Methods for Antimicrobial Susceptibility Testing for Human Mycoplasmas; Approved Guideline (original) (raw)

Abstract

Antimicrobial susceptibility testing is indicated for any organism that contributes to an infectious process warranting antimicrobial chemotherapy, if its susceptibility cannot be reliably predicted from knowledge of the organism’s identity. Standardized in vitro antimicrobial susceptibility tests are also needed in order to evaluate new antimicrobials against specific groups of organisms in comparison with existing agents. Acquired resistance to one or more classes of antimicrobial agents has now emerged in the major mycoplasmal and ureaplasmal species that infect humans, hence the need to establish accurate and reproducible methods to measure antimicrobial activities in vitro with these organisms.

This document provides guidelines for performance, interpretation, and quality control of in vitro broth microdilution and agar dilution susceptibility tests for several antimicrobial agents suitable for use against Mycoplasma pneumoniae (M. pneumoniae), Mycoplasma hominis (M. hominis), and Ureaplasma species (Ureaplasma spp). Information in this document includes designated reference strains and the expected minimal inhibitory concentration ranges for specific drugs that should be obtained when they are tested.

Clinical and Laboratory Standards Institute (CLSI). Methods for Antimicrobial Susceptibility Testing for Human Mycoplasmas; Approved Guideline. CLSI document M43-A (ISBN 1-56238-769-3 [Print]; ISBN 1-56238-770-7 [Electronic]). Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2011.

Committee Membership

Consensus Committee on Microbiology

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Document Development Committee on Antimicrobial Susceptibility Testing of Human Mycoplasmas

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Antimicrobial Susceptibility Testing Quality Control Working Group

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In addition, CLSI and the Document Development Committee on Antimicrobial Susceptibility Testing of Human Mycoplasmas would like to acknowledge the assistance of the following individuals for the performance of studies that generated laboratory data included in this document:

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Acknowledgments

CLSI and the Document Development Committee on Antimicrobial Susceptibility Testing of Human Mycoplasmas acknowledge the experts and their institutions, listed below, for their review, advice, and assistance in determining acceptable limits for quality control within this guideline:

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Foreword

Methods for in vitro susceptibility testing of mycoplasmas were first described in the 1960s. Despite numerous publications over four decades that have reported activities of antimicrobial agents against these organisms using broth- and agar-based methodologies, there has been no universally accepted standardized reference method for testing conditions, media, or quality control (QC) minimal inhibitory concentration (MIC) reference ranges for antimicrobial agents. Lack of a consensus method for MIC determination and the complex in vitro growth conditions required by these fastidious organisms has led to considerable confusion and misinformation regarding antimicrobial activities of various drugs.

The need for standardized antimicrobial susceptibility testing (AST) methods and designated QC parameters for human mycoplasmas is not primarily related to a need for diagnostic laboratories to perform testing for every individual clinical specimen submitted for mycoplasma or ureaplasma culture. Conversely, it is needed because such culture-based testing is not routinely performed; susceptibilities may vary geographically and in response to selective antimicrobial pressure; and clinically significant acquired drug resistance potentially affecting multiple antimicrobial classes occurs in all of the most important mycoplasmal and ureaplasmal human pathogens. Most mycoplasmal and ureaplasmal infections are treated empirically. Thus, standardized AST methods are needed for surveillance of clinical isolates for resistance to currently available drugs due to potential development of resistance, because treatment is usually empirical and individual clinical isolates may need to be tested in special circumstances. Standardized AST methods are also useful to pharmaceutical companies that perform their own testing during drug development, and to reference laboratories that assist in drug development by performing AST. Such testing is required during the initial evaluation of any investigational drug for which an indication for treating infections that may be caused by these organisms is anticipated.

During the past several years, method descriptions and direct comparisons of agar- and broth-based in vitro AST methods for testing human mycoplasmas and ureaplasmas were published.1,2 The Chemotherapy Working Team of the International Research Program on Comparative Mycoplasmology attempted to optimize media selection and testing conditions, and at least one multilaboratory investigation was undertaken to compare results. Many aspects of these procedures were incorporated directly into the protocols described in this document. However, five important factors were lacking in these earlier attempts to develop in vitro AST methods: 1) there was no organizing infrastructure to coordinate multilaboratory testing; 2) no attempt was made to determine intralaboratory reproducibility of testing results; 3) no standardized medium or testing protocol was adopted by all participating laboratories; 4) there were no designated readily available reference strains used; and 5) testing was limited to Ureaplasma spp. These deficiencies were addressed in the work that led to M43.

This guideline is the first publication under the direction of CLSI to describe standardized methods for broth microdilution– and agar dilution–based susceptibility testing of human mycoplasmas and ureaplasmas; the first to designate QC reference strains with defined MIC ranges for various antimicrobial agents; and the first document from any organization to propose interpretive breakpoints for selected antimicrobial agents for use against human mycoplasmas and ureaplasmas. The document was developed using data obtained from six academic microbiology laboratories in the United States, Canada, and France; two US pharmaceutical company microbiology laboratories; a microbiology reference laboratory in the United States; and the Centers for Disease Control and Prevention.

In addition to providing guidelines for performing in vitro AST and listing acceptable MIC ranges for specified reference strains for agar- and broth-based test methods, this document also includes recommendations regarding selection of antimicrobials for testing against mycoplasmas and ureaplasmas as well as recommendations for MIC interpretive criteria for a limited number of drugs. However, this document does not endorse or recommend the use of any specific antimicrobial agent for treatment of mycoplasmal or ureaplasmal infections.

All of the methodology and MIC reference ranges in this document were reviewed and approved by the Antimicrobial Susceptibility Testing Quality Control Working Group and subjected to the CLSI consensus process before finalization and publication. The document development committee expects that this document will provide a valuable educational resource for researchers, clinical microbiologists, and the pharmaceutical industry in the United States and in other countries.

Ken B. Waites, MD

Chairholder, Document Development Committee on Antimicrobial Susceptibility Testing of Human Mycoplasmas

Note that the trade names IsoVitaleX® and Select agar® are included in Appendix A. It is the Clinical and Laboratory Standards Institute’s policy to avoid using a trade name unless the product identified is the only one available, or it serves solely as an illustrative example of the procedure, practice, or material described. In this case, the document development committee and the consensus committee believe the trade names are used to provide instructions for preparation of the agars used for the dilution method for minimal inhibitory concentration assays, because some commercial broths may require special order to ensure they do not contain other antimicrobial agents routinely incorporated to prevent bacterial overgrowth. Because these trade names are important descriptive adjuncts to the document, it is acceptable to use the products’ trade names, as long as the words “or the equivalent” are added to the references. It should be understood that information on these products in this guideline also applies to any equivalent products. Please include in your comments any information that relates to this aspect of M43.

Key Words

Agar dilution, antimicrobial susceptibility testing, broth microdilution, minimal inhibitory concentration, Mycoplasma, Ureaplasma

1. Scope

This document contains standardized protocols for broth microdilution and agar dilution in vitro susceptibility testing for isolates of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma spp. It describes the optimum media formulations for use in broth microdilution and agar dilution assays for each species; provides minimal inhibitory concentration (MIC) quality control (QC) reference ranges for ATCC®a type strains; and offers recommendations for selection of antimicrobials for routine testing and MIC interpretive criteria for a limited number of drugs.

This guideline is intended for use by hospital clinical laboratories; reference microbiology laboratories; and government, industry, and academic research organizations that perform diagnostic testing and/or conduct research in mycoplasmal diseases that affect humans.

2. Introduction

Various methods of antimicrobial susceptibility testing (AST) used for conventional bacteria have been employed for testing mycoplasmas and ureaplasmas. Agar dilution has been used extensively as a reference method. It has the advantages of a relatively stable end point over time, and it allows detection of mixed cultures. However, this technique is not practical for testing small numbers of strains or occasional isolates that may be encountered in diagnostic laboratories.1,3 Agar disk diffusion is not useful for testing mycoplasmas because there has been no correlation between inhibitory zones and MICs, and the relatively slow growth of some of these organisms further limits this technology.3 Broth microdilution is the most widely used method to determine MICs for mycoplasmas and ureaplasmas. It allows several antimicrobials to be tested in the same microdilution plates, but, in addition to being labor intensive, it has a shifting end point over the time required for growth of some Mycoplasma spp.3 Studies using the agar gradient diffusion technique for detection of tetracycline resistance in M. hominis yielded results comparable to broth microdilution.2 Additional comparative studies have also evaluated this method for determination of in vitro susceptibilities of M. hominis to fluoroquinolones and susceptibilities of ureaplasmas to various other antimicrobials.4,5 Agar gradient diffusion has the advantages of simplicity of agar-based testing, has an end point that does not shift over time, does not have a large inoculum effect, and can easily be adapted for testing single isolates.3

Irrespective of methodology, there have been no universally accepted standards for pH, media composition, incubation conditions, or duration of incubation for performing mycoplasmal or ureaplasmal susceptibility tests. Because of inherent differences in their cultivation requirements and growth rates, no single procedure or medium can be considered sufficient for testing all of the clinically important species. No QC organisms, QC interpretive criteria for antimicrobial agents, or MIC breakpoints for use with clinical isolates of Mycoplasma spp. and Ureaplasma spp. have been endorsed by any agency or organization to date. Specific challenges that have hampered previous attempts to develop standardized assays and demonstrate reproducibility of various methods for determining in vitro susceptibilities of these organisms include the difficulty in measuring the concentration of organisms in the inoculum and the detection of growth in liquid medium because their small size does not result in visible turbidity; the low pH necessary for optimum growth of ureaplasmas and generation of an end point in MIC assays; the limited availability (from commercial sources) of complex media formulations necessary to support growth in vitro; the relatively slow growth for some species; and the tendency for broth dilution end points to shift over time.

Commercial MIC panels and kits, which have been available in Europe for several years, consist of microwells containing dried antimicrobials, generally in two concentrations, corresponding to thresholds proposed for conventional bacteria to classify a strain as susceptible, intermediate, or resistant. These kits give results comparable to those obtained by conventional methods of MIC determination.6 Some of these products are now sold in the United States, but they have not yet gained widespread use, have not been rigorously compared to nonproprietary methods for MIC determination, and do not necessarily follow recommendations included in this document.

Procedures for agar- and broth-based in vitro susceptibility assays and the corresponding QC procedures, reference strains, and MIC ranges relevant to both techniques are included in this document. This was done because individual laboratories may have a preference for one or the other assay format, depending on their test volume, frequency, numbers of drugs to test, and other individual needs. Because of the complexity and time-consuming nature of in vitro cultivation of human mycoplasmas and ureaplasmas, and the relative infrequent need and impracticality for performing AST on clinical isolates, the document development committee does not believe that the procedures described in this guideline will be widely used in hospital-based clinical laboratories. However, large-volume regional or national reference laboratories, government public health laboratories that perform surveillance for antimicrobial resistance, and pharmaceutical company laboratories will benefit from having these guidelines for use, should the need arise to evaluate in vitro susceptibilities of human mycoplasmas for existing or investigational antimicrobial agents.

The three separate and distinct procedures for agar and broth microdilution described in this guideline are individualized for M. hominis, M. pneumoniae, and Ureaplasma spp. (Ureaplasma spp. includes both U. parvum and U. urealyticum, which behave in similar manners and for which the same procedures, QC, and interpretive criteria apply.) There has been no attempt to generalize these methods for application to other mycoplasmal species of human or animal origin, which may have very different growth and testing requirements. Therefore, these procedures should be limited to testing only the organisms for which they are described. Despite the efforts of the laboratories participating in this multicenter project to determine MIC ranges for reference strains for all currently available antimicrobial agents relevant for testing against these organisms, there was lack of consensus for assignment of ranges for some drugs by agar and/or broth methods, so these were omitted from Tables 1 and 2 in this document after careful examination of the data by the Document Development Committee on Antimicrobial Susceptibility Testing of Human Mycoplasmas and the Antimicrobial Susceptibility Testing Quality Control Working Group.

2.1. Development of Minimal Inhibitory Concentration Interpretive Criteria or Breakpoints

Even though in vitro susceptibility data of mycoplasmas and ureaplasmas have been reported many times since the 1960s, and various publications have provided interpretations of MIC values, this has been done without authorization of any regulatory or advisory organizations in the United States or other countries. The Document Development Committee on Antimicrobial Susceptibility Testing of Human Mycoplasmas recognizes the fact that if there is a published guideline for performance and QC of in vitro AST for these organisms, it will be more valuable to users if there is some guidance regarding the meaning of the MIC values that are obtained when using the recommended procedures. The process of establishing MIC interpretive criteria or breakpoints for a drug for conventional bacteria is complex, and when such criteria are recognized and approved by the US Food and Drug Administration and/or CLSI, considerable data must be presented and analyzed. This includes evaluation of in vitro MIC determinations that include organisms with and without well-characterized resistance mechanisms that affect the activities of the drug, pharmacokinetic and pharmacodynamic parameters, and clinical and bacteriological outcomes of patients enrolled in large clinical trials. The process of establishing MIC breakpoints is described in CLSI document M23_._7 As was the case for fastidious and uncommon bacteria described in CLSI document M45,8 clinical and microbiological data are not available for human mycoplasmas and ureaplasmas, and such information is not likely to be generated. MIC interpretive criteria are proposed in M43 for drugs for which QC data are included. To the extent possible, the interpretive criteria proposed within this document are based on 1) relating MICs to the presence or absence of resistance determinants such as tet(M) (tetracyclines), ribosomal RNA (rRNA) mutations (erythromycin), or mutations in quinolone resistance determination regions (QRDRs) of DNA (fluoroquinolones); or 2) relating MICs for mycoplasmas and ureaplasmas for specific drugs to those of other bacteria for which interpretive criteria for the same drugs have been established (see Tables 3 through 5).

3. Standard Precautions

Because it is often impossible to know what isolates or specimens might be infectious, all patient and laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard precautions are guidelines that combine the major features of “universal precautions and body substance isolation” practices. Standard precautions cover the transmission of all known infectious agents and thus are more comprehensive than universal precautions, which are intended to apply only to transmission of blood-borne pathogens. Standard and universal precaution guidelines are available from the Centers for Disease Control and Prevention.9 For specific precautions for preventing the laboratory transmission of all known infectious agents from laboratory instruments and materials and for recommendations for the management of exposure to all known infectious diseases, refer to CLSI document M29.10

4. Terminology

4.1. A Note on Terminology

CLSI, as a global leader in standardization, is firmly committed to achieving global harmonization whenever possible. Harmonization is a process of recognizing, understanding, and explaining differences while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the global metrological community have evolved differently in the United States, Europe, and elsewhere; that these differences are reflected in CLSI, International Organization for Standardization, and European Committee for Standardization (CEN) documents; and that legally required use of terms, regional usage, and different consensus timelines are all important considerations in the harmonization process. In light of this, CLSI’s consensus process for development and revision of standards and guidelines focuses on harmonization of terms to facilitate the global application of standards and guidelines.

4.2. Definitions

agar dilution technique – the method of antimicrobial susceptibility testing that is based on preparation of agar plates containing various concentrations of antimicrobial agents on which a defined inoculum of microorganisms is inoculated and then incubated and observed for growth.

antimicrobial susceptibility test interpretive category – a classification based on an in vitro response of an organism to an antimicrobial agent at levels corresponding to blood or tissue levels attainable with usually prescribed doses of that agent.

1)

susceptible – a category that implies that isolates are inhibited by the usually achievable concentrations of antimicrobial agent when the dosage recommended to treat the site of infection is used.

2)

intermediate – a category that includes isolates with antimicrobial agent minimal inhibitory concentrations that approach usually attainable blood and tissue levels and for which response rates may be lower than for susceptible isolates. The intermediate category implies clinical efficacy in body sites where the drugs are physiologically concentrated (eg, quinolones and β-lactams in urine) or when a higher than normal dosage of a drug can be used (eg, β-lactams). This category also includes a buffer zone, which should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharmacotoxicity margins.

3)

resistant – a category that implies that isolates are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules and/or that demonstrate minimal inhibitory concentrations that fall in the range in which specific microbial resistance mechanisms (eg, β-lactamases) are likely, and clinical efficacy of the agent against the isolate has not been reliably shown in treatment studies.

broth microdilution technique – the method of antimicrobial susceptibility testing that is based on preparation of a liquid broth medium containing various concentrations of antimicrobial agents into which a defined inoculum of microorganisms is inoculated and then incubated and observed for growth.

minimal inhibitory concentration (MIC) – the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in an agar or broth dilution susceptibility test.

quality control (QC) – the operational techniques that are used to ensure accuracy and reproducibility in microbiology testing.

reference strain – a particular strain of a bacterial species that can be obtained from a microbiological repository, such as the American Type Culture Collection, which exhibits stable genetic properties and yields reproducible minimal inhibitory concentrations when tested in vitro against designated antimicrobial agents.

4.3. Abbreviations and Acronyms

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5. Indications for Performing Susceptibility Tests on Human Mycoplasmas and Ureaplasmas

Routine performance of antimicrobial susceptibility tests is rarely performed on mycoplasmas and ureaplasmas isolated from clinical specimens. However, the occurrence of tetracycline resistance in M. hominis and Ureaplasma spp. should be considered if any drug in this class is being contemplated for treatment of infections caused by these organisms.11,12 Fluoroquinolone resistance is known to occur in both Ureaplasma spp.13 and _M. hominis._14 Macrolide resistance in Ureaplasma spp.15 and clindamycin resistance in _M. hominis_16 have also been reported. Thus, uniform susceptibility to these drug classes can no longer be assumed and guidance by in vitro susceptibility tests can sometimes be beneficial, particularly if the clinical response to agents usually active against these organisms is not successful. Although most infections caused by M. hominis and Ureaplasma spp. are limited to the urogenital tract, any time these organisms are isolated from a normally sterile and/or extragenital site in a disseminated infection, particularly in a neonate or an immunosuppressed person, performance of in vitro susceptibility testing should be considered to guide patient management, because resistant strains are known to occur in these settings and prolonged treatment may be needed to successfully treat the infection.17

The prolonged time, usually several days, necessary to isolate M. pneumoniae in culture, and the additional several days to obtain MICs, essentially precludes in vitro testing for patient management purposes from a practical standpoint. The increase in macrolide resistance in M. pneumoniae coupled with the difficult and time-consuming nature of culture prompted development of molecular-based assays that can be applied directly to clinical specimens to determine whether M. pneumoniae is present and if rRNA mutations that confer macrolide resistance are present.18-20 Because no other acquired resistance mechanisms have been reported in M. pneumoniae, susceptibility to other agents, such as tetracyclines and fluoroquinolones, can be expected.12 Thus, the primary value for in vitro susceptibility testing of M. pneumoniae is for surveillance purposes and evaluation of new investigational drugs.

6. Antimicrobial Agents Useful for Treatment of Human Mycoplasma and Ureaplasma Infections

The types of antimicrobial agents suitable for use in treatment of mycoplasmal or ureaplasmal infections are very limited. The main drug classes with potential clinical utility include the macrolides, lincosamides, streptogramins, tetracyclines, and fluoroquinolones. Mycoplasmas and ureaplasmas are innately resistant to all β-lactams, sulfonamides, trimethoprim, rifampin, and all other agents acting on bacterial cell wall replication.

M. pneumoniae has historically been predictably susceptible to fluoroquinolones, tetracyclines, and macrolides. However, recent studies from Japan, China, France, and the United States found that the high-level macrolide resistance in M. pneumoniae due to mutations at three locations in domain V on the 23S rRNA gene is increasing in patients with acute respiratory infections.18,19,21,22 This resistance, which can be clinically significant, is greatest in China, where the percentage of resistant organisms has exceeded 90%.21

Tetracycline resistance has been well documented in both M. hominis and Ureaplasma spp. since the mid-1980s, and is mediated by the tet(M) determinant, which codes for a protein that binds to the ribosomes, protecting them from the actions of these drugs.12 The extent to which tetracycline resistance occurs in M. hominis and Ureaplasma spp. varies geographically and according to prior antimicrobial exposure in different populations, but may approach 40% to 50% in some locations in the United States.11

Activity of macrolides and lincosamides varies according to species, with M. hominis being innately resistant to erythromycin and other 14- and 15-membered macrolides, but usually susceptible to lincosamides such as clindamycin.12 For Ureaplasma spp., the reverse is true. Newer macrolides such as azithromycin and clarithromycin and ketolides such as telithromycin have shown in vitro activities comparable to erythromycin for M. pneumoniae. High-level macrolide resistance in U. parvum due to insertions or deletions of amino acids in ribosomal proteins or point mutations in 23S rRNA was recently reported,15,23 but such resistance is currently believed to be a rare occurrence, as is clindamycin resistance in _M. hominis._16

7. Consensus Method for Broth Microdilution Assay

The broth microdilution MIC assay employs a 96-well microdilution plate into which a defined inoculum of the organism to be tested is added to doubling dilutions of antimicrobial agents in small volumes and the plates are incubated until the growth control changes color. The MIC end point can then be determined by lack of color change in broth containing a pH indicator.

7.1. Media

• SP4 broth and SP4 agar for M. pneumoniae
• Mycoplasma broth and agar for M. hominis
• 10B broth and A8 agar for Ureaplasma spp.

A multilaboratory study determined that commercial SP4 and 10B broths yielded similar MICs for QC reference strains of M. pneumoniae and U. urealyticum to those obtained using noncommercial SP4 and 10B broths prepared in each laboratory. A commercially prepared medium for M. hominis was found to be unsuitable.

Complete formulations and instructions for preparation of all broth and agar media used for mycoplasma and ureaplasma susceptibility testing are described in Appendix A.

7.2. Supplies

• Reference powders of antimicrobial agents of known purity
• Conical centrifuge tubes (15-mL and 50-mL polypropylene and/or polystyrene)
• 96-well flat or round bottom microdilution plates with covers
• 12 × 75 mm polystyrene tubes for performing colony-forming unit (CFU) assay
• Acetate sealers for microdilution plates to prevent drying and release of gases that may affect MICs
• Sterile pipette tips (0–200 μL range)
• Sealable plastic bags

7.3. Equipment

• Stereomicroscope
• Multichannel pipetting device
• 37°C aerobic incubator
• 37°C humidified incubator with 5% CO2

7.4. Preparation of Antimicrobial Agents

Weigh out an appropriate amount of each powdered drug to prepare 10 mL of a stock solution. Refer to Table B1 in Appendix B for detailed instructions on how to calculate the amount of drug needed and how to prepare the appropriate dilutions. Dissolve antimicrobial agents according to manufacturers’ instructions. See Appendix C for solvents and diluents for preparation of stock solutions.

Depending on drug stability, stock solutions of some agents may be stored in 1-mL aliquots for variable periods at ≤ –70°C. Others may require preparation on the day of assay depending on the manufacturer’s instructions. It is also permissible to store prediluted microdilution plates at –70°C for up to three months, as long as careful attention is paid to QC measures and MIC results for the control strains of the organisms to be tested to ensure the stability of the antimicrobial agents.

7.5. Preparation of Inoculum for Clinical Isolates and Quality Control Strains

Prepare a stock strain of known titer of the clinical isolate or QC organism to be tested as described in Appendix D. MICs should only be performed on pure isolates.

Thaw vials of the organisms to be tested on the day the assay will be performed and dilute in an appropriate prewarmed medium in 50-mL conical tubes (polypropylene tubes should be used when making M. pneumoniae dilutions because it adheres to polystyrene tubes). If the titer of the stock culture is 107 CFU/mL, prepare a 1:1000 dilution. Theoretically, this should yield organism suspensions containing 104 CFU/mL, but variation sometimes occurs. This means that a 1:1000 dilution requires the addition of 30 µL of stock culture to approximately 30 mL of broth. Prepare a total of 5 mL of organism inoculum for each drug, based on testing eight dilutions in duplicate and appropriate controls. Discard unused stock solutions.

Incubate the inoculated broths aerobically at 37°C for two hours before use to allow organisms to become metabolically active before inoculating microdilution plates. For Ureaplasma spp., incubate for only one hour.

7.6. Performance of Broth Microdilution Assay

Plate design can be modified according to the needs of the laboratory. Below is an example of a plate design. The plate design should include the controls specified in Section 9.9.

A single 96-well microdilution plate can be used for four drugs. Each drug should be tested in duplicate. (Drug 1: rows A, B; Drug 2: rows C, D; Drug 3: rows E, F; and Drug 4: rows G, H). Wells 1–8 will be sufficient in most cases to test the number of dilutions necessary for an end point MIC. Use wells 9, 10, 11, and 12 for solvent, media, drug, and growth controls, respectively. Performing assays in duplicate is recommended in the event there is a pipetting error, multiple skipped wells, contamination, or other QC problems.

Add 0.025 mL of appropriate broth medium in wells 2–8, 10, and 12 of the microdilution plate.

Add 0.025 mL of the highest concentration of drug to be tested to wells 1, 2, and 11 in rows A and B. Well 11 will serve as the drug control. The other drugs to be tested will be added the same way in their respective rows. Prepare the highest drug concentration by performing the appropriate dilution on the stock solution, using information provided in Table B1 in Appendix B.

• Example: If the highest concentration in the range to be tested is 64 μg/mL, add 0.025 mL of a solution containing 512 μg/mL to wells 1 and 2 before serially diluting.

Dilute the antimicrobial agent in serial two-fold dilutions using a 0.025-mL multichannel pipette, beginning at the second well and continuing through well 8. Discard the final 0.025 mL of antimicrobial agent solution.

Prepare a solvent control in well 9 by incorporating 0.025 mL of a 1:10 dilution of the solvent used to dissolve the antimicrobial agent being tested. This 1:10 solution is prepared by adding 0.1 mL of solvent to 0.9 mL of the appropriate diluent as described in Appendix C. This step is necessary for some drugs, such as macrolides, because they are dissolved in alcohol/methanol or dimethyl sulfoxide (DMSO) rather than water. This step is not necessary if water is used as the solvent.

Add 0.175 mL of the desired dilution of the inoculum in wells 1–8 and 12. Well 12 serves as the growth control. Start with well 12 and work backwards to well 1 to prevent drug carryover. Add 0.175 mL of the appropriate uninoculated medium to wells 9, 10, and 11 (total of 0.2 mL) for solvent, media, and drug controls.

Perform a final determination of CFU/mL of the inoculum as described in Appendix D to check that a proper dilution was made and that the inoculum contains 104–105 CFU/mL. This should be done on the inoculum after the preincubation step.

7.7. Incubation

Incubate microdilution plates at 37°C in an ambient air incubator. To maintain the same incubation temperature for all cultures, do not stack microdilution plates more than four high.

Place plastic covers over plates when testing M. pneumoniae and seal them in plastic bags with a piece of paper towel moistened with water to prevent desiccation of the broth during prolonged incubation. Cover plates being tested for Ureaplasma spp. and M. hominis with adhesive back acetate sealers before placing lid to prevent byproducts escaping to other wells and causing color change.

Incubation times differ for the different Mycoplasma and Ureaplasma spp. Read the microdilution plates after 18–24 hours of incubation and then daily for color change in the growth control wells for M. hominis and M. pneumoniae. Ureaplasma spp. will normally grow and produce color change after 16–18 hours of incubation and will need to be checked several times during the first 24 hours, while M. hominis may take 48–72 hours and M. pneumoniae four to six days, or possibly longer, for completion of the assay. Frequent examination of the positive growth control for ureaplasmas is important because of their rapid growth and tendency for the MIC end point to shift over time.

7.8. Determining Minimal Inhibitory Concentration End Points and Assay Validation

Record the MIC as the concentration of antimicrobial agent inhibiting visible color change in broth medium wells (wells 1 through 8) at the time when the organism control well (ie, well 12) first shows color change. This is generally 16–24 hours for Ureaplasma spp., 48–72 hours for M. hominis, and four to six days for M. pneumoniae.

A positive reaction for growth of Ureaplasma spp. in 10B broth will be evidenced by a color change from yellow to pink in the organism control well (ie, well 12). A positive reaction for growth of M. hominis in mycoplasma broth will be evidenced by a color change from pink to deeper red in the organism control well (ie, well 12). A positive reaction for growth of M. pneumoniae in SP4 broth will be evidenced by a color change from pink to yellow in the organism control well (ie, well 12). Turbidity in any test or control well indicates bacterial contamination.

Results are only considered valid if the control plate for organism concentration indicates that there are between 104 and 105 CFU/mL. Refer to Step 7 in Section 7.6.

When a single skipped well occurs in a microdilution test, read the highest MIC. If there is more than one skipped well, the result is uninterpretable and therefore cannot be reported.

If there is a different MIC end point for duplicate rows of the same drug, report the higher end point. If there is a difference of more than one dilution, the assay should be repeated.

Control wells and expected results: well 9 (ie, solvent control) – no color change; well 10 (ie, media control) – no color change; well 11 (ie, drug control) – no color change; well 12 (ie, growth control) – growth and color change according to organism being tested, without turbidity.

8. Consensus Method for Agar Dilution Assay

The agar dilution method for determination of MICs is based on the incorporation of doubling dilutions of antimicrobial agents into molten agar plates, with each plate containing a different concentration. After plates solidify, a defined organism inoculum is added. The plates are incubated and then examined for the presence of colonies.

8.1. Media

• Mycoplasma agar and broth for M. hominis
• SP4 agar and broth for M. pneumoniae
• A8 agar and 10B broth for Ureaplasma spp.

8.2. Supplies

• Reference powders of antimicrobial agents of known purity
• Conical centrifuge tubes (15-mL and 50-mL polystyrene and polypropylene)
• Sterile pipettes

8.3. Equipment

• Multipoint replicator, micropipettor delivering 1 or 10 μL (Steers replicator is preferred)
• 37°C aerobic incubator
• 37°C humidified incubator with 5% CO2
• Stereomicroscope
• Temperature-regulated hot water bath

8.4. Preparing Agar Dilution Plates Containing Antimicrobial Agents

8.4.1. Preparation of Antimicrobial Dilutions

Prepare antimicrobial dilutions for the entire concentration ranges desired for each drug according to Table B2 in Appendix B, using sterile ultrapure clinical laboratory reagent water (CLRW) as the diluent. This is typically eight dilutions. At least 2 mL of each dilution of each drug to be tested are required. Follow instructions in Appendix B for weighing out drug powder, accounting for purity, and preparing stock solutions. Dispense the appropriate dilutions of the drugs in 2-mL volumes in 50-mL sterile polystyrene culture screw-cap tubes.

8.4.2. Procedure for Pouring Plates

To each 2-mL aliquot of antimicrobial dilutions, add 18 mL of molten agar medium, prepared as described in Appendix A and allowed to equilibrate in a water bath to 45 to 50°C. Mix by inverting, taking care to avoid the formation of bubbles. Pour all 20 mL of each agar/antibiotic combination into a 100 × 15-mm Petri dish, and allow to solidify overnight on a level surface covered to protect from light. Allow the plates to dry completely in an inverted position. Label plates with the type of medium, drug name and concentration, and date prepared. Agar plates without antimicrobial agents for growth controls are prepared in the same manner except that 2 mL of ultrapure CLRW replaces the 2 mL of drug.

Agar plates should be stored in sealed plastic bags at 2 to 8°C for no more than three days. Plates should be removed from the refrigerator and allowed to equilibrate to room temperature before use. Ensure that the agar surface is dry before inoculating with bacterial suspensions. If necessary, plates can be placed in an incubator in an inverted position for approximately 30 minutes to hasten drying and temperature equilibration.

8.5. Preparation of Inoculum for Clinical Isolates and Quality Control Strains

It is suggested, although not required, that multiple dilutions be tested, especially when time is critical. If the concentration of the organism in the MIC plate is not 104–105 CFU/mL, the test should be repeated.

Remove frozen stock strains of clinical isolates or QC strains (previously tested for organism concentration and purity) from a –70°C freezer and thaw. Dilute to 104–105 CFU/mL (working stock) in an appropriate medium according to CFU values determined in initial stock concentration. A volume of 10 mL is sufficient to test several drugs. This means that a 1:100 dilution requires the addition of 0.1 mL of stock concentration to approximately 9.9 mL of broth. A 1:1000 dilution requires addition of 10 μL of stock concentration to approximately 10 mL of broth. If a 1:10 dilution is required, add 1 mL of stock culture to 9 mL of broth. Discard unused frozen stock.

Before testing, make 1:10 and 1:100 dilutions of working stock of the clinical isolates or QC strains to be tested in carefully labeled sterile tubes to yield volumes of 1 mL each. Incubate dilutions at 37°C in air for two hours (one hour for ureaplasma). Test samples will include aliquots of the postincubated working stock and of each 1:10 and 1:100 dilution.

8.6. Performance of Agar Dilution Assay

Once the final organism suspensions are prepared for all clinical isolates that are to be tested in the current run, arrange tubes containing suspensions in order in a rack and transfer a small volume (0.5 mL) of each inoculum into the appropriate well of the replicator seed block. If a replicator is not available, the tubes can be sampled directly with a micropipettor or with disposable calibrated loops.

It is helpful to prepare a template or pattern in order to keep track of the organisms and their respective dilutions. Agar plates should be marked for orientation of the inoculum spots. Plates must be inoculated on a level surface.

Using the replicator, pipettor, or calibrated loop, deliver 1 to 10 μL, depending on device used, of each of the three dilutions (1:10, 1:100, and 1:1000) of each strain of organism to the agar plates containing the appropriate antimicrobial concentrations. The goal is to obtain 30–300 CFUs per spot of inoculum on the growth control plate of at least one of the three dilutions tested.

Apply the inoculum directly to the surface of the plate, taking care to avoid splashing. Inoculate the plates containing antimicrobials from the lowest concentration to the highest. Inoculate a drug-free control plate between each series of drug-containing plates.

Inoculate antimicrobial-free plates with spots of each dilution of each clinical isolate being tested as a growth control. Incubate an uninoculated plate as a media sterility control. Prepare a solvent control plate control by incorporating 2 mL of the highest concentration (1:10 dilution) of solvent used to dissolve the antimicrobial being tested in the agar instead of the diluted antimicrobial. This is necessary for some macrolides because they are dissolved in alcohol/methanol or DMSO rather than water. If water is used as the solvent this step is not required.

8.7. Incubation

Allow the inoculated plates to stand at room temperature until the moisture in the inoculum spots has been absorbed into the agar, but not for more than 30 minutes. Invert the plates when placing in the incubator.

Incubate inoculated plates and controls at 37°C in a humidified incubator with 5% CO2 (48–72 hours for M. hominis, four to six or more days for M. pneumoniae, and 24–48 hours for Ureaplasma spp.). High humidity is required to reduce the drying of media during extended incubations and to enhance the growth of the organism.

8.8. Determining Minimal Inhibitory Concentration End Points and Assay Validation

Record the MIC as the lowest concentration of the antimicrobial agent that prevents colony formation when read (with the aid of a stereomicroscope) at the same time the antimicrobial-free control plate demonstrates growth. All three dilutions are examined. A faint haze or growth of only a single colony is read as negative. Use the dilution yielding 30–300 colonies for reporting the MIC and disregard the others.

The media (sterility) control plates should show no evidence of growth.

The solvent control plate for each drug should show approximately the same CFU as the growth control plates. A colony count of 30–300 colonies on at least one dilution of the growth control plates is required in order for the assay to be valid.

9. Quality Control and Quality Assurance Procedures

9.1. Purpose

In AST, QC includes the procedures to monitor the performance of a test system to ensure reliable results. This is achieved by, but not limited to, the testing of QC strains with known susceptibility to the antimicrobial agents tested. The primary purpose of QC testing performed by laboratories (users) is to ensure that the tests are maintained and performed appropriately. The overall goals of a QC program are to assist in monitoring the following:

• The precision (repeatability) and accuracy of susceptibility test procedures
• The performance of reagents used in the tests
• The performance of persons who carry out the tests and read the results

A comprehensive quality assurance (QA) program helps to ensure that testing materials and processes consistently provide quality results. QA includes, but is not limited to, monitoring, evaluating, taking corrective actions (if necessary), recordkeeping, calibration and maintenance of equipment, proficiency testing, training, and QC.

9.2. Quality Control Responsibilities

Modern laboratories rely heavily on pharmaceutical and diagnostic product manufacturers for provision of antimicrobial agents, other reagents, media, or test systems for the performance of antimicrobial susceptibility tests. Manufacturers and users of antimicrobial susceptibility tests have a shared responsibility for quality. The primary purpose of QC testing performed by manufacturers (in-house reference methods or commercial methods) is to ensure that the test or product has been appropriately manufactured. A logical division of responsibility and accountability may be described as follows:

• Manufacturers (in-house or commercial products):
  –
  Antimicrobial stability
  –
  Antimicrobial labeling
  –
  Potency of antimicrobial stock solutions
  –
  Compliance with good manufacturing practices (eg, quality system regulation)
  –
  Integrity of product
  –
  Accountability and traceability to consignee
• Laboratories (users):
  –
  Storage under the environmental conditions recommended by the manufacturer (to prevent drug deterioration)
  –
  Proficiency of personnel performing tests
  –
  Use of current CLSI documents and adherence to the established procedure (eg, inoculum preparation, incubation conditions, interpretation of end points)

9.3. Reference Strains for Quality Control

Use of carefully selected QC strains allows the microbiologist to have confidence that the test is performing within acceptable standards, and thus that the test results are likely to be reliable.

Each QC strain should be obtained from a recognized source (eg, ATCC®). All CLSI-recommended QC strains appropriate for the antimicrobial agent and reference method should be evaluated and expected results established according to the procedures described in M43.

M. hominis ATCC® 23114, M. pneumoniae ATCC® 29342, and U. urealyticum ATCC® 33175 are the QC strains recommended for use based on extensive multilaboratory testing.

A QC strain of the relevant species must be tested with each assay to ensure the test system is working and produces MIC results that fall within specified limits listed in Tables 1 and 2.

9.4. Preparing and Storing Quality Control Strains

Control organisms acquired from the ATCC® should be reconstituted according to ATCC® directions using the appropriate broth and agar for expansion of the organisms (10B, SP4, or Mycoplasma broth). For assurance that there is no loss of viability of the ATCC® stocks, serial ten-fold dilutions should be made and all positive broths should be frozen for future use. U. urealyticum should be monitored carefully because the first sign of color change indicative of active growth may occur in a time period as short as 16 to 24 hours. Evidence of M. hominis growth is usually achieved in 24 to 48 hours. For M. hominis and U. urealyticum, once the original culture is proven viable, it should be propagated to a larger volume, and as soon as a visible color change has been noted, it should be frozen in 1-mL aliquots at –70°C. Perform CFU determination (see procedure in Appendix D) on a frozen stock to enumerate the number of organisms per milliliter. If using growth medium containing 20% serum, no additional cryopreservative is needed and organisms will remain viable indefinitely. Once thawed, isolates must not be refrozen.

Rehydrate M. pneumoniae ATCC® organism stocks according to ATCC® directions and add the liquid to a 25-cm–polystyrene tissue culture flask with 5 mL of SP4 broth. Incubate aerobically at 37°C and examine daily. Once a monolayer has formed and the color indicator has changed from red to orange-yellow, harvest by scraping the organisms with a cell scraper. The culture is further propagated by adding supernatant and organism to a larger tissue culture flask (75 or 150 cm) and repeating the process. After harvesting the monolayer, freeze in 1-mL aliquots and store at –70°C. M. pneumoniae is prone to attach to polystyrene so it is important to scrape the sides of the flask vigorously to remove as many organisms as possible or else recovery of the organism will be poor. If using growth medium containing 20% serum for frozen storage, no additional cryopreservative is needed and organisms will remain viable indefinitely. Once thawed, isolates must not be refrozen.

The number of 1-mL aliquots of ATCC® stock strains to be prepared for frozen storage will depend on the anticipated frequency and numbers of tests that will be performed over time. Additional passages are not recommended from the frozen stocks.

Before MIC testing, remove sample vials of each strain from –70°C frozen stocks and determine the culture concentration in the frozen stock as described in Appendix D. This number will be used to calculate dilutions for working stocks on the day of assay.

9.5. Frequency of Quality Control Testing

QC testing with the appropriate QC strain should be performed each test day.

9.6. Corrective Action

9.6.1. Out-of-Control Result Due to Identifiable Errors

Causes for the out-of-control results may include, but are not limited to:

• QC strain
• Use of the wrong QC strain
  –
  Improper storage
  –
  Contamination
  –
  Nonviability
  –
  Changes in the organism
• Testing supplies
  –
  Improper storage or shipping conditions
  –
  Contamination
  –
  Inadequate volume of broth in tubes or wells
  –
  Use of damaged (eg, cracked, leaking) panels, plates, cards, tubes
  –
  Use of expired materials
• Testing process
  –
  Use of the wrong incubation temperature or conditions
  –
  Inoculum suspensions incorrectly prepared or adjusted
  –
  Inoculum prepared from differential or selective media containing anti-infective agents or other growth-inhibiting compounds
  –
  Use of wrong reagents, ancillary supplies
  –
  Incorrect reading or interpretation of test results
  –
  Transcription error
• Equipment
  –
  Improper functioning or out of calibration (eg, pipettes)

9.6.2. Out-of-Control Result With No Error Identified

If the reason for the out-of-control result cannot be identified, corrective action is required as follows.

9.6.2.1 Immediate Corrective Action

Test the out-of-control antimicrobial agent/organism combination on the day the error is observed and/or as soon as a new working culture or subculture is available. Document all results. See Section 9.7.1.

9.6.2.2 Additional Corrective Action

When immediate corrective action does not resolve the problem, the problem is likely due to a system rather than a random error. Additional investigation and corrective action are required.

If a problem is identified and is corrected, documentation of satisfactory performance should be noted. If a problem is not identified but results go back into control without any specific corrective action, documentation of satisfactory performance should also be noted. See Section 9.7.1.

9.7. Interpretation and Reporting Results

9.7.1. Reporting Results When Quality Control Fails

In the event CFU results from the control strain do not fall into the acceptable range (between 104 and 105 CFU/mL) for a drug or drugs, the MIC for the QC reference control strain as well as the patient isolate must be repeated for the discrepant drug or drugs and the results can only be reported when the controls are within the acceptable range.

If results of growth control, media control, drug control, and solvent controls do not match the expected results described in the relevant broth or agar dilution procedure, the MIC for the QC reference control strain as well as the patient isolate must be repeated for the discrepant drug or drugs and the results can only be reported when all the controls match the expected results.

9.7.2. Macrolide Disclaimer

Some antibiotics, macrolides in particular, may be less active at the low pH (ie, 6.0–6.5) necessary for optimal growth of ureaplasmas. Therefore, true MIC values at physiological pH may be lower than indicated by this assay.24,25 It may be useful to include this comment on susceptibility reports for ureaplasmas when macrolides are tested.

9.8. Verification of Patient Test Results

Multiple test parameters are monitored by following the QC recommendations described in this guideline. However, acceptable results derived from testing QC strains do not guarantee accurate results when testing patient isolates. It is important to review all of the results obtained from all drugs tested on a patient’s isolate before reporting the results. This should include, but not be limited to ensuring that:

• The antimicrobial susceptibility results are consistent with the identification of the isolate.
• The isolate is susceptible to those agents for which resistance has not been documented.

Unusual or inconsistent results should be verified by checking for the following:

• Previous results on the patient (eg, did the patient have the same isolate with an unusual antibiogram previously?)
• Previous QC performance (eg, is there a similar trend or observation with recent QC testing?)
• Problems with the testing supplies, process, or equipment

If a reason for the unusual or inconsistent result cannot be ascertained, a repeat of the susceptibility test or the identification, or both, is in order. Each laboratory must develop its own policy for verification of unusual or inconsistent antimicrobial susceptibility test results. This policy should emphasize those results that may significantly impact patient care.

9.9. Broth Microdilution Quality Control Limits

Acceptable MIC QC limits for a single QC test (single-drug/single-organism combination) are listed in Table 1. The overall performance of the test system should be monitored using these limits by testing appropriate control strains each day the test is performed. Additional parameters must also be documented for each test.

9.9.1. Drug Control

For the broth microdilution method, a well containing medium and the highest concentration of drug tested should be incubated along with the assay to ensure the antibiotic does not cause a color change that would cause false interpretations.

9.9.2. Inoculum Control

Determine CFU/mL of each inoculum in the appropriate medium at the same time as assays are performed to ensure the appropriate number of organisms was used in the test.

9.9.3. Growth Control

Each microdilution broth tray should include a growth control of medium without antimicrobial agent to assess viability of the test organisms. It should be used as a comparison to determine positive wells and the time to read the MIC results. With the broth tests, the growth control serves as a color indicator to base reading of end points.

• U. urealyticum 10B broth: color change yellow to pink
• M. hominis mycoplasma broth: color change pink to deeper red
• M. pneumoniae SP4 broth: color change pink to yellow

9.9.4. Sterility Control

For broth microdilution method, a well of media should be incubated and should show no color change; it is used as a comparison to determine negative wells and to verify sterility of the medium.

9.9.5. Solvent Control

Solvent controls should be run to ensure that the solvent used to dissolve the antimicrobial agent is not inhibitory to the organisms being tested. This control is necessary only when a new agent not previously tested that requires a solvent other than water is being evaluated.

9.9.6. Purity Control

By performing a CFU quantification on all isolates, purity of organisms can be determined. A8 agar yields brown granular colonies for U. urealyticum and will also detect contaminating M. hominis or bacteria. SP4 or mycoplasma agar detects contaminants or mixed cultures with Mycoplasma spp. when inoculated with M. pneumoniae. M. hominis and commensal respiratory Mycoplasma spp. usually produce “fried egg” colonies whereas M. pneumoniae produces small spherical colonies.

9.10. Agar Dilution Quality Control Limits

Acceptable MIC QC limits for a single QC test (single-drug/single-organism combination) are listed in Table 2. The overall performance of the test system should be monitored using these limits by testing the appropriate control strains each day the test is performed.

9.10.1. Inoculum Control

Perform CFU of each inoculum in the appropriate medium at the same time as assays are performed to ensure the appropriate number of organisms was used in the test.

9.10.2. Growth Control

Inoculate antimicrobial-free plates with duplicate spots of each dilution of each strain being tested as a growth control for MIC analysis.

9.10.3. Sterility Control

Incubate an uninoculated plate as a media sterility control plate, which should show no evidence of bacterial growth.

9.10.4. Solvent or Diluent Control

Prepare a solvent plate control by incorporating 2 mL of a 1:10 dilution of the solvent used to dissolve the antimicrobial being tested in the agar instead of the diluted antimicrobial (for diluents other than water). Inoculate with appropriate dilutions of test organisms or QC type strains to ensure solvents are not inhibitory to organisms being tested.

9.10.5. Purity Control

By determining the numbers of CFU/mL on all isolates being tested, purity of organisms can be determined. A8 agar plates yield brown granular colonies for U. urealyticum and would also detect contaminating M. hominis or bacteria. Likewise, SP4 or mycoplasma agar plates would detect contaminants or mixed cultures with Mycoplasma spp. when inoculated with M. pneumoniae. M. hominis and commensal respiratory Mycoplasma spp. usually produce the traditional “fried egg” colonies, whereas M. pneumoniae produces a small spherical colony.

References

Kenny GE, Cartwright FD, Roberts MC. Agar dilution method for determination of antibiotic susceptibility of Ureaplasma urealyticum. Pediatr Infect Dis. 1986;5(6 Suppl):S332-4. [PubMed: 2948164]

Waites KB, Figarola TA, Schmid T, Crabb DM, Duffy LB, Simecka JW. Comparison of agar versus broth dilution techniques for determining antibiotic susceptibilities of Ureaplasma urealyticum. Diagn Microbiol Infect Dis. 1991;14(3):265-271. [PubMed: 1889178]

Waites KB, Bébéar CM, Robertson JA, Talkington DF, Kenny GE. Cumitech 34: Laboratory Diagnosis of Mycoplasmal Infections. Washington, DC: ASM Press; 2001.

Waites KB, Canupp KC, Kenny GE. In vitro susceptibilities of Mycoplasma hominis to six fluoroquinolones as determined by E test. Antimicrob Agents Chemother. 1999;43(10):2571-2573. [PMC free article: PMC89525] [PubMed: 10508049]

Dósa E, Nagy E, Falk W, Szöke I, Ballies U. Evaluation of the Etest for susceptibility testing of Mycoplasma hominis and Ureaplasma urealyticum. J Antimicrob Chemother. 1999;43(4):575-578. [PubMed: 10350390]

Waites KB, Talkington DF, Bébéar CM. Mycoplasmas. In: Truant AL, ed. Manual of Commercial Methods in Clinical Microbiology. Washington, DC: ASM Press; 2002:201-224.

CLSI. Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters; Approved Guideline—Third Edition. CLSI document M23-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.

CLSI. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline—Second Edition. CLSI document M45-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2010.

CDC. 2007 Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings. http://www.cdc.gov/ncidod/dhqp/pdf/guidelines/isolation2007.pdf. Accessed October 18, 2011.

CLSI_. Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—Third Edition_. CLSI document M29-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2005.

Bébéar CM, Kempf I. Antimicrobial therapy and antimicrobial resistance. In: Blanchard A, Browning G, eds. Mycoplasmas: Molecular Biology Pathogenicity and Strategies for Control. Norfolk, UK: Horizon Bioscience; 2005:535-568.

Duffy L, Glass J, Hall G, et al. Fluoroquinolone resistance in Ureaplasma parvum in the United States. J Clin Microbiol. 2006;44(4):1590-1591. [PMC free article: PMC1448668] [PubMed: 16597903]

Bébéar CM, Renaudin H, Charron A, Gruson D, Lefrancois M, Bébéar C. In vitro activity of trovafloxacin compared to those of five antimicrobials against mycoplasmas including Mycoplasma hominis and Ureaplasma urealyticum fluoroquinolone-resistant isolates that have been genetically characterized. Antimicrob Agents Chemother. 2000;44(9):2557-2560. [PMC free article: PMC90107] [PubMed: 10952617]

Beeton ML, Chalker VJ, Maxwell NC, Kotecha S, Spiller OB. Concurrent titration and determination of antibiotic resistance in ureaplasma species with identification of novel point mutations in genes associated with resistance. Antimicrob Agents Chemother. 2009;53(5):2020-2027. [PMC free article: PMC2681543] [PubMed: 19273669]

Pereyre S, Gonzalez P, De Barbeyrac B, et al. Mutations in 23S rRNA account for intrinsic resistance to macrolides in Mycoplasma hominis and Mycoplasma fermentans and for acquired resistance to macrolides in M. hominis. Antimicrob Agents Chemother. 2002;46(10):3142-3150. [PMC free article: PMC128781] [PubMed: 12234836]

Waites KB, Taylor-Robinson D. Mycoplasma and Ureaplasma. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, eds. Manual of Clinical Microbiology. 9th ed. Washington, DC: ASM Press; 2007:1004-1020.

Li X, Atkinson TP, Hagood J, Makris C, Duffy LB, Waites KB. Emerging macrolide resistance in Mycoplasma pneumoniae in children: detection and characterization of resistant isolates. Pediatr Infect Dis J. 2009;28(8):693-696. [PubMed: 19633515]

Peuchant O, Ménard A, Renaudin H, et al. Increased macrolide resistance of Mycoplasma pneumoniae in France directly detected in clinical specimens by real-time PCR and melting curve analysis. J Antimicrob Chemother. 2009;64(1):52-58. [PubMed: 19429926]

Wolff BJ, Thacker WL, Schwartz SB, Winchell JM. Detection of macrolide resistance in Mycoplasma pneumoniae by real-time PCR and high-resolution melt analysis. Antimicrob Agents Chemother. 2008;52(10):3542-3549. [PMC free article: PMC2565909] [PubMed: 18644962]

Liu Y, Ye X, Zhang H, et al. Antimicrobial susceptibility of Mycoplasma pneumoniae isolates and molecular analysis of macrolide-resistant strains from Shanghai, China. Antimicrob Agents Chemother. 2009;53(5):2160-2162. [PMC free article: PMC2681541] [PubMed: 19273684]

Morozumi M, Iwata S, Hasegawa K, et al; Acute Respiratory Diseases Study Group. Increased macrolide resistance of Mycoplasma pneumoniae in pediatric patients with community-acquired pneumonia. Antimicrob Agents Chemother. 2008;52(1):348-350. [PMC free article: PMC2223908] [PubMed: 17954691]

Xiao L, Crabb DM, Duffy LB, et al. Mutations in ribosomal proteins and ribosomal RNA confer macrolide resistance in human Ureaplasma spp. Int J Antimicrob Agents. 2011;37(4):377-379. [PubMed: 21353494]

Kenny GE, Cartwright FD. Effect of pH, inoculum size, and incubation time on the susceptibility of Ureaplasma urealyticum to erythromycin in vitro. Clin Infect Dis. 1993;17 Suppl 1:S215-8. [PubMed: 8399919]

Waites KB, Crouse DT, Cassell GH. Antibiotic susceptibilities and therapeutic options for Ureaplasma urealyticum infections in neonates. Pediatr Infect Dis J. 1992;11(1):23-29. [PubMed: 1549404]

Table 1

Minimal Inhibitory Concentration (µg/mL): Quality Control Ranges for Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma urealyticum (Broth Microdilution Method).

Table 2

Minimal Inhibitory Concentration (µg/mL): Quality Control Ranges for Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma urealyticum (Agar Dilution Method).

Table 3

Mycoplasma hominis Information and Minimal Inhibitory Concentration Interpretive Criteria for Broth Microdilution and Agar Dilution.

Table 4

Mycoplasma pneumoniae Information and Minimal Inhibitory Concentration (MIC) Interpretive Criteria for Broth Microdilution and Agar Dilution.

Table 5

Ureaplasma urealyticum Information and Minimal Inhibitory Concentration (MIC) Interpretive Criteria for Broth Microdilution and Agar Dilution.

Appendix A. Media Formulations

Broth microdilution procedures for Mycoplasma pneumoniae were compared among multiple laboratories using noncommercial and commercially prepared SP4 broth with comparable results for minimal inhibitory concentrations (MICs) of quality control (QC) reference strains. Broth microdilution procedures for Ureaplasma urealyticum were also compared using noncommercial and commercially prepared 10B broth with comparable results. Whether a laboratory performing in vitro susceptibility testing chooses to use commercially or noncommercially prepared media is a matter of choice, as long as this same basic formulation is used and the QC reference strains yield MICs within the designated ranges. No commercial broth medium was found to produce acceptable MIC end points for testing Mycoplasma hominis. All agar dilution MIC assays use noncommercially prepared agars to which antimicrobials are added. Commercial 10B and SP4 broths used for antimicrobial susceptibility testing may require special order to ensure they do not contain other antimicrobial agents routinely incorporated to prevent bacterial overgrowth.

10B Urea Broth (Shepard’s Media)

To prepare 1 L, add the following ingredients to a flask. Adjust the pH to 5.5 with 2N HCl. Autoclave for 15 minutes at 121°C and cool to room temperature before adding supplements.

Base:

View in own window

Supplements:

Prepare and filter sterilize (0.02 µm pore size filter) each supplement separately and add supplements to autoclaved base. Adjust pH to 6.0 with 8 N HCl. Store at 2–8°C in the dark for up to one month.

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A8 Agar for Ureaplasma spp.

To prepare 1 liter, mix the following ingredients in a flask in the order specified. Adjust pH to 5.5 with 1N HCl. Autoclave for 15 minutes at 121°C. Cool in a 45 to 50°C water bath.

Base:

View in own window

Supplements:

Prepare and filter sterilize (0.2 µm pore size filter) each supplement separately (except serum). Mix and add supplements to autoclaved base. Adjust pH to 6.0 with 8 N HCl. Pour 20 mL into each Petri dish. Invert after two hours and keep at room temperature overnight. Store plates in plastic bags at 2–8°C in the dark. Use plates containing antibiotics within 72 hours.

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Cool agar in a 45 to 50°C water bath. Add antimicrobial agents in desired concentrations to molten agar. For control plates, add the same volume of ultrapure CLRW.

SP4 Broth and Agar

To prepare 1 liter, add the following ingredients to a flask. Autoclave for 15 min at 121°C and cool before adding supplements. For agar, cool in a 45 to 50°C water bath.

Base:

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Supplements:

Prepare and filter sterilize (0.2 µm pore size filter) each supplement separately. Mix and add supplements to autoclaved base. Adjust pH to 7.4–7.6 with 8 N NaOH. For agar preparation, pour 20 mL into each Petri dish. Invert after two hours and keep at room temperature overnight. Store plates in plastic bags at 2–8°C in the dark. Use plates containing antibiotics within 72 hours. Broth can be stored at 2–8°C for up to one month.

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Cool agar in a 45 to 50°C water bath. Add antimicrobial agents in desired concentrations to molten agar. For control plates, add the same volume of ultrapure CLRW.

Mycoplasma Broth and Agar

To prepare 1 liter, add the following ingredients to a flask. Autoclave for 15 min at 121°C and cool before adding supplements. For agar, cool in a 45 to 50°C water bath.

Base:

View in own window

Supplements:

Prepare and filter sterilize (0.2 µm pore size filter) each supplement separately (except serum). Mix and add supplements to autoclaved base. Adjust pH to 7.2–7.4 with 8 N NaOH. For agar preparation, pour 20 mL into each Petri dish. Invert after two hours and keep at room temperature overnight. Store plates in plastic bags at 2–8°C in the dark. Use plates containing antibiotics within 72 hours. Broths can be stored at 2–8°C for up to one month.

View in own window

Cool agar in a 45 to 50°C water bath. Add antimicrobial agents in desired concentrations to molten agar. For control plates, add the same volume of ultrapure CLRW.

Preparation of media supplement solutions

1% phenol red

Mix water and propanol together and then add phenol red salt.

(Water-propanol ratio – 8 parts water: 12 parts 2-propanol)

Filter sterilize (0.2 µm filter) and store at 2–8°C.

Expiration: one month

2% yeastolate

Filter sterilize (0.2 µm filter) and store at 2–8°C.

Expiration: six months

10% urea

Add urea to water and stir until dissolved.

Filter sterilize (0.2 µm filter) and store at 2–8°C.

Expiration: six months

2% cysteine

Filter sterilize (0.2 µm filter) using syringe filter.

Prepare fresh on day of use.

50% glucose

Add 50 mL of water to beaker and begin to heat, stirring constantly. Add glucose and heat until glucose is dissolved. Adjust to 100 mL with the remaining water. Filter sterilize using a 0.2 µm filter. Store at 2–8°C.

Expiration: six months

50% arginine

Dissolve 50 g of arginine in 50 mL of ultrapure CLRW. Adjust to 100 mL with the remaining water. Filter sterilize using a 0.2 µm filter. Store at 2–8°C.

Expiration: six months

GHL tripeptide 20 µg/mL

Reconstitute 10 mg GHL tripeptide in 1 mL of ultrapure CLRW. Add water to small vial so no powder is lost. Dilute to 1:500. Dispense in 1-mL aliquots and freeze at –70°C.

Expiration: six months

Reference for Appendix A

1 CLSI. Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline—Fourth Edition. CLSI document C03-A4. Wayne, PA: Clinical and Laboratory Standards Institute; 2006.

Appendix B. Preparing Stock Solutions of Antimicrobial Agents

Source

Obtain reference powders of the drugs to be tested from the manufacturer or other commercial sources. Acceptable powders will bear a label that states the generic name, lot number, potency expressed in micrograms per milligrams, international units per milligrams of powder (or percent purity), and expiration date. Powders should be stored refrigerated or frozen at < –20°C in a desiccator. Always allow cold stored antimicrobial agents in powder form to return to room temperature before weighing.

Weighing Antimicrobial Powders

Before preparing solutions of antimicrobials, it is necessary to determine the proper amount to be dissolved, taking into account the stated purity or potency of the powder provided by the manufacturer. In all cases, consider directions provided by the drug’s manufacturer as part of determining solubility and selection of the appropriate solvent. Antimicrobial powders should be weighed on an analytical balance that has been calibrated by approved reference weights from a national metrology organization. For greatest accuracy, weigh out larger amounts of powder and then prepare dilutions as needed for the actual testing.

If the antibiotic powder potency is expressed as percent purity, it is useful to convert this to potency expressed as micrograms per milligrams for calculating the amount needed to prepare a stock solution.

Antimicrobial purity = 98.0% or 980 μg active drug per milligrams of powder.

To calculate the amount of drug powder to weigh out for preparation of a stock solution, use the following formula:

Example: To prepare 10 mL of a stock solution of drug with a potency of 980 μg/mg at a final concentration of 2048 μg/mL, a total of 20.90 mg is needed.

Weigh out 20.90 mg of antimicrobial agent, dilute in 1 mL of the appropriate solvent and adjust to 10 mL by adding 9 mL of ultrapure CLRW. Aliquot in 1-mL portions and freeze in a cryovial at –70°C until day of use for up to six months. If using a concentration range of 256 μg/mL–0.008 μg/mL or below, which is sufficient for all antimicrobial agents useful for testing against mycoplasmas and ureaplasmas, a stock solution of 2048 μg/mL is sufficient. The number of milliliters of the stock solution required will depend on the number of bacterial isolates to be tested in a single run. On the day of assay, further dilutions can be made in the appropriate medium as needed. Discard any unused stock solution that has been thawed for preparing dilutions for minimal inhibitory concentration testing.

Some drugs must be dissolved in solvents other than water. In such cases, a minimum amount of solvent should be used to solubilize the antimicrobial powder. The final stock concentration can then be made with water or appropriate diluent. Because microbial contamination is extremely rare, solutions that have not been sterilized are generally acceptable. If desired, solutions may be sterilized by membrane filtration.

Table B1

Preparation of Antimicrobial Stock Solutions for Broth Microdilution Susceptibility Tests.

Table B2

Scheme for Preparing Dilutions of Antimicrobial Agents for Agar Dilution Susceptibility Tests.

Appendix C. Solvents and Diluents for Preparation of Stock Solutions of Antimicrobial Agents

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* These compounds are potentially toxic. Consult the material safety data sheets available from the product manufacturer before using any of these materials.

† For glacial acetic acid, use 1/2 volume of water, then add glacial acetic acid dropwise until dissolved, not to exceed 2.5 μL/mL.

Appendix D. Determining Organism Inocula for Broth Microdilution and Agar Dilution Assays for Clinical Isolates and Quality Control Strains

Determination of Culture Concentration (Colony-Forming Units [CFUs])

1. Agar plates to be used for plating should be dried inverted for 30 minutes at 37°C in air before use.

2. Prepare culture dilutions: Five to six serial dilutions are usually sufficient. They are prepared by pipetting accurately a 0.1-mL volume of the frozen stock or working dilution into 0.9 mL of broth medium. Vortex vigorously for 10 seconds. Use a clean pipette tip for each transfer. This process is continued to tube six. Avoid using polystyrene tubes, owing to the potential of organisms sticking to the plastic.

3. Plating: With a permanent marker, mark plates on the bottom into six segments. Using a new tip for each dilution, plate a 20-μL aliquot of each dilution in the corresponding segment on the plate. The tip should be touched to the agar surface (while taking care not to go beneath the surface) after each delivery to transfer the remaining droplet. Plates should be allowed to remain undisturbed in a biological safety cabinet or laminar flow hood until all of the inoculum has been absorbed into the agar. Wrap edges of plates with gas-permeable tape to prevent drying out during incubation.

4. Incubate plates inverted at 37°C in air plus 5% CO2. Colonies appear usually within 24–48 hours for Ureaplasma spp., at 48–72 hours for Mycoplasma hominis, and four to six days for Mycoplasma pneumoniae.

5. Count colonies with the aid of a stereoscope. Count the dilution containing from 30–300 colonies. Counting is simplified if a grid is produced by scoring the bottom of the Petri dish with a razor blade and counting in a manner similar to what would be done with a hemocytometer.

6. Calculate the number of CFU/mL by multiplying the number of colonies counted by 50 and that product by the dilution (formula = colonies counted × 50 × dilution = CFU/mL). For example, if 50 colonies were counted in the 102 dilution, the CFU/mL is 50 × 50 × 102 = 2.5 × 105.

Verification of CFU in Inocula Prepared From Clinical Isolates or Quality Control Strains

Follow the same procedure as above for working dilutions of inocula of clinical isolates to be tested to verify that the correct number of organisms (104–105 CFU/mL) was inoculated into the microdilution trays or onto the agar plates.

The Quality Management System Approach

Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the development of standards and guidelines, which facilitates project management; defines a document structure via a template; and provides a process to identify needed documents. The quality management system approach applies a core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any health care service’s path of workflow (ie, operational aspects that define how a particular product or service is provided). The QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The QSEs are as follows:

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M43-A addresses the QSE indicated by an “X.” For a description of the other documents listed in the grid, please refer to the Related CLSI Reference Materials section on the following page.

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Path of Workflow

A path of workflow is the description of the necessary processes to deliver the particular product or service that the organization or entity provides. A laboratory path of workflow consists of the sequential processes: preexamination, examination, and postexamination and their respective sequential subprocesses. All laboratories follow these processes to deliver the laboratory’s services, namely quality laboratory information.

M43-A addresses the clinical laboratory path of workflow steps indicated by an “X.”

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Related CLSI Reference Materials*

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About the Series

CLSI publication / Clinical and Laboratory Standards Institute

ISSN: 1558-6502 (Print)

ISSN: 2162-2914 (Electronic)

Suggested citation:

CLSI. Methods for Antimicrobial Susceptibility Testing for Human Mycoplasmas; Approved Guideline. CLSI document M43-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2011.

About this book

This document provides guidelines for the performance and quality control of agar and broth microdilution antimicrobial susceptibility tests on human mycoplasmas and ureaplasmas.

A guideline for global application developed through the Clinical and Laboratory Standards Institute consensus process.

M43-A Vol. 31 No. 19

Volume 31 Number 19

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The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in the CLSI catalog and posted on our website at www.clsi.org. If your organization is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail: customerservice@clsi.org; Website: www.clsi.org.

Copyright ©2011 Clinical and Laboratory Standards Institute. Except as stated below, any reproduction of content from a CLSI copyrighted standard, guideline, companion product, or other material requires express written consent from CLSI. All rights reserved. Interested parties may send permission requests to permissions@clsi.org.

CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of this publication for use in its laboratory procedure manual at a single site. To request permission to use this publication in any other manner, e-mail permissions@clsi.org.

Suggested Citation

CLSI. Methods for Antimicrobial Susceptibility Testing for Human Mycoplasmas; Approved Guideline. CLSI document M43-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2011.

Approved Guideline

October 2011

ISBN 1-56238-769-3 (Print)

ISBN 1-56238-770-7 (Electronic)

ISSN 1558-6502 (Print)

ISSN 2162-2914 (Electronic)