By David Haldane
The antibiotic era can be said to have started in 1935 with the publishing of the discovery of prontosil, and a few months later the discovery of sulfanilamide, which was the active component of prontosil. Sulfonamides became widely used and were joined after 1941 by penicillin. For many organisms the early antimicrobials were impressively effective. Yet, even at this stage of use, Abraham and Chain detected “a substance destroying the growth inhibiting property of penicillin”, which they called “penicillinase.” (1) Furthermore, it was found that infections might initially respond and then fail. The discovery of streptomycin in 1944 by Schatz and Waksman was a breakthrough in the treatment of tuberculosis. But soon it was found that some patients who were being treated, were getting better and then relapsing. Mycobacterium tuberculosis was becoming resistant to streptomycin during treatment. (2) It was recognized that some organisms did not respond to antimicrobials at all. We now know that these organisms lack the appropriate target, or have mechanisms that prevent the antimicrobial from reaching effective concentrations and so are intrinsically resistant. For example, Mycobacterium tuberculosis has enzymes that destroy penicillin and so the agent is ineffective for treatment.
Still, the rapid discovery of many of the classes of antimicrobials that we continue to rely on, resulted in a feeling of optimism so that by the late 1960s, there was a feeling that “it is time to close the book on infectious diseases, and declare the war against pestilence won” a quotation which was incorrectly attributed to the Surgeon General, Dr. William H. Stewart (1921-2008), who had in fact said the opposite. (3) Nevertheless, the words reflect the general feeling at the time. Yet in 2014, Dr. Tom Frieden, CDC Director, in a blog entitled “The end of antibiotics. Can we come back from the brink?” quoted Dr. Margaret Chan, Director General of the World Health Organization, as saying “a post antibiotic era means, in effect, an end to modern medicine as we know it.” (4) What went wrong? How did we get to this point from the optimism of the 1960s? One of the main contributors has been antimicrobial resistance. Over the next months we will have a series of articles on the mechanisms of bacterial resistance to various classes of antimicrobial agents. This article will start the series with general mechanisms of resistance.
Why does the mechanism of resistance matter?
For the individual clinician treating a specific organism it may not seem to, but knowledge of it can affect decision making. The development of resistance during treatment is a concern for any patient being treated either for long courses of antimicrobials or for severe infection. Organisms that are able to become resistant by inducing enzymes to destroy antimicrobials, for example the ampC beta lactamases, necessitate either using a different agent that is unaffected, or monitoring for the development of resistance.
Some organisms do not develop resistance in the course of treatment. Methicillin susceptible Staphylococcus aureus (MSSA) will not become MRSA despite a long course of a beta lactam antimicrobial, cloxacillin or cefazolin, because the genes required to change the organism are rarely acquired de novo and resistance does not arise from a simple mutation or induction of an enzyme. The same MSSA, however, might become resistant to a quinolone over a long course of treatment as a result of mutations.
An understanding of the mechanisms of resistance is key to the effective use of antimicrobials. Knowledge of the mechanism is required to effectively test for resistance. An example is the D- test. Examination of the zones allows the detection of inducible resistance to clindamycin or the presence of an efflux pump that confers resistance to erythromycin but does not affect clindamycin. More broadly, it is by understanding the mechanisms that we can hope to determine strategies to circumvent resistance. For example the addition of inhibitors of beta lactamase has greatly increased the utility of drugs like amoxicillin and piperacillin.
The intelligent use of antimicrobials can allow us to avoid some of the pitfalls of resistance and, even in the absence of new classes of agents, prolong the usefulness of the drugs at our disposal. Antimicrobial stewardship is the means by which we hope to attain this goal.
Wild type organisms and their antibiotic susceptibility
In order to study organisms and understand how they interact with antimicrobial agents it is useful to distinguish organisms that have not acquired resistance, referred to as the “wild type”, from those that have acquired resistance mechanisms. Bacterial populations that have not been exposed to an antimicrobial show a range of susceptibility to the agent, just as any population shows variation in any characteristic. This range can be defined by the minimal inhibitory concentration (MIC), the lowest concentration required to inhibit the growth of a strain of the bacteria. Some strains of the wild type population will be inhibited more readily than others. This distribution of MICs allows the development of the “epidemiological cutoff value”, sometimes referred to as the “ECOFF”, which defines a concentration of the antimicrobial at which the “wild type” population would be expected to be inhibited. This concentration, when defined by many strains, enables us to suspect when a mechanism of resistance is present in a new strain because its MIC exceeds the ECOFF value.
When resistance mechanisms occur in a population, the strains that have them tend to extend the distribution of the MICs of the strains that make up the population. As the number of resistant strains grows, the resistant population develops its own variation in distribution of MICs, which generally exceed the ECOFF value, but may have some overlap.
The ECOFF is different from a breakpoint as it is an in vitro concentration. Breakpoints are devised with reference to the concentration of antimicrobials that can be achieved at the site of infection and are used to predict whether treatment with a standard dose is likely to be effective. ECOFFs are measurements of the MIC of an agent that will be effective at inhibiting isolates that have not developed mechanisms of resistance. They are not a substitute for breakpoints. (5)
Even amongst organisms that have not been exposed to antimicrobial pressure, resistance mechanisms can be found as shown by Abraham and Chain. Antibiotics exist in nature and resistance genes have been in existence for millions, perhaps even billions, of years. (6) Organisms can be resistant because the antimicrobial is unable to reach an effective concentration inside the cell, or because the site of action is no longer susceptible to the antimicrobial’s activity.
Mechanisms of antibiotic resistance
- Prevention of an antimicrobial from reaching an effective concentration
The first principle mechanism of resistance is prevention of an antimicrobial from reaching an effective concentration within the cell. There are several strategies organisms use to achieve this goal. The organism can prevent the agent from penetrating the cell. Many agents are dependent on channels that cross the outer membrane of the organism to allow them to get past the outer membrane of gram negative organisms. These channels are termed porins and characteristically organisms have multiple types that differ in their size, number and charge. When the number of a particular porin decreases it becomes difficult for agents that use it to penetrate the cell membrane to enter the cell. Organisms may decrease the number of porins in response to stress and so reduce the concentration of antimicrobial in the cell. This mechanism can act in concert with destruction of the antimicrobial to enable an organism to become resistant. For example, the impermeability of the gram negative outer membrane makes most gram negatives intrinsically resistant to vancomycin. Other agents, such as ertapenem, are normally able to enter the cell readily, via porins, in concentrations sufficient to cause cell death. If the cell down-regulates the porin channel it requires, only small amounts of ertapenem can enter the cell. If the cell has an enzyme that has even poor activity to degrade ertapenem, it may be sufficient to render the cell resistant to its antimicrobial action. (7)
Porins are not the only mechanisms preventing agents from entering the bacterial cell. The thick cell wall of vancomycin intermediately resistant S. aureus is thought to trap vancomycin. Resistance to polymyxins may arise form changes to the cell envelope and lipid A in gram negatives. (8)
In addition to allowing substances into the cell, the cell membrane may have efflux pumps that actively remove substances from the cell. Some efflux pumps are not specific in the types of substance that they remove from the cell, so that an organism expressing them can be resistant to multiple agents. As the antimicrobial enters the cell, it is actively removed and prevented from reaching an effective concentration. Two groups of agents affected by this mechanism are the tetracyclines and the quinolones. Organisms with these broad spectrum efflux pumps are not forced to evolve a specific type of pump to become resistant. This mechanism is remarkable because it affects even completely synthetic agents like quinolones which organisms had never been exposed to until the last few decades.(9)
Once antimicrobials have entered the cell, a common mechanism to prevent them from being effective is to destroy them, or at least make them inactive. Many organisms have enzymes that will break down antimicrobials. The agents most commonly affected are the beta lactams and the aminoglycosides.
Ernest Boris Chain (1906 –1979)
The beta lactams, for example, penicillins, cephalosporins and carbapenems, can be inactivated by breaking open the beta lactam ring by hydrolyzing it. The substance recognized by Abraham and Chain was the first penicillinase to be recognized, and a large number have been recognized since, requiring their own taxonomy so they can be categorized. There are four molecular classes of beta lactamase: the first is the Class A penicillinases; the second the Class B metallo-beta-lactamases; the third are the Class C cephalosporinases; and the fourth, the Class D oxacillinases. The agents can also be classified based on their activity. Mutations in some enzymes have altered the spectrum of activity and given broader activity against antimicrobials. Some of the genes that allow the production of these enzymes are on the bacterial chromosome and some have been mobilized onto plasmids and have become widely disseminated in bacteria. This mechanism is one of the most frequently recognized causes of resistance. (9)
Aminoglycosides are also degraded enzymatically. The mechanism is different from beta lactams as aminoglycosides are inactivated by adding chemical groups that changes the shape of the antimicrobial molecule so that it cannot interact with the active site on the ribosome, and thereby interfere with protein synthesis. A number of enzymes are found that may phosphorylate, adenylate, or acetylate the antimicrobial molecule. The different enzymes have differing specificities for different aminoglycoside molecules so that one enzyme may inactivate some agents but not others. (9)
For some antimicrobial agents, a fourth mechanism is used to prevent the development of an effective concentration: the effective concentration of the antimicrobial required to inhibit growth is increased. The cell compensates for the interference with the metabolic pathway affected by the treatment by increasing production of the substrate. For example Neisseria gonorrhoeae can increase production of para-aminobenzoic acid that competes with and dilutes sulfonamide. S. aureus can increase the concentration of certain penicillin binding protein (PBP) to increase the concentration of beta lactam required for inhibition. The role of this mechanism is small in comparison to the others and is likely most important as an additive effect that potentiates other mechanisms. (10)
2. Target modification
The second main principle is target modification, which removes the possibility of effective concentrations by making the organism intrinsically resistant to the antimicrobial. The target is altered or substituted so that the organism is able to continue to maintain the function of the target molecule, but the antimicrobial is unable to bind and so hinder the activity. For example, MRSA produce a new PBP which has the ability to perform the functions of the enzyme targeted by beta lactams but which does not bind to them. The result is that almost all beta lactams agents are no longer effective for these organisms. This target substitution mechanism can develop as a result of a rare event: the acquisition of a number of genes that enable the organism to make a new protein. For MRSA, the mecA gene encodes a novel PBP, but the mecA gene is regulated by other genes that control its expression, and allow the gene to be induced in the presence of beta lactam agents. These genes are located on a mobile genetic element, called the staphylococcal cassette chromosome mec (SCCmec). S. aureus may have originally acquired the cassette from Staphylococcus sciuri, perhaps via Staphylococcus epidermidis. The acquisition of these genes has probably happened only a few times, as there are now five SCCmec types that have been recognized. Some SCCmec types (types II and III) include genes conferring resistance to other agents in addition to beta lactams, so that strains descended from this clone are multidrug resistant. (11)
Target alteration can also occur by a slow process of evolution as a result of antibiotic pressure. N. gonorrhoeae was exquisitely susceptible to penicillin when it was first used in the early 1940s. Within five years, strains were accumulating mutations that altered the affinity of the PBP that interacted with penicillin and the MIC to penicillin increased. Increasing doses of penicillin were required for treatment success, until isolates developed chromosomally mediated penicillin resistance making them resistant. These strains remained rare, and were overshadowed when N. gonorrhoeae acquired plasmid mediated beta lactamase production (giving “penicillinase producing N. gonorrhoeae” or “PPNG”), however the slow development of resistance by accumulation of mutations has continued and now, in combination with genes acquired from other species, threaten to make the organism resistant even to the third generation cephalosporins which have been the mainstay of treatment in recent years. (10)
In the same way that penicillin resistance developed in N. gonorrhoeae, tetracycline resistance has also increased as a result of mutations that have altered the ribosomal target, decreased the entry of tetracycline to the cell, and increased efflux in chromosomally mediated tetracycline resistant strains. Each of these mechanisms of resistance can operate in concert with other mechanisms. For example, for N. gonorrhoeae, the mutations have led to chromosomal resistance to penicillin by affected the target PBP and also the expression of an efflux pump that eliminates penicillin. (10)
Transmission of resistance
One of the difficulties in controlling resistance is its transmissibility. Intrinsic resistance is “built into” the organism and the genes encoding for it are chromosomal. Examples include the ampC beta lactamase found in some members of the Enterobacteriaceae, or the efflux pumps found in many organisms. Acquired resistance is a result of mutation or horizontal transmission of genes between organisms which confer the means to develop resistance. Genetic material can be transferred between organisms by transfer of plasmids or transposons (conjugation), infection with temperate bacteriophages that do not lyse the infected bacteria (transduction), or by taking up and incorporating free DNA released by disrupted organisms (transformation). (8)
Plasmids may contain multiple genes, including resistance mediating genes. Multiple plasmids may occur in an organism and their genes contribute to the phenotype. They multiply independently of the organism and when it replicates they are found in the offspring. They are transferred between organisms by conjugative pili, elongated proteinaceous tubes that connect two organisms. Plasmids may not be restricted to a single species or even genus. Some transfer may be made between distantly related organisms, for example, plasmids conferring resistance to tetracycline in N. gonorrhoeae are also found in Escherichia coli and Haemophilus influenzae. Transposons differ from plasmids by having an enzyme that allows them to integrate into the host bacterium’s chromosome, or into plasmids. Integrons are larger and carry multiple genes, such as the gene cassettes that confer resistance to beta lactams for MRSA, or resistance to vancomycin for vancomycin resistant enterococci (VRE). They also have enzymes that allow them to be integrated into the bacterial chromosome. One of the great fears of microbiologists has been that the integron that encodes the VanA gene cassette would transfer from enterococci to S. aureus. This transfer occurs easily in the lab and has now occurred in vivo giving rise to vancomycin resistant S. aureus (VRSA), which fortunately remains rare. (8, 9)
Transmission of resistance by transduction is thought to be rare, although bacteriophages may be important in mobilizing resistance genes by inserting genetic elements that enable genes to be transferred by plasmids for example. (12) This mobilization may have been common in S. aureus. Phages carrying antimicrobial resistance genes have been found in agricultural runoffs, and so may play a greater role in horizontal transfer of resistance genes than currently recognized. (13) Transformation is most important in streptococci and Neisseria spp. which are able to take up DNA from the environment and integrate it, a process likely to have contributed to the development of penicillin resistance in Streptococcus pneumoniae. Acinetobacter is also able to integrate DNA from the environment, a route that has been speculated may be how resistance genes are introduced from environmental organisms into clinically significant species.(12)
Bacterial resistance is a property of bacteria that predates modern use of antimicrobial agents. The pressure of antimicrobials on bacteria has revealed the ability of organisms to develop and acquire multiple resistance elements that are able to operate simultaneously. Mobilization of resistance genes has resulted in the rapid spread of resistance to some of our most potent antimicrobials. Each course of antimicrobials contributes to resistance in the patient who is treated. (14)
We would like to thank Dr. Haldane for his contribution to CMPT’s newsletter.
Dr. Haldane is a member of CMPT’s Clinical Bacteriology Expert Committee. He is the Director of Bacteriology and Special Pathogens in the Division of Microbiology, Department of Pathology and Laboratory Medicine at the Queen Elizabeth II Health Sciences Center in Halifax
References
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(http://thehealthcareblog.com/blog/2014/05/05/the-end-of-antibiotics-can-we-come-back-from-the-brink/)
5. Clinical and Laboratory Standards institute Performance Standards for antimicrobial susceptibility testing. 26th Ed. CLSI supplement M100S. Clinical and Laboratory Standards Institute, 950 West Valley Road, suite 2500, Wayne Pennsylvania 19087 USA, 2016.
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11. Deurenberg RH, C Vink, S Kalenic, AW Friedrich, CA Bruggeman, EE Stobberingh. 2006. Clinical Microbiology and Infection. 13: 222-235.
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