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Antimicrobial Resistance Learning Site
Bacterial Resistance StrategiesTo survive in the presence of an antibiotic, bacteria must disrupt a step in the action of the antimicrobial agent (see Pharmacology Module, Mechanisms of Action). This may involve preventing antibiotic access into the bacterial cell or perhaps removal or even degradation of the active component of the antimicrobial agent. No single mechanism of resistance can explain why all bacteria are resistant to a particular antibiotic. In fact, several different mechanisms may work together to confer resistance to a single antimicrobial agent, or multiple mechanisms in different bacteria may achieve the same results. Let's learn what causes antibiotic resistance. Watch below.
Image source: http://file.scirp.org/Html/19-8202738_43142.htm The image above describes the mechanisms of antibiotic resistance. There are multiple examples of mechanisms of antibiotic resistance. These examples include inactivation of a drug by enzymes, activation of drug efflux pumps, inhibition of drug uptake, and alteration of drug target. Several different mechanisms may work together to confer resistance to a single antimicrobial agent.Strategy 1: Preventing AccessAntimicrobial compounds almost always require access into the bacterial cell to reach their target site, where they can interfere with the normal function of the bacterial organism. Porin channels are the passageways by which these antibiotics would normally cross the bacterial outer membrane of Gram-negative bacteria. Some bacteria protect themselves by prohibiting these antimicrobial compounds from entering past their cell walls. For example, one variety of Gram-negative bacteria reduces the uptake of certain antibiotics—such as aminoglycosides and ß-lactams—by modifying the cell membrane porin channel frequency, size, and selectivity. Prohibiting entry in this manner will prevent these antimicrobials from reaching their intended targets that, for aminoglycosides and ß-lactams, are the ribosomes and the penicillin-binding proteins (PBPs), respectively. This mechanism has been observed in:
Strategy 2: Eliminating Antimicrobial Agents from the Cell by Expulsion Using Efflux PumpsTo be effective, antimicrobial agents must also be present at a sufficiently high concentration within the bacterial cell. Some bacteria possess membrane proteins that act as an export or efflux pump for certain antimicrobials, extruding the antibiotic out of the cell as fast as it can enter. This results in low intracellular concentrations that are insufficient to elicit an effect. Some efflux pumps selectively extrude specific antibiotics such as macrolides, lincosamides, streptogramins, and tetracyclines, whereas others (referred to as multiple drug resistance pumps) expel a variety of structurally diverse anti-infectives with different modes of action. This strategy has been observed in:
Strategy 3: Inactivation of Antimicrobial Agents via Modification or DegradationAnother means by which bacteria preserve themselves is by destroying the active component of the antimicrobial agent. A classic example is the hydrolytic deactivation of the ß-lactam ring in penicillins and cephalosporins by the bacterial enzymes called ß-lactamases. The process inactivates penicilloic acid, causing it to be ineffective in binding to PBPs, thereby protecting the process of cell wall synthesis. Want to watch in action? Let's watch! This strategy has been observed in:
Strategy 4: Modification of the Antimicrobial TargetSome resistant bacteria evade antimicrobials by reprogramming or camouflaging critical target sites to avoid recognition. Therefore, despite the presence of an intact and active antimicrobial compound, no subsequent binding or inhibition will take place. This strategy has been observed in:
Other Sources Related to This SectionVarious antibiotics with their mode of action and bacterial mechanism of resistance.
Mechanisms of Resistance Against Different Antimicrobial Classes (Forbes, et al., 1998; Berger-Bachi, 2002).
Molecular Mechanisms of ResistanceBacteria are genetically encoded to use intrinsic or acquired resistance mechanisms to combat antimicrobial agents. Intrinsic resistance may also be seen when comparing clinical susceptibility levels of two different species to a common drug. For example, penicillin G may have greater binding affinity for the penicillin-binding proteins of Streptococcus agalactiae than for those of Enterococcus faecalis. We know that the methicillin resistance of S. aureus (MRSA) is primarily due to changes that occur in the PBP, which is the protein that ß-lactam antibiotics bind to and inactivate, to inhibit cell wall synthesis. This change is caused by the expression of a certain mecA gene in some strains of S. aureus which arise following a history of penicillin and other antimicrobial use. Expression of the mecA gene results in an alternative PBP (PBP2a) that has a low affinity for most ß-lactam antibiotics, thereby allowing these strains to replicate in the presence of methicillin and related antibiotics. Some antimicrobial resistance is caused by multiple changes in the bacterial genome. For example, isoniazid resistance of Mycobacterium tuberculosis results from changes in the following genes: katG gene which encodes a catalase, inhA gene which is the target for isoniazid, the neighboring oxyR and aphC genes and their intergenic region. Biological Versus Clinical ResistanceBiological resistance refers to changes that result in the organism being less susceptible to a particular antimicrobial agent. When antimicrobial susceptibility has been lost to such an extent that the drug is no longer effective for clinical use, the organism is said to have achieved clinical resistance. It is important to note that biologic resistance and clinical resistance do not necessarily coincide. From a clinical laboratory and public health perspective, biologic development of antimicrobial resistance is an ongoing process, while clinical resistance is dependent on current laboratory methods and established cutoffs. Our inability to reliably detect biological resistance with current laboratory procedures and criteria should not be perceived as evidence that it is not occurring (Forbes, et al., 1998). Intrinsic ResistanceIntrinsic resistance is the innate ability of a bacterial species to resist activity of a particular antimicrobial agent through its inherent structural or functional characteristics, which allow tolerance of a particular drug or antimicrobial class. This can also be called “insensitivity” since it occurs in organisms that have never been susceptible to that particular drug. Such natural insensitivity can be due to:
Examples of intrinsic resistance and their respective mechanisms(From Forbes, et al., 1998, Giguere, et al., 2006).
Biofilms, which are an aggregation of bacterial cells firmly attached to a surface via tendrils or filaments, exemplify several forms of intrinsic resistance.
Some examples of bacteria that are capable of forming biofilms that impact animals include Neisseria spp. as dental plaque on teeth, Staphylococcus intermedius on orthopedic implants and pacemakers, and Salmonella spp. on environmental surfaces. Clinical implications: Intrinsic ResistanceKnowledge of intrinsic resistance is important in clinical practice to avoid inappropriate and ineffective therapies. For bacterial pathogens that are naturally insensitive to a large number of antimicrobial classes, such as Mycobacterium tuberculosis and Pseudomonas aeruginosa, this consideration can pose a limitation in the range of treatment options and thus increase the risk for acquired resistance. Acquired ResistanceAcquired resistance is said to occur when a particular microorganism obtains the ability to resist the activity of a particular antimicrobial agent to which it was previously susceptible. This can result from the mutation of genes involved in normal physiological processes and cellular structures, from the acquisition of foreign resistance genes, or from a combination of these two mechanisms. Successful gene change and/or exchange may involve mutation or horizontal gene transfer by transformation, transduction, or conjugation. Unlike intrinsic resistance, traits associated with acquired resistance are found only in some strains or subpopulations of a bacterial species and require laboratory methods for detection. These same methods are used for monitoring rates of acquired resistance as a means of combating the emergence and spread of acquired resistance traits in pathogenic and nonpathogenic bacterial species. Mechanism of acquired resistance via gene change or exchange Antibiotics exert selective pressure on bacterial populations by killing susceptible bacteria, allowing strains with resistance to an antibiotic to survive and multiply. These traits are vertically passed on to subsequently reproduced cells and become sources of resistance. Because resistance traits are not necessarily eliminated or reversed, resistance to a variety of antibiotics may be accumulated over time. This can lead to strains with multiple drug resistance, which are more difficult to eliminate due to limited effective treatment options. In this section, we’ll be discussing acquired resistance as it pertains to:
MutationsImage: A normal bacterial genome results in normal cellular structure and function whereas a mutation in the bacterial genome results in altered cellular structure and function and ultimately modified susceptibility. A mutation is a spontaneous change in the DNA sequence that may lead to a change in the trait for which it’s coded. Any change in a single base pair may lead to a corresponding change in one or more of the corresponding amino acids, which can then change the enzyme or cell structure and consequently affect the affinity or effectiveness activity of related antimicrobials. In prokaryotic genomes, mutations frequently occur due to base changes caused by exogenous agents, DNA polymerase errors, deletions, insertions, and duplications (Gillespie, 2002). Horizontal Gene TransferHorizontal gene transfer, or the process of swapping genetic material between neighboring bacteria, is another means by which resistance can be acquired. Many of the antibiotic resistance genes are carried on plasmids, transposons, or integrons that act as vectors to transfer genes to other similar bacterial species. Horizontal gene transfer may occur via three main mechanisms: transformation, transduction, or conjugation. Mechanisms of Gene Exchange: Conjugation Gene exchange via conjugation involving plasmid transfer Transformation involves the process in which bacteria uptake short fragments of DNA. Transduction involves transfer of DNA from one bacterium into another via bacteriophages. Conjugation involves transfer of DNA via sex pilus and requires cell-to-cell contact. Watch a short video about horizontal gene transfer.
Examples of acquired resistance through mutations and horizontal gene transfer, including resistance observed and mechanism involved.
Detecting Antimicrobial ResistanceHistorically, veterinary practitioners prescribed antibiotics based on expected mode of action, spectrum of activity, and clinical experience. With the emergence and spread of antimicrobial resistance, treatment of bacterial infections has become increasingly difficult and is no longer as straightforward as it was many years prior. Practitioners now need to consider that the organisms being treated may be resistant to some or all antimicrobial agents. These considerations require antimicrobial susceptibility testing as a standard procedure. Antimicrobial susceptibility testing methods are in vitro procedures used to detect antimicrobial resistance and susceptibility in individual bacterial isolates to a wide array of antimicrobial therapy options. These same methods can also be used for monitoring the emergence and spread of resistant microorganisms in the population. Clinical breakpoints are threshold values established for each pathogen-antibiotic-host combination indicating at what level of antibiotic the isolate is sensitive, intermediate, or resistant to standard manufacturer-recommended treatment regimens. The interpretative criteria for these are based on extensive studies that correlate laboratory resistance data with serum-achievable levels for each antimicrobial agent and a history of successful and unsuccessful therapeutic outcomes. Although veterinary laboratories originally based interpretations on standards established using human pathogens, it became apparent by the early 1980s that such an approach did not reliably predict clinical outcomes when applied to veterinary practice. Subsequently, groups were established to develop veterinary-specific standards.
Organizations Publishing StandardsLab Approaches and StrategiesSome points to consider when deciding whether or not to conduct antimicrobial susceptibility testing should include:
Most often, interpretation is reduced to whether the isolate is classified as susceptible, intermediately susceptible, or resistant to a particular antibiotic. It should, however, be remembered that these in vitro procedures are only approximations of in vivo conditions, which can be very different depending on the nature of the drug, the nature of the host, and the conditions surrounding the interaction between the antibiotic and the target pathogen. One critical aspect is following standardized, quality-controlled procedures that can generate reproducible results. Aspects of quality control include:
Because of the required culture time, antimicrobial susceptibility testing by the above methods may take several days, which is not ideal, particularly in critical clinical cases demanding urgency. Often practitioners may use locally established antibiograms as a guideline for therapy. An antibiogram is a compiled susceptibility report or table of commonly isolated organisms in a particular hospital, farm, or geographic area, which can serve as a useful guideline in therapy before actual culture and susceptibility data becomes available for reference. In some cases, specific resistance gene detection by PCR or direct enzyme testing can provide earlier susceptibility information (Example: mecA detection in methicillin-resistant staphylococci). To learn more, read About Antibiograms.
Testing Methods for Detection of Antimicrobial ResistanceThere are several antimicrobial susceptibility testing methods available today and each one has its respective advantages and disadvantages. They all have the same goal, which is to provide a reliable prediction of whether an infection caused by a bacterial isolate will respond therapeutically to a particular antibiotic treatment. These data may be used as guidelines for treatment, or as indicators of emergence and spread of resistance on a population level based on passive or active surveillance. Some examples of antibiotic susceptibility testing methods are:
Selection of the appropriate method will depend on the intended degree of accuracy, convenience, urgency, availability of resources, availability of technical expertise, and cost. Interpretation should be based on veterinary standards whenever possible rather than on human medical standards due to applicability. Among these available tests, the two most commonly used methods in veterinary laboratories are the agar disk-diffusion method and the broth microdilution method. Examples of Antibiotic Sensitivity Testing Methods1. Dilution (broth and agar) The broth dilution method involves placing the isolate into several separate broth solutions containing an antimicrobial agent in a series of varying concentrations. Microdilution testing uses about 0.05 to 0.1 ml total broth volume and can be conveniently performed in a microtiter format. Macrodilution testing uses broth volumes at about 1.0 ml in standard test tubes. For both of these broth dilution methods, the lowest concentration at which the isolate is completely inhibited, as evidenced by the absence of visible bacterial growth, is recorded as the minimal inhibitory concentration (MIC). The test is only valid if the positive control shows growth and the negative control shows no growth. A procedure similar to broth dilution is agar dilution. The agar dilution method follows the same principle of establishing the lowest concentration of a serially diluted antibiotic for which bacterial growth is still inhibited. 2. Disk-diffusion Because of convenience, efficiency, and cost, the disk diffusion method is probably the most widely used method for determining antimicrobial resistance in private veterinary clinics. A growth medium—usually Mueller-Hinton agar—is first evenly seeded throughout the plate with the isolate of interest that has been diluted to a standard concentration (approximately 1−2 x 108 colony forming units per ml). Commercially prepared disks, each of which is preimpregnated with a standard concentration of a particular antibiotic, are evenly dispensed and lightly pressed onto the agar surface. The antibiotic being tested diffuses outward from the diffusion disk and creates an antibiotic concentration gradient in the agar. The highest concentration of antibiotic is found closest to the diffusion disk with decreasing amount of antibiotic present, further and further from the disk. The zone around an antibiotic disk that has no growth is referred to as the zone of inhibition. This approximates the minimum antibiotic concentration sufficient to prevent growth of the test isolate. The zone is measured in mm and compared to a standard interpretation chart used to categorize the isolate as susceptible, intermediately susceptible, or resistant. The MIC measurement cannot be determined from this qualitative test, which simply classifies the isolate as susceptible, intermediate, or resistant. To help your understanding of testing, watch this video example.
3. Gradient diffusion (E-test) The e-test is a commercially available test that uses a plastic test strip impregnated with a gradually decreasing concentration of a particular antibiotic. The strip also displays a numerical scale that corresponds to the antibiotic concentration. This method is a convenient quantitative test of antibiotic resistance. However, a separate strip is needed for each antibiotic, and therefore the cost of this method can be high. Let's watch a video on e-test for antibiotic susceptibility. 4. Automated systems Several commercial systems provide conveniently prepared and formatted microdilution panels, instrumentation and automated plate readings. These methods are intended to reduce technical errors and lengthy preparation times. Most automated antimicrobial susceptibility testing systems provide automated inoculation, reading, and interpretation. Although these systems are rapid and convenient, one major limitation for most laboratories is the cost associated with the purchase, operation, and maintenance of the machinery. 5. Mechanism-specific tests Resistance may also be established through tests that directly detect the presence of a particular resistance mechanism. For example, ß-lactamase detection can be accomplished using an assay such as the chromogenic cephalosporinase test. 6. Resistance gene detection (PCR and DNA hybridization) Since resistance traits are genetically encoded, we can sometimes test for the specific genes that confer antibiotic resistance. Even though nucleic acid-based detection systems are generally rapid and sensitive, it is important to remember that the presence of a resistance gene does not necessarily equate to treatment failure, as resistance is also dependent on the mode and level of expression of these genes. Some of the most common molecular techniques used for antimicrobial resistance detection are as follows:
References and Suggested Readings
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