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Journal of Applied Bacteriology 1990.68, 307-318
A Review Techniques used for the determination of antimicrobial resistance and sensitivity in bacteria L A U R AJ . V . P I D D O C K Antimicrobial Agents Research Group, Department of Medical Microbiology, The Medical School, University of Birmingham, Birmingham B15 ZTJ, U K Received 21 October 1989 1. Introduction, 307 2. Procedures for testing the activity of antimicrobial agents, 308 2.1 Methods using solid media, 309 2.2 Methods using liquid media, 310 2.3 Automated methods, 31 1 2.4 Other methods, 312 2.5 Advantages and disadvantages-which procedure?, 313 3. Designation of sensitive or resistant (interpretation of tests), 313 4. References, 316
1. Introduction
Susceptibility determination is one of the most important procedures undertaken in clinical microbiology laboratories. Whilst the action of a product from one organism inhibiting the growth of another, antibiosis, was first observed by van Leeuwenhoek in 1676 (Balows 1974), Fleming in 1924 was the first to record an antimicrobial susceptibility test (for Staphylococcus aureus and penicillin). Several procedures utilizing the ability of an antimicrobial agent to diffuse through agar and inhibit the growth of the test bacterium (on the agar surface or in the agar matrix giving a clear area or zone of no growth) were described in the early days of development and use of new antimicrobial agents. Most variations in the procedure were the application of the antimicrobial agents to the agar, such as cutting wells into the agar (Reddish 1929) and cylinder plate (Abraham et al. 1941). In 1944, Vincent & Vincent used filter paper discs impregnated with penicillin, and in 1945 Mohs described the radial streak method using 15 mm discs and the use of a sensitive control organism. Other disc diffusion methods were also described at this time (Copeland 1945; Morley 1945; Kokko 1947; Kolmer 1947). Paper discs, 6.5 mm diam., as generally used today, was first described by Bondi et al. in 1947. The use of tablets instead of filter paper discs was first described by Hoyt & Levine in 1947. In the early years of antimicrobial susceptibility testing there was no standardization between methods or even between laboratories. This problem was recognized by Could & Bowie in 1952, who described a technique comparing the zone diameters produced by varying concentrations of antimicrobial agent incorporated on paper discs against control organisms. The test organism was then examined against a disc containing a single concentration, and the zone diameter compared with those produced by the control strains. In 1955, Stokes described a disc diffusion procedure whereby the zone diameter of an antibiotic against a test organism and control organism could be compared on the same agar plate. Variations of the ‘Stokes test’ are still the most commonly used method for routine susceptibility testing in clinical laboratories in Britain today. In 1966 Bauer et al. described standard procedures for performing disc susceptibility tests, and compared zone diameters with the minimum inhibitory concentrations (mic.). The continued need to standardize procedures not just between different laboratories but between different countries has led to various groups describing standard methods: WHO (Anon. 1961); Ericsson & Sherris (1971); Stokes & Waterworth (1972); Swedish Reference Group (Anon. 1981); Deutsches Institut fur Normung (Anon. 1984); National Committee for Clinical Laboratory Standards (NCCLS) (Anon.
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1985). In the USA, the NCCLS has an approved standard procedure for performing disc diffusion antibiotic susceptibility tests which is updated every three years. During external quality assessment of antimicrobial susceptibility testing in the United Kingdom, no significant differences were observed between the ‘Stokes Test’ and the procedure outlined by Bauer-Kirby type procedures (Snell et al. 1982, 1984).
During the development of disc diffusion procedures, techniques to determine the m.i.c. of an antimicrobial agent were also being developed. The early techniques utilized dilutions (usually twofold) of the antibiotic in the liquid media in which the organisms were grown (Fleming 1929). An equivalent volume of the test organism would be added to a set of tubes that covered a concentration range of the antibiotic, and after incubation the m.i.c. was described as the first tube in which there was no visible growth. Variations of this technique were described throughout the 1940s (Fleming 1942; Rammelkamp & Maxon 1942; Schmidt & Sesler 1943; Spink & Ferris 1945; Buggs et al. 1946). The determination of m.i.c. of solid media was first described by Schmith & Reymann in 1940, and subsequently other workers also incorporated antibiotics into agar (Frisk 1945; Frank et al. 1950). It was found that the determination of m.i.c. with either broth or agar dilution procedures was time consuming, costly and technically demanding. However, the use of pre-prepared microtitre trays containing antimicrobial agents and multichannel micropipettes (broth microdilution procedure) and of multipoint inoculators for the agar dilution technique have simplified both techniques. In recent years new techniques for assessing antimicrobial activity have been described, usually aimed at decreasing the time taken for the test. These techniques included the incorporation of fluorogenic substrates for the detection of growth, optical density changes, use of radiolabelled nutrients in the growth media, and electrical impedance.
2. Procedures for testing the activity of antimicrobial agents 2.1
METHODS U S I N G S O L I D MEDIA
The gelling agent in solid media usually employed for antibiotic sensitivity determination is agar to which selected nutrients are added depending on the bacterial species to be grown. Agar is a complex, natural substance derived from seaweed and contains two types of polysaccharide (agarose and agaropectin), a variety of metallic cations and other trace elements. Although synthetic gelling agents (which avoid the batch-to-batch variation of a natural product) have been described, none have achieved widespread production and hence use. Antimicrobial agents with cationic molecular structures, such as polymyxins or aminoglycosides, may be electrostatically bound to acid or sulphate groups in the agar, causing a slow rate of diffusion through the matrix, and because of batch variability, standard results may be difficult to obtain for some species against some antibiotics. Companies that produce media are aware of the problem and usually attempt to standardize products which they sell for sensitivity testing, e.g. by supplementing the media with calcium and magnesium to approximate physiological concentrations (Thornsberry et al. 1977). Although an agar medium appears to be solid it is actually a gel composed primarily of water and it therefore allows diffusion of substances from areas of high concentration to low. To determine the activity of an antimicrobial agent many clinical laboratories perform agar diffusion tests. The antibiotic can be applied to the seeded agar plate in several ways: six dry or freshly prepared wet discs containing a precise amount of drug, or by adding a solution of the antibiotic to a well cut in the agar. Glass or metal cylinders applied to the surface of the agar are rarely used nowadays. The antibiotic diffuses essentially in two dimensions away from the well or disc, forming a concentration gradient which inhibits the growth of bacteria and hence causes a zone of inhibition. The zone extends until the concentration of the drug is insufficient to inhibit the growth of the organism. The zone of inhibition is affected by the rate at which the drug diffuses through the agar and the rate of growth of the bacterium. There is, therefore, a critical time at which the edge of the zone is formed. Likewise there is a critical bacterial cell population: usually large zones of inhibition will be formed when the bacteria are slow growing (e.g. due to low temperature, minimal media, etc.) and small zones when the bacteria grow rapidly.
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Because of the nature of the diffusion test there are various factors that can affect the test. As the drug content of the disc is fixed, the most important variable is the size of the inoculum, as this will influence the final area of the zone of inhibition. The inoculum is also important in tests on organisms that produce drug-inactivating enzymes, as such strains may appear susceptible to the test compound if a light inoculum is used. With heavy inocula the organism may appear less susceptible. For routine susceptibility testing in clinical laboratories an inoculum of lo6 cfu/ml which gives semiconfluent growth is usually used. The zones of inhibition are measured in millimetres. The optimal depth of agar is about 4 mm (Barry & Fay 1973). As discussed earlier, the growth medium employed can profoundly influence the zone size. Different media, however, must be employed for the culture of fastidious organisms. For most susceptibility tests Mueller-Hinton or agar specifically formulated for sensitivity tests is used and can be supplemented where necessary with blood or blood products which have no effect on the activity of the majority of antimicrobial agents (Brenner & Sherris 1972). The medium of Wilkins & Chalgren (1976) is frequently employed for testing anaerobic bacteria. Most susceptibility tests are incubated at 35-37°C for 18 h for the optimal growth of most human pathogens, although there are certain exceptions such as when testing the activity of methicillin against Staphylococcus aureus. The simplicity of the disc diffusion method, the fact that most organisms can be easily designated sensitive or resistant, and the availability of commercially prepared discs has caused this technique to be the major method of evaluation of antibiotic susceptibility throughout the world. The concentration of the drug in the discs is such that a zone diameter of 30-35 mm is usually indicative of susceptibility, and one of 15-20 mm o r less (dependent on the agent), resistance. The appropriate drug concentration is usually selected after many strains of bacteria (at least one hundred) with known and different m.i.c.s are tested against discs covering a range of drug concentrations. The diameter of the zone of inhibition is plotted graphically against the logarithm of antibiotic concentration, and the most appropriate disc concentration chosen. Within a chemical class of antibiotics the similarity between spectrum and activity has resulted in discs of the same drug content being used. For example 30 pg of a cephalosporin is usually used when testing Enterobacteriaceae. Zone-Diameter Interpretive standards and approximate m.i.c. correlates can be found in several recent publications (Acar 1980). The technique has been further modified such that up to six different antibiotics can be tested against one organism on one petri dish, by applying the discs in a ring approximately 3 cm apart. The rings of discs are also commercially available. In the UK, the Stokes method of disc diffusion assay is most frequently used. In this method a control organism and test organism are examined against the same antibiotic discs on the same plate, usually by inoculating the control organism into the centre portion of the plate (using a cotton-wool swab and a rotary spreading device) and the test organism to the outer portion; the antibiotic discs are placed such that half the disc lies on the control inoculum and half on the test. The designation of sensitive or resistant is made by comparing the zone sizes of the test and control strains. If the zone radius of the test is equal to, or not more than 3 mm smaller than the control, the organism is designated sensitive. If the zone radius is greater than 3 mm smaller than the control, the test organism is resistant. The control organisms are usually the ‘Oxford’ Staphylococcus aureus NCTC 6571, for staphylococci, streptococci and Haemophilus; Escherichia coli NCTC 10418 for coliform bacteria; and Pseudomonas aeruginosa NCTC 10662 for pseudomonads. For some antibiotics the control organism may not be appropriate, especially for newer agents which may exhibit very high activity against the control organism, thereby causing many organisms to be falsely designated resistant. Laboratories must therefore keep a cautious eye on sensitivity test results of new agents and alter their procedures as necessary. Likewise, it should be remembered that agents have different spectra of activity, and that the control organism may be less susceptible than the test, thereby causing some organisms to be falsely designated sensitive, e.g. ampicillin and Haemophilus influenme. The testing of staphylococci against beta-lactams can often be a problem, as heterogeneous expression of resistance to agents such as methicillin is observed. The sensitivity test conditions are therefore optimized so that resistance can be detected. Methicillin resistance in staphylococci is due to a protein that is regulated by temperature and osmotic pressure, and maximum expression is seen with 5% sodium
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chloride in the media and incubation at 30°C; therefore, sensitivity tests should be carried out under these conditions. To determine the m.i.c. on solid media the antibiotic can be incorporated into the liquefied agar medium (at 42”C), and by preparing a series of plates containing increasing concentrations of the drug (usually in twofold dilutions). With a multipoint inoculator up to 100 strains per plate may be tested. In general the inoculum is applied as a spot of approximately 5 mm diameter and each spot contains about 1 x lo4 or 1 x lo6 viable cells. The spots should be 5-7 mm apart as some organisms produce substances that inhibit the growth of others. To prevent the swarming of organisms such as Proteus spp. interfering with the determination of m.i.c., 30 mg/l 1-(4-nitrophenyl)-glycerol(PNPG) is added to the agar. Control strains of known susceptibility are always inoculated as a method of quality control and an antibiotic-free plate is inoculated at the beginning and end of a series of plates to ensure adequate inoculation and growth. The m.i.c. is the lowest concentration of antibiotic that completely inhibits growth at an inoculum of lo4 cfu, and for the higher inoculum the lowest concentration that inhibits growth of the spot substantially (ignoring a faint haze of growth or a couple of colonies). The agar dilution susceptibility test is usually employed when many organisms are to be tested, e.g. when new agents are to be screened for the spectrum of activity. An abbreviated form of the agar dilution procedure, known as the ‘breakpoint’ method, is gaining in popularity. In this instance, only one or two concentrations of antibiotic called the breakpoint concentration are used (please see Section 3), and the spots of growth are compared with those on an antibiotic-free control. No growth indicates a susceptible strain and growth resistant.
2.2
METHODS USING LIQUID M E D I A
The major disadvantage of agar dilution m.i.c. tests is that plate cultures are not easily subcultured in order to determine the minimum bactericidal concentration (m.b.c.) of a drug. Although a replica transfer method can be used it is cumbersome and technically demanding. The broth dilution procedure generally uses 1-2 ml of liquid media. This method has been scaled down, however, and is now usually performed in microtitre trays and final volumes of 1W200 PI. The m.i.c. is the concentration at which there is no visible growth. Subcultures are made on agar media from the last wells or tubes that show visible growth and from all successive tubes. The minimum bactericidal concentration (m.b.c.) causing 99.9% kill is the lowest drug concentration that gives no growth on the agar. The liquid medium employed for m.i.c. testing is usually the same as that used in solid media without the agar. The effect of cations upon the activity of certain antimicrobial agents against certain species such as Pseudomonas spp. may be reduced by using liquid media. One of the disadvantages of susceptibility testing with liquid media is that the supplements for growth required by fastidious organisms are often opaque. Newer additives such as Fildes reagent or Levinthal’s broth provide clearer media (Thornsberry et al. 1976; Sonnenwirth 1970). In tests that employ liquid media the inoculum density is very important. The usual inoculum is lo5 cfu/ml. If a heavy inoculum is used it is possible that it will contain a few mutant cells that suggest decreased susceptibility to the test agent. During growth these few cells will multiply, cause turbidity, and hence a high m.i.c. value. Additional problems can occur when a bacterial strain exhibits heterogeneity in its susceptibility, such as methicillin-resistant staphylococci with betalactams. One of the advantages of growing bacteria in liquid media is that the effect of the antimicrobial agent may be observed directly by microscopy. The minimum amount of drug that produces morphological changes has been called the minimum antibacterial concentration (m.a.c.) (Lorian 1975). Such values are qualitative, however, and meaningful interpretation is difficult; also, only agents such as beta-lactams and quinolones cause obvious morphological changes and usually only to Gramnegative bacteria. The kinetics of bactericidal action may also be observed when bacteria are grown in liquid media, either by subculturing to antibiotic-free agar plates or by monitoring the growth of the bacterium by optical density or nephelometry. Such methods utilize the light-scattering ability of a turbid broth and can detect early changes in the growth of bacteria (although > lo6 cfu/ml are required to be visible), and were the first techniques adapted for mechanization and automation.
Susceptibility testing 2.3
31 1
AUTOMATED METHODS
Many laboratories are using semi-automated or automated methods for both rapid bacterial identification and antimicrobial susceptibility testing. Automation not only shortens the time taken for the test but also offers the potential for reduced technical errors and increased endpoint accuracy. Several methods have been evaluated for automation and include photometry, microcalorimetry, radiometric, electrical impedance and bioluminescence. The first rapid susceptibility system marketed was the Autobac broth disc elution system (originally manufactured by Pfizer Diagnostics, now General Diagnostics) (McKie et a/. 1974). The original machine was designated to provide rapid (5 h) qualitative susceptibility test results for Enterobacteriaceae, Pseudomonas spp., staphylococci and enterococci. The new machine can now also perform 5 h m.i.c. determinations, urine screening and rapid identification of Gram-negative bacilli. The system consists of a test cuvette, a disc dispenser, a shaker incubator and a photometer. The test cuvette contains 13 chambers so that 12 drugs can be tested with the 13th chamber as an antibiotic-free control. The inoculum contains 1-3 x 10’ cfu/ml and is determined by the standardization meter in the photometer. The inoculated broth is dispensed to the chambers, and the elution disc placed on a ledge in the chamber so that it contacts the broth but does not enter the chamber and interfere with the light beam. The antimicrobial agent in the disc therefore elutes into the broth, which is enhanced by shaking the chamber during incubation at 35°C. The standardization of the inoculum and the susceptibility test endpoint are determined by light-scatter readings of the test compared with the control, as calculated by the computer. In studies that compared the Autobac I with the agar dilution test and agar disc diffusion test the overall agreement was similar and exceeded 90% (Thornsberry et al. 1975). Some discrepancies were noted, however, including those between nitrofurantion and Proteus spp., due to incomplete elution of the drug from the disc (Butler & Gavan 1977), and some drug-organism combinations should not be tested, e.g. cephalothin and enterococci, kanamycin and Pseudornonas spp., and methicillin and staphylococci. The MS-2 Microbiology System (Abbott) is an automated system for simultaneous antimicrobial susceptibility testing, urine screening and identification of Gram-negative bacilli (Spencer et al. 1977). It operates by photometrically monitoring turbidity or colorimetric changes in the broth in which the test organism is growing. This machine is similar to the Autobac in that it uses a multichannel cuvette, shaking incubation and light transmission nephelometry, but it is more sophisticated. The 11-chambered cuvette is preloaded with elution discs. An unstandardized inoculum in broth is inoculated into a common upper chamber and incubated with shaking in the analysis module. When the inoculum reaches a preset density it is automatically transferred into the test chambers by a vacuum mechanism. The optical density in each cuvette is then automatically recorded at timed intervals, and kinetic analyses are performed by the computer to determine susceptibility of the test organism. The MS-2 system has been shown to be accurate for susceptibility testing of many species (Thornsberry et a/. 1980) but, as with the Autobac system, results obtained for methicillin and staphylococci were unreliable. Problems were also encountered with Enterohacter species. Another automated procedure that utilizes the ability of a growing culture to scatter light is the AMS (Automicrobic) system (developed by McDonnell Douglas Corp.; Aldridge et ul. 1977). The system consists of a diluent dispenser, a vacuum card filling module, a card sealing apparatus, a reader-incubator and computer with printer. Small plastic cards with microchambers containing nutrients and antibiotics are used for culture. A plastic straw is inserted into the card which is placed in a rack that holds the inoculum in a tube. The rack (which can hold 10 cards) is placed in the vacuum-filling module, which fills the microchambers with the inoculum, thereby rehydrating the biochemicals in the chamber and the straw entry port is heat-sealed. The card is then placed in the reader-incubator module. A printed report is then generated by the system after a period of time. The AMS system has been found to give susceptibility results that are similar to those of the MS-2 system (Malloy et a / . 1983). Four automated procedures are currently available for performing microtitre tray susceptibility tests: TouchSCAN/AutoSCAN (American Micro Scan), Sceptor (Becton Dickinson), Sensititre
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(Gibco-Diagnostics) and Micro-Coder (Micro-Media Systems). The Sensititre and Sceptor systems are supplied with trays containing dehydrated reagents and the other two supply trays containing frozen reagents. The dehydrated trays have a shelf life of up to 24 months whereas the frozen trays can be stored for up to five months at -20°C. The dehydrated trays can also be inoculated automatically, unlike the frozen trays. All systems require an incubation time of 15-20 h. In the Autoscan system the plates are read automatically, whereas for all the other systems the tray is placed in a ‘reading box’ and the operator records the m.i.c. endpoint with a joy-stick or sensor. The system will then generate a report. Several studies have shown the automated microtitre m.i.c.s to correlate well with conventional procedures (Barry et al. 1978; Gavan et a/. 1980; Jones et a / . 1981).
2.4 O T H E R M E T H O D S In recent years several investigators have developed alternative methods for determining bacterial growth, and hence susceptibility to antimicrobial agents. The other methods have been based on a variety of bacterial metabolic activities, such as pH changes (Rogers et a/. 1955), changes in redox potential (Sellers 1970), electrical conductivity or impedance (Colvin & Sherris 1977), radiometric (Deland & Wagner 1970), bioluminescence (Velland et al. 1974) and microcalorimetry (Binford et a / . 1973). The use of pH changes to monitor growth depends on the ability of an organism to ferment a carbohydrate, usually glucose, and produce acid. A phenol red indicator that clearly turns yellow when growth occurs is used to monitor the pH change. For the strains studied the incubation times required to detect differences between antibiotic-free controls and tests varied from 1-8 h. This method has not been developed further as there has been a lack of quantitative endpoints and of procedural and interpretative standardization. The other problem is that the concentration of acid required to cause indicator dye changes may also alter the activity of certain agents. Indicator dyes are also used to detect changes in redox potential. Many such dyes also have inherent antibacterial activity, however, so resazurin or a n indophenol reagent (0.15% dichlorophenol indophenol in phosphate buffer pH 7.4) are usually used. Haemoglobin has also been used as an indicator of oxygen consumption, giving results within 2 h. The procedures that require indicator dyes have never been widely evaluated or accepted, and as none have received commercial research, and hence development of a standard method it is unlikely that these methods will be improved. It is possible, however, that indicator dyes may be incorporated into automated techniques for the early reading of microdilution tests. The electrical impedance (or relative conductivity) or resistance to the flow of alternating current through a broth medium changes as the growth of bacteria produces metabolic byproducts. If the bacterial population exceeds lo6 cfu/ml the impedance change can be measured reliably and used as a predictable measure of growth. Impedance is monitored by incorporating electrodes into each tube or cell and the culture is monitored continuously. In the impedance instrument changes in the test chambers containing an antibiotic compared with a control are recorded as ratios. If a lo7 cfu/ml inoculum is used a 6 h impedance test has a correlation of 74% with a standard overnight M I C test. False low m.i.c.s have been obtained for polymyxin and tetracycline and false high m.i.c.s for some beta-lactams. When results were analysed according to resistant, sensitive or intermediate and compared with a disc diffusion test the agreement was 85% (Thornsberry et a/. 1975). In the Bactec instrument (Becton Dickinson) organisms grow in media containing 14C-palmitic acid o r 14C-glucose as a carbon source, and monitors growth by the measurement of the concentration of 14C0, released into the atmosphere in a closed chamber (Deland 1972). This instrument was originally developed as a monitor for the early detection of growth in blood cultures. However, inhibition of growth can also be quantified. The concentration of antibiotic required t o produce 50% inhibition of 14C0, release (compared with an antibiotic-free control) after 3 h correlates well with results from broth MIC tests. Discrepancies were noted for staphylococci. The detection of bacterial growth using ATP-dependent bioluminescence has also been developed for studying the effect of antimicrobial agents. The method is based on the reaction of fire-fly luciferase and reduced luciferin and ATP and magnesium which forms a luciferin-AMP complex. This
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complex dissociates, releasing light which can be measured with a photometer, thereby assessing the amount of bacterial ATP in the culture. By comparing the luminescence of tubes with and without antibiotic after a period of time a ratio can be obtained. These ratios have been shown to have good agreement with broth dilution and agar diffusion assays (Velland et al. 1974). Discrepancies have been noted for Proteus mirabilis, Pseudomonas aeruginosa and enterococci.
2.5
ADVANTAGES A N D D!SADVANTAGES--WHICH
PROCEDURE?
The agar dilution technique for susceptibility testing has several advantages when compared with the broth dilution procedure. Inoculating devices allow up to 100 strains per plate to be tested at one time, so many organisms can be compared in parallel under the same conditions against the same antibiotic. Contamination of the agar can be readily detected in situ, unlike a broth culture where the turbidity due to growth of the test organism cannot usually be distinguished from that of a contaminant. A clear liquid media is necessary for broth dilution assays, and for fastidious organisms this may not be available, therefore these species can be tested only by agar procedures. The major disadvantages of agar dilution procedures is that the minimum bactericidal concentration (m.b.c.) cannot be easily determined, and for certain infections such as bacterial endocarditis the m.b.c. is often more relevant than the m.i.c. Automated methods also do not assess microbial killing well, and for many organisms 99-99.9% kill (such as for an m.b.c.) cannot be detected. All the tests are affected by media composition, pH, incubation temperature and length of incubation although some tests for some antibiotics are more affected than others. One critical consideration in all tests is the size of the inoculum. Whilst the clinical laboratory is always striving to perform a test that has clinical relevance, because of constraints of time and technological ease, certain parameters such as inoculum size in susceptibility testing have become standardized. Whilst an inoculum of 103-104 organisms/ml may represent the concentration of bacteria infecting the blood in patients with endocarditis, there may be 107-108organisms/ml of cerebrospinal fluid in patients with meningitis. However, if a low inoculum is used in susceptibility tests a false-sensitive result may be obtained, and if a high inoculum is used a false-resistant result may be obtained, particularly if a broth dilution procedure has been used. If a high inoculum is used it is possible to select a subpopulation of bacteria that have spontaneously mutated to become antibiotic resistant, and it is these organisms that grow up with a higher m.i.c. As they usually only occur at a frequency of 1 in lo7 (the frequency of a mutation in a gene) the test provides an ideal environment for their selection and growth, unlike the human body where factors other than the presence of an antibiotic contribute towards bacterial killing. For these reasons an inoculum of lo5 cfu/ml is usually employed for broth dilution tests and lo4 and lo6 cfu/ml in agar dilution tests. The disc diffusion susceptibility test has the advantage of readily detecting antagonism or synergy between two antibiotics. If the discs are placed so that antibiotics which are known to cause these effects are placed in close proximity to each other then antagonism can be observed by a blunting of the zone of inhibition of one antibiotic caused by the other. Synergy can be seen by the enhancement of the zone of inhibition. Antagonism can be seen when one antibiotic interferes with the mechanism of another, such as with tetracycline and gentamicin. It can also occur when one antibiotic induces enzymes that can hydrolyse another antibiotic, such as erythromycin and spiromycin with erythromycin-resistant Staph. aureus, and cefoxitin and cefotaxime in Enterobacter cloacae. Whether the observed antagonism correlates with failure of therapy is dependent upon the type and site of infection and the pharmacokinetics of the antibiotic. Automatic procedures that rely upon optical density or nephelometry for detecting antibacterial activity can give false-resistant results for some antibiotics. Beta-lactams and quinolones cause filamentation of bacterial cells (as well as other morphological changes), and filamentation can cause an increase in turbidity when, in fact, the number of viable cells is decreasing.
3. Designation of sensitive or resistant (interpretation of tests) The disc diffusion test is the most frequeptly used procedure for determining the susceptibility of clinical strains to antimicrobial agents. The diameters of the zone of inhibition of each agent are
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usually interpreted as ‘sensitive’, ‘resistant’ or ‘intermediate’. The term ‘sensitive’ implies an organism that will respond to the test antibiotic; ‘resistant’, that the therapy is unlikely to be effective; and ‘intermediate’ where the organism is only likely to respond where high concentrations of the agent are achievable. The three categories are established from data known about the relationship between the m.i.c. (usually from agar dilution tests) and the zone size, the distribution of the strains into each class of susceptibility and the pharmacokinetics of the antibiotic. As might be anticipated, different species and different antibiotics have separate interpretive values. To interpret the zone size, it must first be related to an m.i.c. This is done by performing m.i.c. tests and disc diffusion tests in parallel for a particular agent under well-defined conditions. Each zone diameter is then plotted graphically against the m.i.c. for that strain (on a logarithmic scale). A regression line is constructed with the method of least squares to establish the relationship between the zone size and the m.i.c. The zone size breakpoint is usually obtained by applying the attainable therapeutic concentration of the agent (usually in serum) to the regression line. The calculation of breakpoint concentrations is discussed in more detail later in this section. The use of regression line analysis has been criticized by Krasemann & Hildenbran (1980). Firstly, individual strains of the same species may have a wide variety of zone sizes, and a wide range of m.i.c.s may be obtained by using twofold dilution steps in the m.i.c. determination. The construction of the regression line may mask the variation and will not indicate the probability of false-sensitive or false-resistant data. The use of regression analysis in this fashion also neglects certain mathematical dogma. Often, data for sensitive and resistant organisms of different species are plotted on the same graph, therefore those for resistant Ent. cloacae and sensitive staphylococci will not necessarily give data for resistant staphylococci. Linearity of the data may not occur and therefore a mathematical error will be introduced by constructing a regression line; in addition, the regression line is constructed on the assumption that the m.i.c. data are correct. As stated earlier, however, both the zone size and the m.i.c. can be variables: therefore, two lines should be calculated (one for zone sizes based on m.i.c.s and the other for m.i.c.s based on zone sizes). In most instances, the two lines will be very similar. To overcome the problems of regression analysis and to obtain a more valid breakpoint zone size, the rate of false-sensitive and false-resistant is calculated by the error rate-bound method (Brownlee 1965; Metzler & Daham 1974). A scattergram of the m.i.c. data is plotted as a log, value against the zone diameter. A horizontal line, M, , is drawn corresponding to a therapeutically achievable concentration in serum and tissues. A vertical line, Z,, is selected for the zone diameter that is equal to or less than the m.i.c.s of resistant strains, giving a rate of error for false-resistant of less than or equal to 5%. Another vertical line, Z,, is selected for the zone diameter that is equal to or greater than the m.i.c.s of sensitive strains, giving a rate of error for false-susceptible of less than or equal to 1‘51.The scatter-gram of the data is therefore divided into six areas (Fig. 1). The top left area is the area of true resistant, the bottom left the false resistant (
Journal of Applied Bacteriology 1990.68, 307-318
A Review Techniques used for the determination of antimicrobial resistance and sensitivity in bacteria L A U R AJ . V . P I D D O C K Antimicrobial Agents Research Group, Department of Medical Microbiology, The Medical School, University of Birmingham, Birmingham B15 ZTJ, U K Received 21 October 1989 1. Introduction, 307 2. Procedures for testing the activity of antimicrobial agents, 308 2.1 Methods using solid media, 309 2.2 Methods using liquid media, 310 2.3 Automated methods, 31 1 2.4 Other methods, 312 2.5 Advantages and disadvantages-which procedure?, 313 3. Designation of sensitive or resistant (interpretation of tests), 313 4. References, 316
1. Introduction
Susceptibility determination is one of the most important procedures undertaken in clinical microbiology laboratories. Whilst the action of a product from one organism inhibiting the growth of another, antibiosis, was first observed by van Leeuwenhoek in 1676 (Balows 1974), Fleming in 1924 was the first to record an antimicrobial susceptibility test (for Staphylococcus aureus and penicillin). Several procedures utilizing the ability of an antimicrobial agent to diffuse through agar and inhibit the growth of the test bacterium (on the agar surface or in the agar matrix giving a clear area or zone of no growth) were described in the early days of development and use of new antimicrobial agents. Most variations in the procedure were the application of the antimicrobial agents to the agar, such as cutting wells into the agar (Reddish 1929) and cylinder plate (Abraham et al. 1941). In 1944, Vincent & Vincent used filter paper discs impregnated with penicillin, and in 1945 Mohs described the radial streak method using 15 mm discs and the use of a sensitive control organism. Other disc diffusion methods were also described at this time (Copeland 1945; Morley 1945; Kokko 1947; Kolmer 1947). Paper discs, 6.5 mm diam., as generally used today, was first described by Bondi et al. in 1947. The use of tablets instead of filter paper discs was first described by Hoyt & Levine in 1947. In the early years of antimicrobial susceptibility testing there was no standardization between methods or even between laboratories. This problem was recognized by Could & Bowie in 1952, who described a technique comparing the zone diameters produced by varying concentrations of antimicrobial agent incorporated on paper discs against control organisms. The test organism was then examined against a disc containing a single concentration, and the zone diameter compared with those produced by the control strains. In 1955, Stokes described a disc diffusion procedure whereby the zone diameter of an antibiotic against a test organism and control organism could be compared on the same agar plate. Variations of the ‘Stokes test’ are still the most commonly used method for routine susceptibility testing in clinical laboratories in Britain today. In 1966 Bauer et al. described standard procedures for performing disc susceptibility tests, and compared zone diameters with the minimum inhibitory concentrations (mic.). The continued need to standardize procedures not just between different laboratories but between different countries has led to various groups describing standard methods: WHO (Anon. 1961); Ericsson & Sherris (1971); Stokes & Waterworth (1972); Swedish Reference Group (Anon. 1981); Deutsches Institut fur Normung (Anon. 1984); National Committee for Clinical Laboratory Standards (NCCLS) (Anon.
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1985). In the USA, the NCCLS has an approved standard procedure for performing disc diffusion antibiotic susceptibility tests which is updated every three years. During external quality assessment of antimicrobial susceptibility testing in the United Kingdom, no significant differences were observed between the ‘Stokes Test’ and the procedure outlined by Bauer-Kirby type procedures (Snell et al. 1982, 1984).
During the development of disc diffusion procedures, techniques to determine the m.i.c. of an antimicrobial agent were also being developed. The early techniques utilized dilutions (usually twofold) of the antibiotic in the liquid media in which the organisms were grown (Fleming 1929). An equivalent volume of the test organism would be added to a set of tubes that covered a concentration range of the antibiotic, and after incubation the m.i.c. was described as the first tube in which there was no visible growth. Variations of this technique were described throughout the 1940s (Fleming 1942; Rammelkamp & Maxon 1942; Schmidt & Sesler 1943; Spink & Ferris 1945; Buggs et al. 1946). The determination of m.i.c. of solid media was first described by Schmith & Reymann in 1940, and subsequently other workers also incorporated antibiotics into agar (Frisk 1945; Frank et al. 1950). It was found that the determination of m.i.c. with either broth or agar dilution procedures was time consuming, costly and technically demanding. However, the use of pre-prepared microtitre trays containing antimicrobial agents and multichannel micropipettes (broth microdilution procedure) and of multipoint inoculators for the agar dilution technique have simplified both techniques. In recent years new techniques for assessing antimicrobial activity have been described, usually aimed at decreasing the time taken for the test. These techniques included the incorporation of fluorogenic substrates for the detection of growth, optical density changes, use of radiolabelled nutrients in the growth media, and electrical impedance.
2. Procedures for testing the activity of antimicrobial agents 2.1
METHODS U S I N G S O L I D MEDIA
The gelling agent in solid media usually employed for antibiotic sensitivity determination is agar to which selected nutrients are added depending on the bacterial species to be grown. Agar is a complex, natural substance derived from seaweed and contains two types of polysaccharide (agarose and agaropectin), a variety of metallic cations and other trace elements. Although synthetic gelling agents (which avoid the batch-to-batch variation of a natural product) have been described, none have achieved widespread production and hence use. Antimicrobial agents with cationic molecular structures, such as polymyxins or aminoglycosides, may be electrostatically bound to acid or sulphate groups in the agar, causing a slow rate of diffusion through the matrix, and because of batch variability, standard results may be difficult to obtain for some species against some antibiotics. Companies that produce media are aware of the problem and usually attempt to standardize products which they sell for sensitivity testing, e.g. by supplementing the media with calcium and magnesium to approximate physiological concentrations (Thornsberry et al. 1977). Although an agar medium appears to be solid it is actually a gel composed primarily of water and it therefore allows diffusion of substances from areas of high concentration to low. To determine the activity of an antimicrobial agent many clinical laboratories perform agar diffusion tests. The antibiotic can be applied to the seeded agar plate in several ways: six dry or freshly prepared wet discs containing a precise amount of drug, or by adding a solution of the antibiotic to a well cut in the agar. Glass or metal cylinders applied to the surface of the agar are rarely used nowadays. The antibiotic diffuses essentially in two dimensions away from the well or disc, forming a concentration gradient which inhibits the growth of bacteria and hence causes a zone of inhibition. The zone extends until the concentration of the drug is insufficient to inhibit the growth of the organism. The zone of inhibition is affected by the rate at which the drug diffuses through the agar and the rate of growth of the bacterium. There is, therefore, a critical time at which the edge of the zone is formed. Likewise there is a critical bacterial cell population: usually large zones of inhibition will be formed when the bacteria are slow growing (e.g. due to low temperature, minimal media, etc.) and small zones when the bacteria grow rapidly.
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Because of the nature of the diffusion test there are various factors that can affect the test. As the drug content of the disc is fixed, the most important variable is the size of the inoculum, as this will influence the final area of the zone of inhibition. The inoculum is also important in tests on organisms that produce drug-inactivating enzymes, as such strains may appear susceptible to the test compound if a light inoculum is used. With heavy inocula the organism may appear less susceptible. For routine susceptibility testing in clinical laboratories an inoculum of lo6 cfu/ml which gives semiconfluent growth is usually used. The zones of inhibition are measured in millimetres. The optimal depth of agar is about 4 mm (Barry & Fay 1973). As discussed earlier, the growth medium employed can profoundly influence the zone size. Different media, however, must be employed for the culture of fastidious organisms. For most susceptibility tests Mueller-Hinton or agar specifically formulated for sensitivity tests is used and can be supplemented where necessary with blood or blood products which have no effect on the activity of the majority of antimicrobial agents (Brenner & Sherris 1972). The medium of Wilkins & Chalgren (1976) is frequently employed for testing anaerobic bacteria. Most susceptibility tests are incubated at 35-37°C for 18 h for the optimal growth of most human pathogens, although there are certain exceptions such as when testing the activity of methicillin against Staphylococcus aureus. The simplicity of the disc diffusion method, the fact that most organisms can be easily designated sensitive or resistant, and the availability of commercially prepared discs has caused this technique to be the major method of evaluation of antibiotic susceptibility throughout the world. The concentration of the drug in the discs is such that a zone diameter of 30-35 mm is usually indicative of susceptibility, and one of 15-20 mm o r less (dependent on the agent), resistance. The appropriate drug concentration is usually selected after many strains of bacteria (at least one hundred) with known and different m.i.c.s are tested against discs covering a range of drug concentrations. The diameter of the zone of inhibition is plotted graphically against the logarithm of antibiotic concentration, and the most appropriate disc concentration chosen. Within a chemical class of antibiotics the similarity between spectrum and activity has resulted in discs of the same drug content being used. For example 30 pg of a cephalosporin is usually used when testing Enterobacteriaceae. Zone-Diameter Interpretive standards and approximate m.i.c. correlates can be found in several recent publications (Acar 1980). The technique has been further modified such that up to six different antibiotics can be tested against one organism on one petri dish, by applying the discs in a ring approximately 3 cm apart. The rings of discs are also commercially available. In the UK, the Stokes method of disc diffusion assay is most frequently used. In this method a control organism and test organism are examined against the same antibiotic discs on the same plate, usually by inoculating the control organism into the centre portion of the plate (using a cotton-wool swab and a rotary spreading device) and the test organism to the outer portion; the antibiotic discs are placed such that half the disc lies on the control inoculum and half on the test. The designation of sensitive or resistant is made by comparing the zone sizes of the test and control strains. If the zone radius of the test is equal to, or not more than 3 mm smaller than the control, the organism is designated sensitive. If the zone radius is greater than 3 mm smaller than the control, the test organism is resistant. The control organisms are usually the ‘Oxford’ Staphylococcus aureus NCTC 6571, for staphylococci, streptococci and Haemophilus; Escherichia coli NCTC 10418 for coliform bacteria; and Pseudomonas aeruginosa NCTC 10662 for pseudomonads. For some antibiotics the control organism may not be appropriate, especially for newer agents which may exhibit very high activity against the control organism, thereby causing many organisms to be falsely designated resistant. Laboratories must therefore keep a cautious eye on sensitivity test results of new agents and alter their procedures as necessary. Likewise, it should be remembered that agents have different spectra of activity, and that the control organism may be less susceptible than the test, thereby causing some organisms to be falsely designated sensitive, e.g. ampicillin and Haemophilus influenme. The testing of staphylococci against beta-lactams can often be a problem, as heterogeneous expression of resistance to agents such as methicillin is observed. The sensitivity test conditions are therefore optimized so that resistance can be detected. Methicillin resistance in staphylococci is due to a protein that is regulated by temperature and osmotic pressure, and maximum expression is seen with 5% sodium
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chloride in the media and incubation at 30°C; therefore, sensitivity tests should be carried out under these conditions. To determine the m.i.c. on solid media the antibiotic can be incorporated into the liquefied agar medium (at 42”C), and by preparing a series of plates containing increasing concentrations of the drug (usually in twofold dilutions). With a multipoint inoculator up to 100 strains per plate may be tested. In general the inoculum is applied as a spot of approximately 5 mm diameter and each spot contains about 1 x lo4 or 1 x lo6 viable cells. The spots should be 5-7 mm apart as some organisms produce substances that inhibit the growth of others. To prevent the swarming of organisms such as Proteus spp. interfering with the determination of m.i.c., 30 mg/l 1-(4-nitrophenyl)-glycerol(PNPG) is added to the agar. Control strains of known susceptibility are always inoculated as a method of quality control and an antibiotic-free plate is inoculated at the beginning and end of a series of plates to ensure adequate inoculation and growth. The m.i.c. is the lowest concentration of antibiotic that completely inhibits growth at an inoculum of lo4 cfu, and for the higher inoculum the lowest concentration that inhibits growth of the spot substantially (ignoring a faint haze of growth or a couple of colonies). The agar dilution susceptibility test is usually employed when many organisms are to be tested, e.g. when new agents are to be screened for the spectrum of activity. An abbreviated form of the agar dilution procedure, known as the ‘breakpoint’ method, is gaining in popularity. In this instance, only one or two concentrations of antibiotic called the breakpoint concentration are used (please see Section 3), and the spots of growth are compared with those on an antibiotic-free control. No growth indicates a susceptible strain and growth resistant.
2.2
METHODS USING LIQUID M E D I A
The major disadvantage of agar dilution m.i.c. tests is that plate cultures are not easily subcultured in order to determine the minimum bactericidal concentration (m.b.c.) of a drug. Although a replica transfer method can be used it is cumbersome and technically demanding. The broth dilution procedure generally uses 1-2 ml of liquid media. This method has been scaled down, however, and is now usually performed in microtitre trays and final volumes of 1W200 PI. The m.i.c. is the concentration at which there is no visible growth. Subcultures are made on agar media from the last wells or tubes that show visible growth and from all successive tubes. The minimum bactericidal concentration (m.b.c.) causing 99.9% kill is the lowest drug concentration that gives no growth on the agar. The liquid medium employed for m.i.c. testing is usually the same as that used in solid media without the agar. The effect of cations upon the activity of certain antimicrobial agents against certain species such as Pseudomonas spp. may be reduced by using liquid media. One of the disadvantages of susceptibility testing with liquid media is that the supplements for growth required by fastidious organisms are often opaque. Newer additives such as Fildes reagent or Levinthal’s broth provide clearer media (Thornsberry et al. 1976; Sonnenwirth 1970). In tests that employ liquid media the inoculum density is very important. The usual inoculum is lo5 cfu/ml. If a heavy inoculum is used it is possible that it will contain a few mutant cells that suggest decreased susceptibility to the test agent. During growth these few cells will multiply, cause turbidity, and hence a high m.i.c. value. Additional problems can occur when a bacterial strain exhibits heterogeneity in its susceptibility, such as methicillin-resistant staphylococci with betalactams. One of the advantages of growing bacteria in liquid media is that the effect of the antimicrobial agent may be observed directly by microscopy. The minimum amount of drug that produces morphological changes has been called the minimum antibacterial concentration (m.a.c.) (Lorian 1975). Such values are qualitative, however, and meaningful interpretation is difficult; also, only agents such as beta-lactams and quinolones cause obvious morphological changes and usually only to Gramnegative bacteria. The kinetics of bactericidal action may also be observed when bacteria are grown in liquid media, either by subculturing to antibiotic-free agar plates or by monitoring the growth of the bacterium by optical density or nephelometry. Such methods utilize the light-scattering ability of a turbid broth and can detect early changes in the growth of bacteria (although > lo6 cfu/ml are required to be visible), and were the first techniques adapted for mechanization and automation.
Susceptibility testing 2.3
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AUTOMATED METHODS
Many laboratories are using semi-automated or automated methods for both rapid bacterial identification and antimicrobial susceptibility testing. Automation not only shortens the time taken for the test but also offers the potential for reduced technical errors and increased endpoint accuracy. Several methods have been evaluated for automation and include photometry, microcalorimetry, radiometric, electrical impedance and bioluminescence. The first rapid susceptibility system marketed was the Autobac broth disc elution system (originally manufactured by Pfizer Diagnostics, now General Diagnostics) (McKie et a/. 1974). The original machine was designated to provide rapid (5 h) qualitative susceptibility test results for Enterobacteriaceae, Pseudomonas spp., staphylococci and enterococci. The new machine can now also perform 5 h m.i.c. determinations, urine screening and rapid identification of Gram-negative bacilli. The system consists of a test cuvette, a disc dispenser, a shaker incubator and a photometer. The test cuvette contains 13 chambers so that 12 drugs can be tested with the 13th chamber as an antibiotic-free control. The inoculum contains 1-3 x 10’ cfu/ml and is determined by the standardization meter in the photometer. The inoculated broth is dispensed to the chambers, and the elution disc placed on a ledge in the chamber so that it contacts the broth but does not enter the chamber and interfere with the light beam. The antimicrobial agent in the disc therefore elutes into the broth, which is enhanced by shaking the chamber during incubation at 35°C. The standardization of the inoculum and the susceptibility test endpoint are determined by light-scatter readings of the test compared with the control, as calculated by the computer. In studies that compared the Autobac I with the agar dilution test and agar disc diffusion test the overall agreement was similar and exceeded 90% (Thornsberry et al. 1975). Some discrepancies were noted, however, including those between nitrofurantion and Proteus spp., due to incomplete elution of the drug from the disc (Butler & Gavan 1977), and some drug-organism combinations should not be tested, e.g. cephalothin and enterococci, kanamycin and Pseudornonas spp., and methicillin and staphylococci. The MS-2 Microbiology System (Abbott) is an automated system for simultaneous antimicrobial susceptibility testing, urine screening and identification of Gram-negative bacilli (Spencer et al. 1977). It operates by photometrically monitoring turbidity or colorimetric changes in the broth in which the test organism is growing. This machine is similar to the Autobac in that it uses a multichannel cuvette, shaking incubation and light transmission nephelometry, but it is more sophisticated. The 11-chambered cuvette is preloaded with elution discs. An unstandardized inoculum in broth is inoculated into a common upper chamber and incubated with shaking in the analysis module. When the inoculum reaches a preset density it is automatically transferred into the test chambers by a vacuum mechanism. The optical density in each cuvette is then automatically recorded at timed intervals, and kinetic analyses are performed by the computer to determine susceptibility of the test organism. The MS-2 system has been shown to be accurate for susceptibility testing of many species (Thornsberry et a/. 1980) but, as with the Autobac system, results obtained for methicillin and staphylococci were unreliable. Problems were also encountered with Enterohacter species. Another automated procedure that utilizes the ability of a growing culture to scatter light is the AMS (Automicrobic) system (developed by McDonnell Douglas Corp.; Aldridge et ul. 1977). The system consists of a diluent dispenser, a vacuum card filling module, a card sealing apparatus, a reader-incubator and computer with printer. Small plastic cards with microchambers containing nutrients and antibiotics are used for culture. A plastic straw is inserted into the card which is placed in a rack that holds the inoculum in a tube. The rack (which can hold 10 cards) is placed in the vacuum-filling module, which fills the microchambers with the inoculum, thereby rehydrating the biochemicals in the chamber and the straw entry port is heat-sealed. The card is then placed in the reader-incubator module. A printed report is then generated by the system after a period of time. The AMS system has been found to give susceptibility results that are similar to those of the MS-2 system (Malloy et a / . 1983). Four automated procedures are currently available for performing microtitre tray susceptibility tests: TouchSCAN/AutoSCAN (American Micro Scan), Sceptor (Becton Dickinson), Sensititre
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(Gibco-Diagnostics) and Micro-Coder (Micro-Media Systems). The Sensititre and Sceptor systems are supplied with trays containing dehydrated reagents and the other two supply trays containing frozen reagents. The dehydrated trays have a shelf life of up to 24 months whereas the frozen trays can be stored for up to five months at -20°C. The dehydrated trays can also be inoculated automatically, unlike the frozen trays. All systems require an incubation time of 15-20 h. In the Autoscan system the plates are read automatically, whereas for all the other systems the tray is placed in a ‘reading box’ and the operator records the m.i.c. endpoint with a joy-stick or sensor. The system will then generate a report. Several studies have shown the automated microtitre m.i.c.s to correlate well with conventional procedures (Barry et al. 1978; Gavan et a/. 1980; Jones et a / . 1981).
2.4 O T H E R M E T H O D S In recent years several investigators have developed alternative methods for determining bacterial growth, and hence susceptibility to antimicrobial agents. The other methods have been based on a variety of bacterial metabolic activities, such as pH changes (Rogers et a/. 1955), changes in redox potential (Sellers 1970), electrical conductivity or impedance (Colvin & Sherris 1977), radiometric (Deland & Wagner 1970), bioluminescence (Velland et al. 1974) and microcalorimetry (Binford et a / . 1973). The use of pH changes to monitor growth depends on the ability of an organism to ferment a carbohydrate, usually glucose, and produce acid. A phenol red indicator that clearly turns yellow when growth occurs is used to monitor the pH change. For the strains studied the incubation times required to detect differences between antibiotic-free controls and tests varied from 1-8 h. This method has not been developed further as there has been a lack of quantitative endpoints and of procedural and interpretative standardization. The other problem is that the concentration of acid required to cause indicator dye changes may also alter the activity of certain agents. Indicator dyes are also used to detect changes in redox potential. Many such dyes also have inherent antibacterial activity, however, so resazurin or a n indophenol reagent (0.15% dichlorophenol indophenol in phosphate buffer pH 7.4) are usually used. Haemoglobin has also been used as an indicator of oxygen consumption, giving results within 2 h. The procedures that require indicator dyes have never been widely evaluated or accepted, and as none have received commercial research, and hence development of a standard method it is unlikely that these methods will be improved. It is possible, however, that indicator dyes may be incorporated into automated techniques for the early reading of microdilution tests. The electrical impedance (or relative conductivity) or resistance to the flow of alternating current through a broth medium changes as the growth of bacteria produces metabolic byproducts. If the bacterial population exceeds lo6 cfu/ml the impedance change can be measured reliably and used as a predictable measure of growth. Impedance is monitored by incorporating electrodes into each tube or cell and the culture is monitored continuously. In the impedance instrument changes in the test chambers containing an antibiotic compared with a control are recorded as ratios. If a lo7 cfu/ml inoculum is used a 6 h impedance test has a correlation of 74% with a standard overnight M I C test. False low m.i.c.s have been obtained for polymyxin and tetracycline and false high m.i.c.s for some beta-lactams. When results were analysed according to resistant, sensitive or intermediate and compared with a disc diffusion test the agreement was 85% (Thornsberry et a/. 1975). In the Bactec instrument (Becton Dickinson) organisms grow in media containing 14C-palmitic acid o r 14C-glucose as a carbon source, and monitors growth by the measurement of the concentration of 14C0, released into the atmosphere in a closed chamber (Deland 1972). This instrument was originally developed as a monitor for the early detection of growth in blood cultures. However, inhibition of growth can also be quantified. The concentration of antibiotic required t o produce 50% inhibition of 14C0, release (compared with an antibiotic-free control) after 3 h correlates well with results from broth MIC tests. Discrepancies were noted for staphylococci. The detection of bacterial growth using ATP-dependent bioluminescence has also been developed for studying the effect of antimicrobial agents. The method is based on the reaction of fire-fly luciferase and reduced luciferin and ATP and magnesium which forms a luciferin-AMP complex. This
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complex dissociates, releasing light which can be measured with a photometer, thereby assessing the amount of bacterial ATP in the culture. By comparing the luminescence of tubes with and without antibiotic after a period of time a ratio can be obtained. These ratios have been shown to have good agreement with broth dilution and agar diffusion assays (Velland et al. 1974). Discrepancies have been noted for Proteus mirabilis, Pseudomonas aeruginosa and enterococci.
2.5
ADVANTAGES A N D D!SADVANTAGES--WHICH
PROCEDURE?
The agar dilution technique for susceptibility testing has several advantages when compared with the broth dilution procedure. Inoculating devices allow up to 100 strains per plate to be tested at one time, so many organisms can be compared in parallel under the same conditions against the same antibiotic. Contamination of the agar can be readily detected in situ, unlike a broth culture where the turbidity due to growth of the test organism cannot usually be distinguished from that of a contaminant. A clear liquid media is necessary for broth dilution assays, and for fastidious organisms this may not be available, therefore these species can be tested only by agar procedures. The major disadvantages of agar dilution procedures is that the minimum bactericidal concentration (m.b.c.) cannot be easily determined, and for certain infections such as bacterial endocarditis the m.b.c. is often more relevant than the m.i.c. Automated methods also do not assess microbial killing well, and for many organisms 99-99.9% kill (such as for an m.b.c.) cannot be detected. All the tests are affected by media composition, pH, incubation temperature and length of incubation although some tests for some antibiotics are more affected than others. One critical consideration in all tests is the size of the inoculum. Whilst the clinical laboratory is always striving to perform a test that has clinical relevance, because of constraints of time and technological ease, certain parameters such as inoculum size in susceptibility testing have become standardized. Whilst an inoculum of 103-104 organisms/ml may represent the concentration of bacteria infecting the blood in patients with endocarditis, there may be 107-108organisms/ml of cerebrospinal fluid in patients with meningitis. However, if a low inoculum is used in susceptibility tests a false-sensitive result may be obtained, and if a high inoculum is used a false-resistant result may be obtained, particularly if a broth dilution procedure has been used. If a high inoculum is used it is possible to select a subpopulation of bacteria that have spontaneously mutated to become antibiotic resistant, and it is these organisms that grow up with a higher m.i.c. As they usually only occur at a frequency of 1 in lo7 (the frequency of a mutation in a gene) the test provides an ideal environment for their selection and growth, unlike the human body where factors other than the presence of an antibiotic contribute towards bacterial killing. For these reasons an inoculum of lo5 cfu/ml is usually employed for broth dilution tests and lo4 and lo6 cfu/ml in agar dilution tests. The disc diffusion susceptibility test has the advantage of readily detecting antagonism or synergy between two antibiotics. If the discs are placed so that antibiotics which are known to cause these effects are placed in close proximity to each other then antagonism can be observed by a blunting of the zone of inhibition of one antibiotic caused by the other. Synergy can be seen by the enhancement of the zone of inhibition. Antagonism can be seen when one antibiotic interferes with the mechanism of another, such as with tetracycline and gentamicin. It can also occur when one antibiotic induces enzymes that can hydrolyse another antibiotic, such as erythromycin and spiromycin with erythromycin-resistant Staph. aureus, and cefoxitin and cefotaxime in Enterobacter cloacae. Whether the observed antagonism correlates with failure of therapy is dependent upon the type and site of infection and the pharmacokinetics of the antibiotic. Automatic procedures that rely upon optical density or nephelometry for detecting antibacterial activity can give false-resistant results for some antibiotics. Beta-lactams and quinolones cause filamentation of bacterial cells (as well as other morphological changes), and filamentation can cause an increase in turbidity when, in fact, the number of viable cells is decreasing.
3. Designation of sensitive or resistant (interpretation of tests) The disc diffusion test is the most frequeptly used procedure for determining the susceptibility of clinical strains to antimicrobial agents. The diameters of the zone of inhibition of each agent are
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usually interpreted as ‘sensitive’, ‘resistant’ or ‘intermediate’. The term ‘sensitive’ implies an organism that will respond to the test antibiotic; ‘resistant’, that the therapy is unlikely to be effective; and ‘intermediate’ where the organism is only likely to respond where high concentrations of the agent are achievable. The three categories are established from data known about the relationship between the m.i.c. (usually from agar dilution tests) and the zone size, the distribution of the strains into each class of susceptibility and the pharmacokinetics of the antibiotic. As might be anticipated, different species and different antibiotics have separate interpretive values. To interpret the zone size, it must first be related to an m.i.c. This is done by performing m.i.c. tests and disc diffusion tests in parallel for a particular agent under well-defined conditions. Each zone diameter is then plotted graphically against the m.i.c. for that strain (on a logarithmic scale). A regression line is constructed with the method of least squares to establish the relationship between the zone size and the m.i.c. The zone size breakpoint is usually obtained by applying the attainable therapeutic concentration of the agent (usually in serum) to the regression line. The calculation of breakpoint concentrations is discussed in more detail later in this section. The use of regression line analysis has been criticized by Krasemann & Hildenbran (1980). Firstly, individual strains of the same species may have a wide variety of zone sizes, and a wide range of m.i.c.s may be obtained by using twofold dilution steps in the m.i.c. determination. The construction of the regression line may mask the variation and will not indicate the probability of false-sensitive or false-resistant data. The use of regression analysis in this fashion also neglects certain mathematical dogma. Often, data for sensitive and resistant organisms of different species are plotted on the same graph, therefore those for resistant Ent. cloacae and sensitive staphylococci will not necessarily give data for resistant staphylococci. Linearity of the data may not occur and therefore a mathematical error will be introduced by constructing a regression line; in addition, the regression line is constructed on the assumption that the m.i.c. data are correct. As stated earlier, however, both the zone size and the m.i.c. can be variables: therefore, two lines should be calculated (one for zone sizes based on m.i.c.s and the other for m.i.c.s based on zone sizes). In most instances, the two lines will be very similar. To overcome the problems of regression analysis and to obtain a more valid breakpoint zone size, the rate of false-sensitive and false-resistant is calculated by the error rate-bound method (Brownlee 1965; Metzler & Daham 1974). A scattergram of the m.i.c. data is plotted as a log, value against the zone diameter. A horizontal line, M, , is drawn corresponding to a therapeutically achievable concentration in serum and tissues. A vertical line, Z,, is selected for the zone diameter that is equal to or less than the m.i.c.s of resistant strains, giving a rate of error for false-resistant of less than or equal to 5%. Another vertical line, Z,, is selected for the zone diameter that is equal to or greater than the m.i.c.s of sensitive strains, giving a rate of error for false-susceptible of less than or equal to 1‘51.The scatter-gram of the data is therefore divided into six areas (Fig. 1). The top left area is the area of true resistant, the bottom left the false resistant (
