How do minute microorganisms actually resist antimicrobial actions? What enables them to do this? How do previously susceptible bacteria gain resistance? How is antimicrobial resistance detected in bacterial populations? The Microbiology Module addresses the intricate science behind the antibiotic resistance phenomenon. It will explain what takes place within the bacterial cell to enable antimicrobial resistance and how it can be detected and measured. These basic principles should be a useful resource for client education and for reinforcing the veterinarian’s role in protecting the public’s health.
- Identify bacterial antimicrobial resistance mechanisms for resisting antimicrobial agents.
- Discuss the molecular basis for bacterial antimicrobial resistance.
- explain laboratory methods for detecting and measuring antimicrobial resistance.
- Bacterial Resistance Strategies
- Molecular Mechanisms of Resistance
- Testing Methods for Detection of Antimicrobial Resistance
- Module Summary
Bacterial Resistance Strategies
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.
Examples of bacterial strategies to resist antimicrobial agents. An examination of these strategies is discussed below.
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 Access
Antimicrobial 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:
- Pseudomonas aeruginosa against carbapenems (ß-lactam antibiotics)
- Enterobacter aerogenes and Klebsiella spp. against carbapenems
- Vancomycin intermediate-resistant S. aureus or VISA strains with thickened cell wall trapping vancomycin
- Many Gram-negative bacteria against aminoglycosides
- Many Gram-negative bacteria against quinolones
Strategy 2: Eliminating Antimicrobial Agents from the Cell by Expulsion Using Efflux Pumps
To 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:
- E. coli and other Enterobacteriaceae against tetracyclines
- Enterobacteriaceae against chloramphenicol
- Staphylococci against macrolides and streptogramins
- Staphylococcus aureus and Streptococcus pneumoniae against fluoroquinolones
Did You Know?Efflux pumps are variants of membrane pumps possessed by all bacteria, both pathogenic and nonpathogenic, to move lipophilic or amphipathic molecules in and out of their cells. Some efflux pumps are used by antibiotic-producing bacteria to pump antibiotics out of their cells as fast as the antibiotic is made. This constitutes an immunity protective mechanism for the bacteria to prevent being killed by its own chemical weapon (Walsh, 2000). Want to learn more? Watch the efflux video.
Strategy 3: Inactivation of Antimicrobial Agents via Modification or Degradation
Another 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.
This strategy has been observed in:
- Enterobacteriaceae against chloramphenicol (acetylation)
- Gram-negative and Gram-positive bacteria against aminoglycosides (phosphorylation, adenylation, and acetylation).
Did You Know? The first antibiotic resistance mechanism described was penicillinase. It was first reported by Abraham and Chain in 1940 (Abraham, E. P. and E. Chain. 1940. An enzyme from bacteria able to destroy penicillin. Nature. 146: 837).
Less than 10 years after the clinical introduction of penicillins, penicillin-resistant Staphylococcus aureus was observed in a majority of Gram-positive infections in people. The initial response by the pharmaceutical industry was to develop ß-lactam antibiotics that were unaffected by the specific ß-lactamases secreted by S. aureus. However, as a result, bacterial strains producing ß-lactamases with different properties began to emerge, as well as those with other resistance mechanisms. This cycle of resistance counteracting resistance continues even today (Bush, 1988. Beta-Lactamase Inhibitors from Laboratory to Clinic. Clinical Microbiology Reviews. 1(1):109-123).
Strategy 4: Modification of the Antimicrobial Target
Some 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:
- Staphylococci against methicillin and other ß-lactams (changes or acquisition of different PBPs that do not sufficiently bind ß-lactams to inhibit cell wall synthesis)
- Enterococci against vancomycin (alteration in cell wall precursor components to decrease binding of vancomycin)
- Mycobacterium spp. against streptomycin (modification of ribosomal proteins or 16S rRNA)
- Mutations in RNA polymerase resulting in resistance to the rifamycins
- Mutations in DNA gyrase resulting in resistance to quinolones
Some Examples of Bacterial Resistance Due to Target Site Modification
- Alteration in PBPs reducing affinity of ß-lactam antibiotics (Methicillin-Resistant Staphylococcus aureus, S. pneumoniae, Neisseria gonorrhoeae, Group A streptococci, Listeria monocytogenes)
- Changes in peptidoglycan layer and cell wall thickness reducing activity of vancomycin: Vancomycin-resistant S. aureus
- Changes in vancomycin precursors reducing activity of vancomycin: Enterococcus faecium and E. faecalis
- Alterations in DNA gyrase subunits reducing activity of fluoroquinolones: Many Gram-negative bacteria
- Alteration in topoisomerase IV subunits reducing activity of fluoroquinolones: Many Gram-positive bacteria, particularly S. aureus and Streptococcus pneumoniae
- Changes in RNA polymerase reducing activity of rifampicin: Mycobacterium tuberculosis
Other Sources Related to This Section
Various antibiotics with their mode of action and bacterial mechanism of resistance.
|Antimicrobial Class||Mechanism of Resistance||Specific Means to Achieve Resistance||Examples|
Examples: penicillin, ampicillin, mezlocillin, peperacillin, cefazolin, cefotaxime, ceftazidime, aztreonam, imipenem
|Ensymatic destruction||Destruction of ß-lactam rings by ß-lactamase enzymes.
With the ß-lactam ring destroyed, the antibiotic will no longer have the ability to bind to PBP (penicillin-binding protein), and interfere with cell wall syntheses.
|Resistance of staphylococi to penicillin; resistance of Enterobacteriaceae to penicillins, cephalosporins, and aztreonam|
|Altered target||Changes in penicillin binding proteins.
Mutational changes in original PBPs or acquisition of different PBPs will lead to inability of the antibiotic to bind to the PBP and inhibit cell wall synthesis
|Resistance of staphylococci to methicillin and oxacillin|
|Decreased uptake||Porcin channel formation is decreased.
Since this is where ß-lactams cross the outer membrane to reach the PBP of Gram-negative bacteria, a change in the number or character of these channels can reduce ß-lactam uptake
|Altered target||Alteration in the molecular structure of cell wall precursor components decreases binding of vancomycin so that cell wall synthesis is able to continue||Resistance of enterococci to vancomycin|
Examples: gentamicin, tobramycin, amikacin, netilmicin, streptomycin, kanamycin
|Enzymatic modification||Modifying enzymes alter various sites on the aminoglycoside molecule so that the ability of this drug to bind the ribosome and halt protein synthesis is greatly diminished or lost entirely.||Resistance of many Gram-positive and Gram negative bacteria to aminoglycosides|
|Decreased uptake||Change in number or character of porin channels (through which aminoglycosides cross the outer membrane to reach the ribosomes of gram-negative bacteria) so that aminoglycoside uptake is diminished.||Resistance of variety of Gram-negative bacteria to aminoglycosides|
|Altered target||Modification of ribosomal proteins or of 16s rRNA.
This reduces the ability of aminoglycoside to successfully bind and inhibit protein synthesis
|Resistance of Mycobacterium spp to streptomycin|
Examples: ciprofloxacin, levofloxacin, norfloxacin, lomefloxacin
|Decreased uptake||Alterations in the outer membrane diminishes uptake of drug and/or activation of an "efflux" pump that removes quinolones before intracellular concentration is sufficient for inhibiting DNA metabolism.||Resistance of Gram negative and staphylococci (efflux mechanism only) to various quinolones|
|Altered target||Changes in DNA gyrase subunits decrease the ability of quinolones to bind this enzyme and interfere with DNA processes||Gram-negative and Gram-positive resistance to various quinolones|
Molecular Mechanisms of Resistance
Bacteria 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 Resistance
Biological 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 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:
- lack of affinity of the drug for the bacterial target.
- inaccessibility of the drug into the bacterial cell.
- extrusion of the drug by chromosomally encoded active exporters.
- innate production of enzymes that inactivate the drug.
Examples of intrinsic resistance and their respective mechanisms
|Organisms||Natural Resistance Against||Mechanism|
|Anaerobic bacteria||Aminoglycosides||Lack of oxidative metabolism to drive uptake of aminoglycosides|
|Aerobic bacteria||Metronidazole||Inability to anaerobically reduce metronidazole to its active form|
|Gram-positive bacteria||Aztreonam||Lack of penicillin binding proteins (PBPs) for aztreonam to bind and inhibit|
|Gram-negative bacteria||Vancomycin||Lack of uptake resulting from inability of vancomycin to penetrate outer membrane|
|Klebsiella spp.||Ampicillin||Production of ß-lactamase enzymes that destroy ampicillin before the drug can reach the PBPs|
|Stenotrophomonas maltophilia||Imipenem||Production of ß-lactamase enzymes that destroy imipenem before the drug can reach the PBPs|
|Lactobacilli and Leuconostoc||Vancomycin||Lack of appropriate cell wall precursor target to allow vancomycin to bind and inhibit cell wall synthesis|
|Pseudomonas aeruginosa||Sulfonamides, trimethoprim, tetracycline, or chloramphencicol||Lack of uptake resulting from inability of antibiotics to achieve effective intracellular concentrations|
|Enterococci||Aminoglycosides||Lack of sufficient oxidative metabolism to drive uptake of aminoglycosidess|
|All cephalosporins||Lack of PBPs for cephalosporins to bind and inhibit|
Biofilms, which are an aggregation of bacterial cells firmly attached to a surface via tendrils or filaments, exemplify several forms of intrinsic resistance.
- They are surrounded by a slimy protective coating of DNA, proteins, and polysaccharides that forms a barrier to penetration by antibiotics.
- Electrical charges on the slime surface further bar entry of some antimicrobial drugs.
- The complex three-dimensional structure of biofilms contains transport proteins for nutrient uptake and waste disposal; the latter of these can pump drugs out of cells.
- Biofilms have the ability to reduce the concentration of some antimicrobial drugs reaching bacterial cells, rendering them less effective in disabling bacteria.
- Since the cells deep within a biofilm receive less oxygen and fewer nutrients, they grow relatively slowly and are thus less susceptible to antimicrobial drug action.
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 Resistance
Knowledge 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 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:
- Horizontal Gene Transfer
- Detecting Antimicrobial Resistance
- Lab Approaches and Strategies
- Test Methods in Detecting Antimicrobial Resistance
- Examples of Antibiotic Sensitivity Testing Methods
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 Transfer
Horizontal 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.
Did You Know? Conjugation was first described in 1946 by Lederberg and Tatum, based on studies showing that the intestinal bacteria E. coli use a process resembling sex to exchange circular, extrachromosomal elements, now known as plasmids. (Torrence and Isaacson, 2003)
Examples of acquired resistance through mutations and horizontal gene transfer, including resistance observed and mechanism involved.
|Acquired Resistance Through||Resistance Observed||Mechanism Involved|
|Mutations||Mycobacterium tuberculosis resistance to rifamycins||Point mutations in the rifampin-binding region of rpoB|
|Resistance of many clinical isolates to fluoroquinolones||Predominantly mutation of the quinolone-resistance-determining-region (QRDR) of GyrA and ParC/GrlA|
|E. coli, Hemophilius influenzae resistance to trimethoprim||Mutations in the chromosomal gene specifying dihydrofolate reductase|
|Horizontal gene transfer||Staphylococcus aureus resistance to methicillin (MRSA)||Via acquisition of mecA genes which is on a mobile genetic element called "staphylococcal cassette chromosome" (SCCmec) which codes for penicillin binding proteins (PBPs) that are not sensitive to ß-lactam inhibition|
|Resistance of many pathogenic bacteria against sulfonamides||Mediated by the horizontal transfer of foreign folP genes or parts of it|
|Enterococcus faecium and E. faecalis resistance to vancomycin||Via acquisition of one of two related gene clusters VanA and VanB, which code for enzymes that modify peptidoglycan precursor, reducing affinity to vancomycin|
Detecting Antimicrobial Resistance
Historically, 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.
Watch a short video, produced by the FDA, about antimicrobial resistance.
Organizations Publishing Standards
- United States: Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS).
- European Union: European Committee on Antimicrobial Susceptibility Testing (EUCAST).
- European Union: Office International des Èpizooties (OIE).
- United Kingdom: British Society for Antimicrobial Chemotherapy.
- Germany: Deutsches Institut für Normung.
- France: Société Française de Microbiologie.
- Sweden: Swedish Reference Group for antibiotics.
- Australia: CDS disk diffusion method.
Lab Approaches and Strategies
Some points to consider when deciding whether or not to conduct antimicrobial susceptibility testing should include:
- Clinical relevance of the isolate
- Purity of the isolate
- Logical panel of antimicrobial agents to be tested (e.g., do not include antibiotics to which the isolate is known to have intrinsic resistance)
- Availability of test methodology, resources, and trained personnel
- Standardization of testing
- Valid interpretation of results
- Cost efficiency
- Effective means to communicate results and interpretation to end-users
- Public health impact
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:
- Standardized bacterial inoculum size and physiological state
- Culture medium (nutrient composition, pH, cation concentration, blood and serum supplements and thymidine content)
- Incubation conditions (atmosphere, temperature, duration)
- Concentration of antimicrobials for testing
- Routine testing of prescribed quality control strains
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 Resistance
There 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:
- Dilution (broth and agar)
- Gradient diffusion (E-test)
- Automated systems (Vitek)
- Mechanism-specific tests (such as ß-lactamase detection test and chromogenic cephalosporin test)
- Resistance gene detection (PCR and DNA hybridization)
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 Methods
1. 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.
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.
On this agar plate, a bacterial isolate is tested for resistance to each of twelve different antibiotics. The clear zones around each disc are the zones of inhibition that indicate the extent of the test organism’s inability to survive in the presence of the test antibiotic.
For example, this E. coli isolate on the left has a zone of inhibition of 10.1 mm around ampicillin (AM); since the zone diameter interpretation chart is as follows: Resistant: 13 mm or less; Intermediate: 14−16 mm; Susceptible: 17 mm or more. This particular E. coli isolate is read as resistant to ampicillin.
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.
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:
- Polymerase chain reaction (PCR): One of the most commonly used molecular techniques for detecting certain DNA sequences. This involves several cycles of sample DNA denaturation, annealing of specific primers to the target sequence, if present, and extension of the DNA sequence as facilitated by a thermostable polymerase. This leads to replication of a duplicate DNA sequence, which is visibly detectable by gel electrophoresis via a DNA-intercalating chemical that fluoresces under UV light.
- DNA hybridization: DNA pyrimidines (cytosine and thymidine) specifically pair up with purines (guanine and adenine, or uracil for RNA). To take advantage of this, a labeled probe with a known specific sequence can pair up with opened or denatured DNA from the test sample, as long as their sequences complement each other. If hybridization occurs, the probe labels the DNA hybrid with a detectable radioactive isotope, antigenic substrate, enzyme, or chemiluminescent compound. If no target sequence is present or the isolate does not have the specific gene of interest, no probe attachment will occur, and therefore no signals will be detected.
Modifications of PCR and DNA hybridization: Considering the above principles, several modifications have been introduced which further improve the sensitivity and specificity of standardized procedures. Examples include the use of 5′-fluorescence-labeled oligonucleotides, the development of molecular beacons, development of DNA arrays, and DNA chips, among many others.
- Antimicrobial resistance is the ability of a microorganism to survive and multiply in the presence of an antimicrobial agent that would normally inhibit or kill the microorganism.
- The increasing global incidence and prevalence of antimicrobial resistance have raised concerns. More bacterial pathogens have also developed multiple drug resistance and severely limited therapeutic options for infections in both animals and people.
- Bacteria are able to resist the effects of antimicrobials by preventing intracellular access, immediately removing antimicrobial substances through efflux pumps, modifying the antimicrobial agent through enzymatic breakdown, or modifying the antimicrobial targets within the bacterial cell to render the substance ineffective. Successful development of resistance often results from a combination of two or more of these strategies.
- Antimicrobial resistance traits are genetically coded and can either be intrinsic or acquired.
- Intrinsic resistance is due to innately coded genes which create natural resistance to a particular antibiotic. Innate resistance is normally expressed by virtually all strains of a particular bacterial species.
- Acquired resistance is gained by previously susceptible bacteria either through mutation or horizontally obtained from other bacteria possessing such resistance via transformation, transduction, or conjugation. Acquired resistance is limited to subpopulations of a particular bacterial species and may result from selective pressure exerted by antibiotic usage.
- Antimicrobial susceptibility testing (AST) methods are in vitro procedures used to detect antimicrobial resistance in individual bacterial isolates. Because these laboratory detection methods can determine resistance or susceptibility of an isolate against an array of possible therapeutic options, AST results can be a useful guideline in selecting the best antibiotic treatment for each particular patient.
- Examples of AST methods are broth (and agar) dilution methods, disk-diffusion test, e-test, automated detection using various commercially available detection kits, mechanism-specific enzyme detection methods, and genotypic methods to detect antibiotic resistance genes.
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