into protein, and the enzyme is not made (See Fig. 8).

Mutations or horizontal gene transfer may result in a modulation of gene expression or translational events that favor increased production of the enzyme being tied up or altered by the antimicrobial agent (see Fig. 9). 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.

5. Many pathogenic bacteria, as well as normal flora, form complex bacterial communities as biofilms (def). Bacteria in biofilms are often able to communicate with one another by a process called quorum sensing (def) and are able to interact with and adapt to their environment as a population of bacteria rather than as individual bacteria. By living as a community of bacteria as a biofilm, these bacteria are:

Why bacterium within a biofilm are more antibiotic resistant isn't completely understood but various mechanisms have been preposed. The extracellular polysaccharide may make it more difficult for the antibiotic to reach all of the bacteria. Bacteria within a biofilm are generally in a metabolically more inert state and this could slow down antibacterial action of the drug. Many antibiotics are static, not cidal in action; the body depends on phagocytes to remove the inhibited bacteria. The biofilm structure makes engulfment by phagocytes pretty much impossible.

6. Dormant persisters

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. Antibiotic tolerance is especially significant in terms of bacteria that form biofilms associated with catheters, heart valves, orthopedic devices, and people with cystic fibrosis. These biofilms often contain a small percentage of dormant persisters that, because they are not dividing, tolerate the antibiotics.

Its been found that bacteria simultaneously produce toxins that inhibit their own growth and antitoxins that bind to the toxin and cause its neutralizion. Small numbers of bacteria in the population, however, become persisters because they produce lower levels of antitoxin or the antitoxin is degraded by stress. As a result, the free toxin arrests bacterial growth enabling a persistent state that is able to survive stressors such as antibiotics and starvation.

 

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 horizontal gene transfer (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 bacterial population by increasing the chance of forming an antibiotic-resistant mutant that is able to survive at the expense of the majority of the population. (Remember that most mutations are harmful to a cell.)

 

 

In addition, horizontal gene transfer as a result of transformation (def), transduction (def), and conjugation (def) can transfer antibiotic resistance from one bacterium to another. Horizontal gene transfer enables bacteria to respond and adapt to their environment much more rapidly than mutation by acquiring large DNA sequences from another bacterium in a single transfer.

 

 

 

 

Exposure to the antibiotic typically selects for strains of the organism that have become resistant through these natural processes. Misuse of antibiotics, such as prescribing them for non-bacterial 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.

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. 10A-10F). 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.

In addition to plasmids, conjugative transposons also frequently transmit antibiotic resistance from one bacterium to another. Conjugative transposons (def), like conjugative plasmids, carry the genes that enable mating pairs to form for conjugation. Therefore, conjugative transposons also enable mobilizable plasmids and nonconjugative transposons to be transferred to a recipient bacterium during conjugation.

 

Examples of resistant strains of bacteria of ever increasing medical importance include:

1. Antibiotic-Resistant Neisseria gonorrhoeae

Out of an estimated 820,000 Neisseria gonorrhoeae infections per year, 246,000 are antibiotic-resistant.

2. Carbapenem-Resistant Enterobacteriaceae (CRE)

More recently, carbapenemase-producing Klebsiella pneumoniae (KPC) strains are frequently being identified among nosocomial pathogens globally. Carbapenemase is a broad-spectrum beta-lactamase enzyme first found in K. pneumoniae isolates that results in resistance to all penicillins, cephalosporins, carbapenems (i.e., imipenem, ertapenem, metropenem), and monobactams (i.e., aztreonam). These broad-spectrum beta-lactamases are also known as extended spectrum beta-lactamases or ESBLs. These ESBLs are now being seen in a variety Enterobacteriaceae including Enterobacter spp., E. coli, Serratia spp., and Salmonella enterica. These ESBL-producing Enterobacteriaceae are known as carbapenem-resistant Enterobacteriaceae, or CRE. Almost half of hospital patients who get bloodstream infections from CRE bacteria die from the infection.  

3. Methicillin-Resistant Staphylococcus aureus (MRSA)

Staphylococcus aureus resistance to methicillin confers resistance to all penicillins and cephalosporins.

4. Vancomycin-Resistant Enterococcus (VRE)

Vancomycin-resistant Enterococcus (VRE) are intrinsically resistant to most antibiotics and have acquired resistance to the first line drug of choice, vancomycin.

5. XDR TB

Extensively drug-resistant tuberculosis (XDR TB), a relatively rare type of multidrug-resistant Mycobacterium tuberculosis that is resistant to almost all drugs used to treat TB, including the two best first-line drugs: isoniazid and rifampin. XDR TB is also resistant to the best second-line medications: fluoroquinolones and at least one of three injectable drugs i.e., amikacin, kanamycin, or capreomycin.

6. Clostridium difficile infections

A 2015 CDC study found that "Clostridium difficile caused almost half a million infections among patients in the United States in a single year. An estimated 15,000 deaths are directly attributable to C. difficile infections, making it a substantial cause of infectious disease death in the United States" with an excess medical cost of $1,000,000,000 per year.

 

Bacteria such as E. coli, Proteus, Enterobacter, Serratia, Pseudomonas, Staphylococcus aureus, and Enterococcus mentioned above, are the leading cause of health care-associated infections (def). According to the Centers for Disease Control and Prevention (CDC) Healthcare-associated infection's website, "In American hospitals alone, healthcare-associated infections account for an estimated 1.7 million infections and 99,000 associated deaths each year" in the U.S. The CDC also estimates that “more than two million people in the United States get infections that are resistant to antibiotics and at least 23,000 people die as a result.”

Finally, 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. Examples include anthrax (Bacillus anthracis), tetanus (Clostridium tetani), botulism (Clostridium botulinum), gas gangrene (Clostridium perfringens), and pseudomembranous colitis (Clostridium difficile).

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