what can you conclude about the susceptibility of the bacteria to the antibiotics?

Abstruse

An of import task of the clinical microbiology laboratory is the performance of antimicrobial susceptibility testing of significant bacterial isolates. The goals of testing are to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of choice for particular infections. The most widely used testing methods include broth microdilution or rapid automated musical instrument methods that use commercially marketed materials and devices. Manual methods that provide flexibility and possible cost savings include the disk diffusion and slope diffusion methods. Each method has strengths and weaknesses, including organisms that may be accurately tested by the method. Some methods provide quantitative results (eg, minimum inhibitory concentration), and all provide qualitative assessments using the categories susceptible, intermediate, or resistant. In general, current testing methods provide accurate detection of common antimicrobial resistance mechanisms. However, newer or emerging mechanisms of resistance crave constant vigilance regarding the ability of each test method to accurately detect resistance.

Emergence of Antimicrobial Resistance and the Rationale for Performing Susceptibility Testing

The operation of antimicrobial susceptibility testing by the clinical microbiology laboratory is important to confirm susceptibility to chosen empirical antimicrobial agents, or to detect resistance in individual bacterial isolates. Empirical therapy continues to be effective for some bacterial pathogens because resistance mechanisms have not been observed e.chiliad., continued penicillin susceptibility of Streptococcus pyogenes . Susceptibility testing of private isolates is important with species that may possess caused resistance mechanisms (eg, members of the Enterobacteriaceae, Pseudomonas species, Staphylococcus species, Enterococcus species, and Streptococcus pneumoniae ).

Overview of Commonly Used Susceptibility Testing Methods

Broth dilution tests. Ane of the earliest antimicrobial susceptibility testing methods was the macrobroth or tube-dilution method [1]. This procedure involved preparing two-fold dilutions of antibiotics (eg, 1, 2, 4, 8, and 16 µg/mL) in a liquid growth medium dispensed in test tubes [1, 2]. The antibiotic-containing tubes were inoculated with a standardized bacterial suspension of one–5×10^fiveCFU/mL. Following overnight incubation at 35°C, the tubes were examined for visible bacterial growth every bit evidenced by turbidity. The lowest concentration of antibiotic that prevented growth represented the minimal inhibitory concentration (MIC). The precision of this method was considered to be plus or minus 1 2-fold concentration, due in large part to the practice of manually preparing serial dilutions of the antibiotics [three]. The advantage of this technique was the generation of a quantitative result (ie, the MIC). The master disadvantages of the macrodilution method were the tedious, manual job of preparing the antibiotic solutions for each test, the possibility of errors in preparation of the antibody solutions, and the relatively large amount of reagents and space required for each exam.

The miniaturization and mechanization of the examination by utilise of small-scale, disposable, plastic "microdilution" trays (Figure ane) has made broth dilution testing practical and pop. Standard trays contain 96 wells, each containing a volume of 0.1 mL that allows approximately 12 antibiotics to be tested in a range of 8 two-fold dilutions in a single tray [2, 4]. Microdilution panels are typically prepared using dispensing instruments that aliquot precise volumes of preweighed and diluted antibiotics in goop into the private wells of trays from large book vessels. Hundreds of identical trays can exist prepared from a single chief set up of dilutions in a relatively brief period. Few clinical microbiology laboratories gear up their ain panels; instead frozen or dried microdilution panels are purchased from one of several commercial suppliers. The toll of the preprepared panels range from approximately $10 to $22 each. Inoculation of panels with the standard 5×10⁵CFU/mL is accomplished using a dispensable device that transfers 0.01 to 0.05 mL of standardized bacterial suspension into each well of the microdilution tray or by use of a mechanized dispenser. Following incubation, MICs are determined using a manual or automated viewing device for inspection of each of the panel wells for growth [2].

Effigy 1

A goop microdilution susceptibility console containing 98 reagent wells and a disposable tray inoculator

The advantages of the microdilution process include the generation of MICs, the reproducibility and convenience of having preprepared panels, and the economy of reagents and space that occurs due to the miniaturization of the exam. There is also assistance in generating computerized reports if an automated panel reader is used. The main disadvantage of the microdilution method is some inflexibility of drug selections available in standard commercial panels.

Antimicrobial gradient method. The antimicrobial slope diffusion method uses the principle of institution of an antimicrobial concentration slope in an agar medium as a means of determining susceptibility. The Etest (bioMérieux AB BIODISK) (Effigy 2) is a commercial version available in the United States. It employs sparse plastic test strips that are impregnated on the underside with a dried antibiotic concentration gradient and are marked on the upper surface with a concentration scale. As many every bit 5 or 6 strips may be placed in a radial manner on the surface of an advisable 150-mm agar plate that has been inoculated with a standardized organism suspension like that used for a disk diffusion examination. After overnight incubation, the tests are read by viewing the strips from the tiptop of the plate. The MIC is determined by the intersection of the lower part of the ellipse shaped growth inhibition area with the exam strip.

Figure 2

A Staphylococcus aureus isolate tested past the Etest gradient diffusion method with vancomycin (VA), daptomycin (DM), and linezolid (LZ) on Mueller-Hinton agar. The minimum inhibitory concentration of each agent is determined by the intersection of the organism growth with the strip as measured using the scale inscribed on the strip.

The gradient improvidence method has intrinsic flexibility by existence able to test the drugs the laboratory chooses. Etest strips price approximately $2-$3 each and tin represent an expensive approach if more than a few drugs are tested. This method is all-time suited to situations in which an MIC for only 1 or 2 drugs is needed or when a fastidious organism requiring enriched medium or special incubation atmosphere is to exist tested (eg, penicillin and ceftriaxone with pneumococci) [v-7]. Generally, Etest results have correlated well with MICs generated by goop or agar dilution methods [5-9]. However, in that location are some systematic biases toward higher or lower MICs adamant past the Etest when testing certain organism-antimicrobial agent combinations [6, 10]. This tin can stand for a potential shortcoming when standard MIC interpretive criteria derived from goop dilution testing [10] are applied to Etest MICs that may non exist identical.

Deejay improvidence test. The deejay diffusion susceptibility method [2, 11, 12] is simple and practical and has been well-standardized. The test is performed past applying a bacterial inoculum of approximately 1–2×10⁸CFU/mL to the surface of a large (150 mm diameter) Mueller-Hinton agar plate. Up to 12 commercially-prepared, fixed concentration, paper antibiotic disks are placed on the inoculated agar surface (Effigy 3). Plates are incubated for sixteen–24 h at 35°C prior to determination of results. The zones of growth inhibition effectually each of the antibody disks are measured to the nearest millimeter. The diameter of the zone is related to the susceptibility of the isolate and to the improvidence rate of the drug through the agar medium. The zone diameters of each drug are interpreted using the criteria published by the Clinical and Laboratory Standards Institute (CLSI, formerly the National Commission for Clinical Laboratory Standards or NCCLS) [13] or those included in the US Nutrient and Drug Administration (FDA)-approved product inserts for the disks. The results of the disk diffusion exam are "qualitative," in that a category of susceptibility (ie, susceptible, intermediate, or resistant) is derived from the test rather than an MIC. However, some commercially-available zone reader systems merits to calculate an judge MIC with some organisms and antibiotics by comparing zone sizes with standard curves of that species and drug stored in an algorithm [14, 15].

Figure 3

A deejay diffusion exam with an isolate of Escherichia coli from a urine civilization. The diameters of all zones of inhibition are measured and those values translated to categories of susceptible, intermediate, or resistant using the latest tables published past the CLSI.

The advantages of the disk method are the test simplicity that does not require whatever special equipment, the provision of chiselled results hands interpreted past all clinicians, and flexibility in selection of disks for testing. It is the least costly of all susceptibility methods (approximately $2.50-$5 per test for materials). The disadvantages of the disk test are the lack of mechanization or automation of the test. Although not all fastidious or slow growing leaner can be accurately tested by this method, the disk exam has been standardized for testing streptococci, Haemophilus influenzae, and N. meningitidis through employ of specialized media, incubation conditions, and specific zone size interpretive criteria [12].

Automated musical instrument systems. Use of instrumentation tin standardize the reading of terminate points and often produce susceptibility test results in a shorter flow than manual readings considering sensitive optical detection systems allow detection of subtle changes in bacterial growth. There are 4 automatic instruments presently cleared by the FDA for use in the United States. Three of these can generate rapid (3.five–xvi h) susceptibility exam results, while the fourth is an overnight system [16]. The MicroScan WalkAway (Siemens Healthcare Diagnostics) is a large self-independent incubator/reader device that can incubate and clarify forty–96 microdilution trays. The WalkAway utilizes standard size microdilution trays that are hydrated and inoculated manually and so placed in i of the incubator slots in the musical instrument. The musical instrument incubates the trays for the appropriate period, examining them periodically with either a photometer or fluorometer to make up one's mind growth development. Gram-negative susceptibility examination panels containing fluorogenic substrates can exist read in 3.v–seven h. Split up gram-positive and gram-negative panels read using turbidimetric end points are ready in 4.5–eighteen hours.

The BD Phoenix Automated Microbiology Organization (BD Diagnostics) has a large incubator reader with a capacity to procedure 99 exam panels that contain 84 wells devoted to antibiotic doubling dilutions and are inoculated manually. The Phoenix monitors each console every twenty min using both turbidometric and colorimetric (oxidation-reduction indicator) growth detection. Test panels for gram-negative, gram-positive, Due south. pneumoniae, β-hemolytic, and viridans grouping streptococci are bachelor. MIC results are generated in 6–sixteen h.

The Vitek ii Organization (bioMérieux) is highly automated and uses very compact plastic reagent cards (credit card size) that contain microliter quantities of antibiotics and test media in a 64-well format. The Vitek 2 employs repetitive turbidimetric monitoring of bacterial growth during an abbreviated incubation period. The instrument can be configured to accommodate thirty–240 simultaneous tests. The susceptibility cards allow testing of mutual, apace growing gram-positive, and gram-negative aerobic leaner, and S. pneumoniae in a period of 4–10 h. An older, less automated, Vitek 1 System is still used in some laboratories. The system is more limited with a 45-well card and does not include S. pneumoniae .

The Sensititre ARIS 2X (Expedition Diagnostic Systems) is an automated, overnight, incubation and reading system with a 64-panel capacity. The test panels are standard 96-well microdilution plates that tin can be inoculated with a Sensititre Autoinculator. Growth is determined by fluorescence measurement subsequently 18–24 h of incubation. Test panels are available for gram-positive and gram-negative bacteria, S. pneumoniae, Haemophilus species, and nonfermentative gram-negative bacilli.

The Phoenix, Sensititre ARIS 2X, Vitek 1 and ii, and WalkAway instruments take enhanced figurer software used to translate susceptibility results including "adept systems" for analyzing test results for singular patterns and unusual resistance phenotypes [16]. 2 studies [17, 18] take shown that providing rapid susceptibility test results tin can lead to more timely changes to appropriate antimicrobial therapy, substantial direct price savings owing to ordering of fewer additional laboratory tests, performance of fewer invasive procedures, and a shortened length of stay. These benefits are all-time realized when coupled with extended laboratory staffing schedules, and existent-time, electronic transmission of verified results. One of the early shortcomings of rapid susceptibility testing methods was a lessened ability to detect some types of antimicrobial resistance including inducible β-lactamases and vancomycin resistance. However, the recently FDA-cleared instruments have made significant improvements in large part through modifications of the instruments' calculator software to either provide extended incubation for problematic organism-drug combinations, or by editing of susceptibility results using expert software to foreclose unlikely results from existence reported. In some cases these modifications result in prolonged incubation (ie, >10 h) of test panels to clinch accurate results, thus rendering them less "rapid."

Selection of Drugs for Routine Testing

The laboratory must test and report the antimicrobial agents that are most appropriate for the organism isolated, for the site of the infection, and the institution's formulary [13, nineteen]. The CLSI provides tables that list the antimicrobial agents advisable for testing members of the Enterobacteriaceae, Pseudomonas, and other gram-negative glucose nonfermenters, staphylococci, enterococci, streptococci, Haemophilus species, etc. [13]. The listings include recommendations for agents that are important to test routinely, and those that may exist tested or reported selectively based on the institution's formulary.

The availability of antimicrobial agents for testing by the laboratory'south routine testing methodology must next be adamant. The deejay improvidence and gradient diffusion procedures offer the greatest flexibility including testing of newly bachelor drugs. Near broth microdilution or automated exam panels contain ⩽96 wells, effectively limiting the number of agents tested or the range of dilutions of each drug that can exist included. Manufacturers of commercially prepared panels have attempted to bargain with this trouble by offering a number of different standard panel configurations, or by including fewer dilutions of each drug in a single panel [nineteen]. Another solution to this problem is testing antimicrobial agents that have activities that are essentially the same equally the desired formulary drugs. The CLSI susceptibility testing document [13] lists groups of some antimicrobial agents with nearly identical activities that can provide practical alternatives for testing.

Interpretation of Susceptibility Examination Results

The results of a susceptibility test must exist interpreted by the laboratory prior to communicating a written report to a patient's physician. Optimal interpretation of MICs requires knowledge of the pharmacokinetics of the drug in humans, and information on the likely success of a particular drug in eradicating bacteria at diverse trunk sites [20]. This is best accomplished past referring to an expert source such as the CLSI, which publishes interpretive criteria for MICs of all relevant antibiotics for most bacterial genera [13]. Indeed, both MIC values and deejay improvidence zone diameters must exist interpreted using a table of values that relate to proven clinical efficacy of each antibody and for diverse bacterial species [12]. The CLSI zone size and MIC interpretive criteria are established by analysis of 3 kinds of information: (1.) microbiologic information, including a comparing of MICs and zone sizes on a big number of bacterial strains, including those with known mechanisms of resistance that have been defined either phenotypically or genotypically; (two) pharmacokinetic and pharmacodynamic data; and (three) clinical studies results (including comparisons of MIC and zone diameter with microbiological eradication and clinical efficacy) obtained during studies prior to FDA approval and marketing of an antibody [20].

A "susceptible" result indicates that the patient's organism should respond to therapy with that antibiotic using the dosage recommended normally for that type of infection and species [thirteen, 20]. Conversely, an organism with a MIC or zone size interpreted as "resistant" should not exist inhibited by the concentrations of the antibiotic achieved with the dosages normally used with that drug [13, 20]. An "intermediate" outcome indicates that a microorganism falls into a range of susceptibility in which the MIC approaches or exceeds the level of antibiotic that can ordinarily be achieved and for which clinical response is likely to exist less than with a susceptible strain. Exceptions can occur if the antibiotic is highly full-bodied in a torso fluid such equally urine, or if higher than normal dosages of the antibiotic tin be safely administered (eg, some penicillins and cephalosporins). At times, the "intermediate" result can too mean that certain variables in the susceptibility examination may not have been properly controlled, and that the values have fallen into a "buffer zone" separating susceptible from resistant strains [13, 20]. Generally, reporting of a category upshot of susceptible, intermediate, or resistant provides the clinician with the information necessary to select appropriate therapy. Reporting of MICs could help a physician is selecting from amongst a group of similar drugs for therapy of infective endocarditis or osteomyelitis, in which therapy is probable to be protracted.

It is important that the tables used for susceptibility examination interpretations represent the most current criteria. Indeed, the CLSI documents are reviewed and updated oftentimes, usually once per yr. Use of old or outdated information from the original editions of FDA-approved drug labels or older CLSI tables could represent a serious shortcoming in the reporting of patients' results.

What Is the Acceptable Accuracy of a Susceptibility Exam Method?

When assessing the accuracy of various susceptibility testing methods as compared to standard reference methods, the terms very major and major errors accept been used to describe false-susceptible or false-resistant results, respectively. In evaluations of new susceptibility testing methods it is of import to examine a representative number of strains that are resistant to various drugs to verify the ability of the new examination to detect resistance and to test a number of susceptible strains to determine the rate of major errors that might be expected in a typical clinical laboratory setting [16, 21]. To be cleared for marketing in the U.s.a., the FDA requires that very major errors attributable to a test device should exist <1.5% for individual species/drug comparisons, major errors should not exceed three%, and an overall essential MIC understanding of >90% of device MICs within ane doubling dilution of a CLSI reference MIC [22]. A contempo, international standard on susceptibility test device evaluation proposes similar simply not identical criteria for acceptable accuracy [23]. The emergence of new antimicrobial resistance mechanisms, including some that may be difficult to detect (eg, vancomycin intermediate susceptibility in S. aureus and carbapenemase production in some gram-negative organisms) requires that the performance of susceptibility devices be constantly reassessed and updated when needed. In some cases, it has been necessary to utilise special coincident testing methods (eg, single concentration screening agars, modified Hodge test for carbapenemase production) [13] to supplement routine testing by a commercial instrument organisation.

Current Test Methods and Future Directions

The antimicrobial susceptibility testing methods described in this article provide reliable results when used according to the procedures defined past the CLSI or past the manufacturers of the commercial products. However, there is considerable opportunity for improvement in the area of rapid and accurate recognition of bacterial resistance to antibiotics. At that place is a need for development of new automated instruments that could provide faster results and as well salvage money past virtue of lower reagent costs and reduced labor requirements. To accomplish this, it will likely be necessary to explore different methodologic approaches for detection of bacterial growth. The direct detection of resistance genes by polymerase concatenation reaction or similar techniques has limited utility, because only a few resistance genes are firmly associated with phenotypic resistance (eg, mecA, vanA, and vanB ) [24]. There are hundreds of β-lactamases, and numerous mutations, acquisitions, and expression mechanisms that consequence in fluoroquinolone, aminoglycoside, and macrolide resistance [25]; too many to be easily detected past electric current molecular techniques. Thus, it seems probable that phenotypic measures of the level of susceptibility of bacterial isolates to antimicrobial agents will go on to be clinically relevant for years to come.

Acknowledgments

Potential conflicts of interest. J.H.J. and Thou.J.F. disclose their membership on microbiology advisory committees for BD Diagnostics and bioMérieux. J.H.J. has advised Accelr8 Technology and has received research support from BD Diagnostics, bioMérieux, Merck, Pfizer, and Siemens Healthcare.

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campbellprole1994.blogspot.com

Source: https://academic.oup.com/cid/article/49/11/1749/344384

what can you conclude about the susceptibility of the bacteria to the antibiotics?

Abstruse

Emergence of Antimicrobial Resistance and the Rationale for Performing Susceptibility Testing

Overview of Commonly Used Susceptibility Testing Methods

Selection of Drugs for Routine Testing

Interpretation of Susceptibility Examination Results

What Is the Acceptable Accuracy of a Susceptibility Exam Method?

Current Test Methods and Future Directions

Acknowledgments

References

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