Microbiology Data for 
Systemic Antibacterial 
Drugs  Development, 
Analysis, and Presentation 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
February 2018 
Clinical/Antimicrobial 
Revision 2 
   
Microbiology Data for 
Systemic Antibacterial 
Drugs  Development, 
Analysis, and Presentation 
Guidance for Industry 
 
 
 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information 
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD  20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353; Email: druginfo@fda.hhs.gov 
https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
February 2018 
Clinical/Antimicrobial 
Revision 2 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
I.  INTRODUCTION..................................................................................................1 
II.  MICROBIOLOGY DEVELOPMENT PROGRAM ..........................................2 
A.  Early Development Nonclinical Considerations ............................................................. 2 
1. Antibacterial Spectrum of Activity....................................................................................... 2 
2. Mechanism of Action ........................................................................................................... 2 
3. Intracellular Antimicrobial Concentration Assessment ...................................................... 3 
4. Resistance Studies ............................................................................................................... 3 
B.  In Vitro Antimicrobial Susceptibility Test Methods During Drug Development ........ 4 
1. Early Clinical Development ................................................................................................ 4 
2. Provisional Antibacterial Susceptibility Test Interpretive Criteria .................................... 4 
3. Establishing In Vitro Antibacterial Susceptibility Test Interpretive Criteria ...................... 5 
4. Quality Control Parameters ................................................................................................ 6 
C.  Other Considerations ........................................................................................................ 6 
1. First and Second Lists of Target Bacteria in Labeling ....................................................... 6 
2. Antibacterial Interactions and Fixed-Combination Studies ................................................ 7 
3. Additional Nonclinical Studies of Antibacterial Drugs ....................................................... 7 
4. Animal Models of Infection ................................................................................................. 7 
5. Microbiology Information Collected in Clinical Trials ...................................................... 8 
6. Electronic Submission of Microbiology Information .......................................................... 8 
7. Location of Microbiology Information ................................................................................ 9 
8. Postmarketing Microbiology Information ........................................................................... 9 
REFERENCES .................................................................................................................11 
APPENDIX A:  GUIDELINES FOR BACTERIAL ISOLATE SELECTION  FOR 
STUDIES OF IN VITRO ANTIBACTERIAL ACTIVITY.........................................12 
APPENDIX B:  STUDY REPORTS OF SPECTRUM OF ACTIVITY AND 
RESISTANCE ..................................................................................................................14 
APPENDIX C:  DATABASE FOR FINAL IN VITRO ANTIBACTERIAL  
SUSCEPTIBILITY TEST INTERPRETIVE CRITERIA ..........................................16 
APPENDIX D:  EXAMPLE FORMAT FOR SECTIONS OF LABELING  THAT 
PERTAIN TO MICROBIOLOGY ................................................................................19 
APPENDIX E:  INFORMATION REGARDING THE SECOND BACTERIA LIST 
IN THE MICROBIOLOGY SUBSECTION OF LABELING ....................................22 
 
 
  Contains Nonbinding Recommendations 
 
Microbiology Data for Systemic Antibacterial Drugs  
Development, Analysis, and Presentation 
Guidance for Industry1 
 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page. 
 
 
 
I.  INTRODUCTION 
 
The purpose of this guidance is to assist sponsors in the development, analysis, and presentation 
of microbiology data during antibacterial drug development.2  Specifically, this guidance 
addresses the Food and Drug Administrations (FDAs) current thinking regarding the overall 
microbiology development program needed to support clinical development and approval of 
antibacterial drugs administered systemically as well as microbiology information collected after 
approval.3 
 
This guidance replaces the guidance for industry Microbiology Data for Systemic Antibacterial 
Drugs  Development, Analysis, and Presentation issued in August 2016.  Changes to this 
guidance compared to the August 2016 version include changes in the presentation of 
microbiology data as required by section 3044 of the 21st Century Cures Act, which added 
section 511A to the Federal Food, Drug, and Cosmetic Act (FD&C Act).  Section 511A created 
new processes for susceptibility test interpretive criteria recognition, required FDA to establish a 
web page for susceptibility test interpretive criteria, and required changes to the labeling for 
antibacterial and antifungal drugs on susceptibility test interpretive criteria, among other 
changes.  FDAs recommendations on implementing section 511A are described in section 
III.C.7., Location of Microbiology Information, section III.C.8., Postmarketing Microbiology 
Information, and Appendix D.  
                                                 
1 This guidance has been prepared by the Division of Anti-Infective Products in the Center for Drug Evaluation and 
Research at the Food and Drug Administration.  You may submit comments on this guidance at any time.  Submit 
comments to Docket No. FDA-2009-D-0408 (available at https://www.regulations.gov/document?D=FDA-2009-D-
0408-0017).   
 
2 For the purposes of this guidance, all references to drugs include both human drugs and therapeutic biological 
products unless otherwise specified. 
 
3 This guidance addresses the types of microbiology information that should be provided to support an 
investigational new drug application (IND), a new drug application (NDA), a biologics license application (BLA), 
and a supplemental NDA or BLA.  The term sponsor is used in this guidance and refers to sponsors submitting an 
IND as well as applicants submitting an NDA or BLA. 
1  Contains Nonbinding Recommendations 
 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
as recommendations, unless specific regulatory or statutory requirements are cited.  The use of 
the word should in Agency guidances means that something is suggested or recommended, but 
not required. 
 
 
II.  MICROBIOLOGY DEVELOPMENT PROGRAM 
 
Microbiology data provide important information to guide clinical development of an 
investigational new drug.  Microbiology data guide clinicians on the use of an antibacterial drug 
for its intended indications. 
 
A.  Early Development Nonclinical Considerations 
 
1.  Antibacterial Spectrum of Activity 
 
Sponsors should evaluate the activity of an antibacterial drug, including its active components 
and major circulating metabolites, against a test panel of relevant bacteria early in clinical 
development.  Sponsors should provide data on a sufficient range of clinically relevant bacteria 
to allow an assessment of the potential clinical efficacy of the antibacterial drug for the intended 
indication.  Appendix A provides the suggested number of genera and species that should be 
tested and the recommended characteristics and diversity of the test isolates. 
 
When conducting studies of the spectrum of activity, sponsors should test in parallel FDA-
approved antibacterial drugs, especially those with the same mechanism of action as the 
investigational drug.  In the case of a drug that acts by a new mechanism of action, we 
recommend that sponsors include FDA-approved antibacterial drugs with a spectrum of activity 
similar to the investigational drug.  In the event there is no FDA-approved antibacterial drug with 
a similar spectrum of activity, we recommend that sponsors discuss with the FDA the approved 
drugs to include in these studies. 
 
Sponsors should evaluate the minimum inhibitory concentrations (MICs) in the relevant target 
bacteria and provide the rationale for an estimate of the epidemiologic cutoff (EC) of the 
investigational drug against target bacteria. 
 
Appendix B provides an example of the recommended elements of the study reports on 
antibacterial spectrum of activity. 
 
2.  Mechanism of Action 
 
Sponsors should evaluate the mechanism of action of an investigational drug (e.g., inhibition of 
cell wall synthesis, lysis of cell membrane, protein synthesis).  Sponsors should provide 
information about the drugs chemical structure and a description of any structural or biological 
similarities to known antibacterial drugs.  Data to substantiate both physiological and 
2  Contains Nonbinding Recommendations 
 
morphological effects on the microbial cells can provide a basis for understanding the 
development of resistance through alterations in the drugs target sites.  Sponsors also should 
provide studies evaluating microbial killing (e.g., microbial kill curves). 
 
3.  Intracellular Antimicrobial Concentration Assessment 
 
The ability of an antibacterial drug to achieve significant intracellular concentrations may have 
clinical importance when the target bacterium can reside within the cell (e.g., Listeria, 
Chlamydophila, Legionella).  In situations where the antibacterial drug is intended to treat 
infections caused by bacteria that reside within the cell, sponsors should provide data on the 
drugs ability to penetrate into host cells and demonstrate the drugs activity against target 
bacteria inside the cell (e.g., assessment of viable intracellular microorganisms following 
exposure of the cells to various concentrations of an antibacterial drug). 
 
4.  Resistance Studies 
 
Characterization of the resistance mechanisms and their distribution within the proposed target 
bacteria may delineate the potential clinical usefulness of the drug.  Mechanisms include 
alterations of the drug by production of enzymes (e.g., beta-lactamases, extended spectrum beta-
lactamases), inability to reach the target, and changes in the affinity of the antibacterial drug for 
the target site.  To determine if there may be a proportion of bacteria in the overall population 
that are resistant to the antibacterial drug (i.e., hetero-resistance), sponsors should conduct testing 
to evaluate for the presence of such bacteria.  When possible, we recommend that sponsors 
provide the genotypic characteristic of resistance mechanisms. 
 
Sponsors should compare the activity of an investigational antibacterial drug to the activity 
profile of approved and other existing antibacterial drugs with the same mechanism of action to 
assess the possibility of cross-resistance. 
 
Under some circumstances, tentative inferences can be drawn about cross-resistance between 
antibacterial drugs within a specific population of isolates from regression analyses (i.e., MIC 
versus MIC, zone diameter versus zone diameter) of one drug compared to another drug.  If 
cross-resistance exists between both the investigational and control drugs, a strong correlation 
between the MICs of both drugs would be expected to be observed, with a majority of the MICs 
clustered on a 45-degree diagonal.  If resistance affects the activity of one drug over the other, 
the cluster is usually skewed in the direction of one drug and away from the expected diagonal. 
 
Detailed information on the mechanism of action, resistance, or cross-resistance for an 
antibacterial drug with a novel mechanism of action may not be available for sponsors to include 
in the initial investigational new drug application.  This information should be provided early in 
drug development and ideally before initiation of phase 2 clinical development. 
 
Appendix B provides an example of the recommended elements of study reports evaluating 
resistance. 
 
3  Contains Nonbinding Recommendations 
 
B.  In Vitro Antimicrobial Susceptibility Test Methods During Drug 
Development 
 
1.  Early Clinical Development 
 
Before conducting clinical trials, sponsors should describe the methods used for generating 
susceptibility data.  Sponsors can reference a standard method4 or evaluate susceptibility by 
other methods including modification of the method.  Sponsors should provide a detailed 
description of the method including the justification for the modification of the method, the 
effect on susceptibility results, and the performance characteristics of the method (i.e., 
sensitivity, specificity, precision, linearity).  Sponsors should discuss any modification of an 
established in vitro susceptibility test method with the FDA before implementation in the drug 
development program. 
 
Modifications can include the addition of any substance (e.g., blood, body fluids, polysorbate).  
In some cases, isolates obtained during clinical trials may need to be tested for their 
susceptibility in the presence and absence of the substance and the results of both methods 
correlated with clinical and microbiological results.  Sponsors also should conduct studies to 
address the influence of the growth medium (e.g., pH, divalent cations), inoculum density, 
incubation conditions (e.g., concentration of carbon dioxide), and additives (e.g., polysorbate), in 
both broth and agar medium on in vitro susceptibility test results. 
 
If a sponsor proposes to use freeze-dried panels to assess the MIC of clinical isolates, the sponsor 
should conduct a comparative study to demonstrate comparability of MIC results for the frozen 
and freeze-dried panels.  The sponsor should discuss this proposed study with the FDA to ensure 
that appropriate data are developed for the equivalency assessment.  The sponsor should submit 
the data to the FDA before initiating phase 2 trials. 
 
2.  Provisional Antibacterial Susceptibility Test Interpretive Criteria 
 
Provisional antibacterial susceptibility test (AST) interpretive criteria usually are based on the 
limited information available before the initiation of phase 3 clinical trials.  In vitro microbiology 
data include distributions of MICs or zone diameters that are obtained by testing the antibacterial 
drug against a population of recent clinical isolates that represent the target bacteria for the 
indications being sought (see Appendix A for guidelines in the selection of target bacteria).  
Sponsors should provide testing data on a sufficient range of clinically relevant bacteria for the 
intended indications.  Sponsors should identify the prominent genotypes, serotypes, biotypes, 
and isolates with known mechanisms of resistance and include these in the test panel.  In 
addition, the mechanism of action of the investigational antibacterial drug and other drugs with 
the same mechanism of action should be considered when establishing susceptibility testing 
methods and provisional AST interpretive criteria. 
 
                                                 
4 Standard methods for susceptibility testing are developed by organizations such as the Clinical and Laboratory 
Standards Institute; information can be found at https://clsi.org.  Sponsors can describe the standard methods that 
they used by referencing recognized testing methodology. 
4  Contains Nonbinding Recommendations 
 
When an investigational drug has a similar mechanism of action to an approved drug, the data 
justifying provisional AST interpretive criteria for the investigational drug should be presented 
as regressions of MIC versus MIC and zone diameter versus zone diameter.  Sponsors should 
examine these data for clusters of isolates that are substantially different from those clusters near 
the expected regression line of MIC versus MIC or zone versus zone plots.  For example, a 
cluster in a position away from the expected regression line suggests that one of the drugs is 
affected by a resistance mechanism that does not affect the other drug.  Therefore, the two drugs 
are not interchangeable and provisional AST interpretive criteria of the investigational drug may 
not be similar to the AST interpretive criteria of an approved drug with a similar mechanism of 
action.  When developing an antibacterial drug with a specific mechanism of action for the 
treatment of bacteria resistant to other antibacterial drugs with the same mechanism of action, 
these types of analyses can be extremely useful in demonstrating the activity of the 
investigational drug. 
 
Sponsors should analyze the data in terms of frequency distributions (e.g., histograms) of AST 
results.  Frequency distribution analyses can help define which populations of isolates harbor 
specific resistance mechanisms that sponsors should identify.  Frequency distributions can be 
analyzed for both dilution and diffusion susceptibility testing methods.  Frequency distributions 
call for evaluation for each target bacteria, especially if there is no clear demarcation between the 
resistant and susceptible populations.  EC values should be estimated for each targeted species or 
bacterial group.   
 
Additional considerations to the provisional AST interpretive criteria include the methodological 
variability between diffusion and dilution susceptibility testing methods.  Sponsors can suggest 
adjustments to the provisional AST interpretive criteria by evaluating scattergrams of dilution 
testing results compared with diffusion testing results of the same isolates tested with both 
methods.  This evaluation can be performed using the error rate bounding method that compares 
diffusion testing to dilution testing.  The computational algorithm generates AST interpretive 
criteria that minimize the number of isolates with diffusion testing results that fall outside these 
criteria.   
 
Finally, evaluation of the frequency distribution analyses relative to the 
pharmacokinetic/pharmacodynamic (PK/PD) characteristics of the investigational antibacterial 
drug can further refine the provisional AST interpretive criteria. 
 
3.  Establishing In Vitro Antibacterial Susceptibility Test Interpretive Criteria 
 
Sponsors should perform an analysis of the correlation between the clinical cure and 
microbiologic eradication rates in the clinical trials with the provisional AST interpretive criteria 
results to determine their clinical relevance.  When appropriate, sponsors should assess the 
clinical response and microbiologic eradication rates as overall rates and as individual rates 
against bacteria with resistance to other antibacterial drugs as well as specific virulence factors.  
The available human PK/PD information, including target attainment analyses, assists in the 
selection of AST interpretive criteria.  These analyses help to form the basis for the final 
selection of the AST interpretive criteria. 
 
5  Contains Nonbinding Recommendations 
 
The purpose of establishing AST interpretive criteria is to guide the selection of appropriate 
antibacterial therapy.  The accuracy and clinical relevance of such tests depend on adherence to 
standardized methods and appropriate consideration of the test results. 
 
Sponsors also should consider the time it takes to develop a susceptibility test.  FDA encourages 
sponsors to coordinate susceptibility test development with drug development  so that 
susceptibility testing is available to inform use of the drug after it is marketed. 
 
Appendix C provides the recommended electronic database format for the data from clinical trials 
as it pertains to AST interpretive criteria.  
 
4.  Quality Control Parameters 
 
Sponsors should establish quality control (QC) parameters for AST before determining the 
activity of the antibacterial drug to ensure the generation of precise, accurate, and reproducible 
results.  Routine QC procedures involve performance testing of designated QC strains that are 
genetically stable and have well-characterized susceptibility characteristics.  Generally, the 
establishment of QC parameters should involve the use of 3 different lots of test medium, frozen 
panels in the case of MICs, 2 different lots of disks in the case of disk diffusion, and 10 
replicates of each QC strain over 3 days in at least 7 different laboratories.  This testing is done 
to generate enough data points to determine appropriate QC parameters. 
 
Sponsors should obtain reference bacteria recognized from a reputable source such as the 
American Type Culture Collection (ATCC).  In the event that sponsors do not use these 
recommended QC bacteria, they should justify the use of other well-characterized bacteria.  If a 
QC bacterium is chosen that is different from these recommended QC bacteria, it should be 
deposited in a recognized culture collection (e.g., ATCC). 
 
The use of established methods and concomitant use of QC strains lends confidence to the in 
vitro susceptibility data generated from the testing of bacteria.  Therefore, sponsors should 
provide QC data with all susceptibility test results done on bacteria at each facility that is 
conducting susceptibility testing for clinical trials.  Alternatively, if a central laboratory performs 
in vitro susceptibility tests, we recommend that sponsors provide the QC data generated by the 
central laboratory.  In addition, we recommend that sponsors analyze the QC data generated 
during the conduct of clinical trials to determine whether adjustments to the QC ranges are 
necessary.   
 
C.  Other Considerations 
 
1.  First and Second Lists of Target Bacteria in Labeling 
 
The Microbiology subsection of labeling contains two lists of bacteria.  Sponsors should format 
this section as described in Appendix D. 
 
The first list is based on bacteria evaluated during clinical trials that are included in the 
INDICATIONS AND USAGE section of labeling (21 CFR 201.57(c)(2)(i)(C)).  
6  Contains Nonbinding Recommendations 
 
 
The second list is based on the relevance of the bacteria to the indication and its susceptibility to 
concentrations of the antibacterial drug that can be achieved using the proposed dosage.  
Appendix E provides a summary of the information needed to support the inclusion of bacteria in 
the second list.  The inclusion of bacteria in the second list is not based on results from adequate 
and well-controlled clinical trials.  Sponsors should provide information in support of the second 
list for each species proposed for inclusion by indication.   
 
2.  Antibacterial Interactions and Fixed-Combination Studies 
 
Drug interaction studies may provide information (e.g., synergy, antagonism, indifference) 
on the effects one antibacterial drug may have on another.  Potential for interaction usually 
can be determined by qualitative or quantitative in vitro studies when the activity cannot be 
accurately anticipated from general knowledge of the drug characteristics.  The preferred 
methods for the characterization of antibacterial interaction are kill curves.  Another method 
is the checkerboard titration analyzed by fractional inhibitory concentration.  
 
For fixed-combination drug products, including drugs that contain an antibacterial drug and a 
component that counteracts a resistance mechanism (e.g., beta-lactam and beta-lactamase 
inhibitor combination), we recommend that sponsors provide the in vitro and/or in vivo data to 
support the contribution of each of the drugs (separately and in combination) to the activity. 
 
3.  Additional Nonclinical Studies of Antibacterial Drugs 
 
Antibacterial drugs may have various effects on target bacteria and/or interactions with the host.  
These phenomena include, but are not limited to, postantibiotic effect, postantibiotic leukocyte 
effect, sub-MIC effects, effects on endotoxin, effects on virulence factors, and interactions with 
the host immune system.  The sponsor should provide data from studies that are designed to 
investigate these effects of the investigational antibacterial drug. 
 
Some antibacterial drugs may be inactive when protein bound, or there may be insufficient free 
active drug at trough concentrations.  Therefore, we recommend that sponsors characterize the 
effects of human serum proteins and other human body fluids (e.g., lung surfactant), when 
appropriate, on the in vitro and in vivo activity of the drug.  The effects of human serum proteins 
and human body fluids on activity of the drug should be evaluated over the range of clinically 
relevant concentrations of the antibacterial drug. 
 
4.  Animal Models of Infection 
 
The goal of the animal models is to investigate the antibacterial activity of the investigational 
drug.  Ideally, the animal model of infection should be similar to the infection of interest in 
humans, and the bacteria used in the animal model should be similar in character (e.g., 
antibacterial resistance, virulence factors) to bacteria that cause human disease.    
 
In addition to measuring survival, sponsors should measure bacterial burden in blood and relevant 
affected organs.  For example, an evaluation of antibacterial activity in relevant tissue sites (e.g., 
7  Contains Nonbinding Recommendations 
 
spleen, kidney, lung, thigh) can be important to characterize the drugs potential in the treatment 
of particular body site infections in humans.  Animal models can provide preliminary 
information on PK/PD parameters as they pertain to microbiology data (e.g., area under the 
plasma concentration time curve over the MIC, maximal concentration over the MIC and time 
above the MIC).5 
 
Sponsors can conduct comparative studies of the investigational antibacterial drug with other 
antibacterial drugs exhibiting the same mechanism of action or drugs with the same spectrum of 
activity.  Results can be reported as 50 percent effective dose (ED50), 50 percent protective dose 
(PD50), or 50 percent curative dose (CD50).  Sponsors should also provide the bacterial burden 
data at baseline and various time points. 
 
5.  Microbiology Information Collected in Clinical Trials 
 
We recommend that a sponsor use a central laboratory for microbiologic testing (including 
confirmation of bacteria identification and AST) during clinical trials.  We recommend that 
sponsors provide clinical trial protocols and laboratory procedure manuals (including details of 
specimen collection, isolate/specimen transport, isolate characterization and identification, 
isolate preservation, and susceptibility testing methods) to the FDA for review before trial 
initiation.  
 
6.  Electronic Submission of Microbiology Information 
 
Sponsors should provide microbiology information in the electronic common technical document 
(eCTD) as described in the guidance for industry Providing Regulatory Submissions in Electronic 
Format  Certain Human Pharmaceutical Product Applications and Related Submissions Using 
the eCTD Specifications.6  Generally, sponsors should provide the information on microbiology in 
two sections of the eCTD as follows: 
 
  Module 2, Section 2.7, Clinical Summary, subsection 2.7.2.4, Special Studies.  This 
section should contain the microbiology summary reports and the information used to 
justify the microbiology information included in the labeling. 
 
  Module 5, Clinical Study Reports, subsection 5.3.5.4, Other Study Reports.  This section 
should contain the nonclinical study and clinical trial reports used in the construction of 
the summary information provided in subsection 2.7.2.4.  All the study reports used to 
construct the summary report presented in section 2.7.2.4 should be cross-linked to the 
summary report.  All sections should be cross-referenced to each other. 
 
                                                 
5 The FDA encourages sponsors to consult the FDA when considering a nonanimal testing method believed to be 
suitable, adequate, validated, and feasible.  The FDA will consider if the alternative method could be assessed for 
equivalency to an animal test method. 
 
6 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the FDA 
Drugs guidance web page at 
https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.  
8  Contains Nonbinding Recommendations 
 
7. Location of Microbiology Information 
 
The AST interpretive criteria recognized by the FDA are listed on the FDA Susceptibility Test 
Interpretive Criteria web page (https://www.fda.gov/STIC).  FDA will update the Interpretive 
Criteria web page at least every six months and upon approval of a new drug application or 
supplement, as applicable.7  Recognized performance standards, method standards, and quality 
control parameters for AST can be found on the FDAs Recognized Consensus Standards web 
page.8  
 
Other microbiology information (e.g., mechanism of action, antimicrobial activity, information 
regarding emergence of resistance) remains in the drug product labeling under Section 12.4 
Microbiology.    
 
8.  Postmarketing Microbiology Information 
 
Over time after approval, additional information regarding the methods for in vitro AST and/or 
the QC parameters used to monitor the performance of the test as well as how the susceptibility 
test results should be interpreted may become available.  Changes in AST interpretative criteria 
may translate into a lack of efficacy and/or safety concerns when out-of-date AST information 
leads to failure to appropriately treat the infectious disease.  Consequently, it is important that the 
in vitro AST methods, the QC parameters, and the AST interpretive criteria be reviewed on a 
regular basis and updated to reflect the most current information.  Application holders should 
review data regarding their drug for any necessary updates and submit the rationale for any 
changes in the in vitro AST methods, QC parameters, spectrum of activity, or AST interpretive 
criteria, as well as new information regarding emergence of resistance, in an applicants annual 
report under the new drug application.  FDA will review these annual report submissions and 
determine whether changes or updates to the currently recognized AST interpretive criteria are 
appropriate.  FDA will then update the Interpretive Criteria web page to reflect these changes, as 
needed.  If the applicant determines that other microbiology information that is contained in the 
drug labeling should be updated, the applicant can submit a labeling supplement.  
 
For applicants submitting an annual report with supporting data to update or change the method 
of determining the AST, we recommend including a description of the old and new methods with 
the changes noted, as well as validation data and QC parameters of the new method.  Any change 
                                                 
7 Section 511A(b) of the FD&C Act required the creation of the FDA Interpretive Criteria web page 
(https://www.fda.gov/STIC) for information about susceptibility test interpretive criteria for antimicrobial drugs.  
The FDA Interpretive Criteria web page recognizes appropriate interpretive criteria standards set by standard 
development organizations and provides exceptions, additions, or entire interpretive criteria when necessary 
(including when no relevant interpretive criteria are included in an FDA-recognized standard).  In accordance with 
section 511A(d), antimicrobial drug labeling must include a reference to the FDA Interpretive Criteria web page.  
More information about AST interpretive criteria labeling can be found in the guidance for industry Systemic 
Antibacterial and Antifungal Drugs: Susceptibility Test Interpretive Criteria Labeling for NDAs and ANDAs.  
 
8 The FDA Recognized Consensus Standards web page is available at 
https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfStandards/search.cfm.  Occasionally, consensus standards for 
methods and quality control parameters may not be established at the time a new drug is approved. If appropriate, 
FDA will list acceptable methods and quality control information for the drug on the Interpretive Criteria web page 
along with the AST interpretive criteria identified for such drug. 
9  Contains Nonbinding Recommendations 
 
to a test method (e.g., microbroth dilution) should be accompanied by data to show that the 
results correlate with other methods (e.g., agar dilution, disk diffusion testing) by which 
susceptibility testing can be done.  When an applicant provides information to change the QC 
parameters, the applicant also should include the results of validation studies. 
 
We recommend applicants include the following information when providing changes to the 
existing AST interpretive criteria: 
 
  Rationale for change 
 
  The MIC and zone diameter distributions against the genera and species of interest; data 
should be from isolates collected in the preceding 3 years of the submission 
 
  Susceptibility to the antibacterial drug to determine microbiologically supported cutoffs 
(e.g., histograms) 
 
  Categorical agreement between MIC and zone diameter breakpoints in graphical form 
 
  Error-rate bounded method of Metzler and DeHaan (Metzler and DeHaan 1974) to 
determine discrepancies between the two methods; the Metzler and DeHaan method 
usually needs to be modified because two MIC values are normally described to define an 
intermediate category (Bruden et al. 1992) 
 
  Relevant human PK data 
 
  Clinical data from adequate and well-controlled trials and literature reports 
 
10  Contains Nonbinding Recommendations 
 
REFERENCES 
 
Bruden, MN, GE Zurenko, et al., 1992, Modification of the Error-Bounded Classification Scheme 
for Use With Two MIC Breakpoints, Diagn Microbiol Inf Dis, 15:135140. 
 
Clinical and Laboratory Standards Institute (CLSI), 1999, Methods for Determining Bactericidal 
Activity of Antimicrobial Agents; Approved Guideline, CLSI document M26A, Clinical and 
Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, 
USA. 
 
CLSI, 2016, Development of In Vitro Testing Criteria and Quality Control Parameters; 
Approved Guideline-Fourth Edition, CLSI document M23-A4, Clinical and Laboratory 
Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA 
 
CLSI, 2012, Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved 
Standard  Eighth Edition, CLSI document M11-A8, Clinical and Laboratory Standards Institute, 
950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA. 
 
CLSI, 2015, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow 
Aerobically; Approved Standard  Tenth Edition, CLSI document M07-A10, Clinical and 
Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, 
USA. 
 
CLSI, 2015, Performance Standards for Antimicrobial Disk Diffusion Susceptibility Tests; 
Approved Standard  Twelfth Edition, CLSI document M02-A12, Clinical and Laboratory 
Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA. 
 
CLSI, 2017, Performance Standards for Antimicrobial Susceptibility Testing; Twenty-seventh 
Informational Supplement, CLSI document M100-S27, Clinical and Laboratory Standards 
Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA. 
 
Metzler, DM and RM DeHaan, 1974, Susceptibility Tests of Anaerobic Bacteria:  Statistical and 
Clinical Considerations, J Infect Dis, 130:588594. 
 
Turnidge, J and DL Paterson, 2007, Setting and Revising Antibacterial Susceptibility 
Breakpoints, Clin Microbiol Rev, 20(3):391408. 
 
11  Contains Nonbinding Recommendations 
 
APPENDIX A:  
GUIDELINES FOR BACTERIAL ISOLATE SELECTION  
FOR STUDIES OF IN VITRO ANTIBACTERIAL ACTIVITY 
 
For the purposes of determining the antibacterial activity of a new molecular entity and for 
establishing correlation of test methods (i.e., minimum inhibitory concentration (MIC) and disk 
diffusion), we recommend that the sponsor provide antibacterial susceptibility test (AST) data for 
bacterial isolates intended for inclusion in labeling.  The sponsor should consult with the division 
to determine an adequate number of tested isolates in those situations, as well as for special types 
of studies where significant information may be obtained using few isolates (e.g., bactericidal 
studies, time-kill studies).   
 
Table A lists the suggested number of bacterial isolates of each species or bacterial group that 
should be tested in nonclinical studies, when those species will be included in labeling for the 
antibacterial drug (i.e., in either the first or second list of target bacteria). 
 
We recommend that sponsors identify the prominent genotypes, serotypes, biotypes, and isolates 
with known mechanisms of resistance and include these in the test panel.  When appropriate, the 
spectrum of activity against hetero-resistant bacteria should be determined (e.g., vancomycin 
hetero-resistant Staphylococcus aureus).  The organisms tested should be recent clinical isolates 
collected within the last 3 years of the new drug application with susceptibility profiles that are 
representative of antibacterial drugs used to treat infections caused by the target bacteria for the 
indication being sought.  Sponsors should provide summary data by subset of organisms 
demonstrating resistance (e.g., methicillin-resistant S. aureus, extended spectrum beta-
lactamases).   
 
We recommend that at least 75 percent of isolates analyzed in these studies be collected in the 
United States.  Sponsors who include isolates from outside the United States should compare the 
characteristics of those isolates with the same species found in the United States (e.g., 
phenotype, genotype, serotype, susceptibility profile, and virulence factors). 
 
Sponsors should perform susceptibility testing using a range of drug dilutions that will minimize 
reporting greater than or equal to or less than or equal to MIC values (e.g., greater than or equal 
to 16 mcg/mL, less than or equal to 0.012 mcg/mL).  All AST data should be determined using 
standard reference methods unless modifications of the standard methods are needed.  Sponsors 
should present a summary of susceptibility testing results as MIC histograms or other frequency 
distributions displaying any proposed in vitro AST interpretive criteria.  All susceptibility test 
data should be accompanied by appropriate quality control data. 
 
12  Contains Nonbinding Recommendations 
 
Table A:  Suggested Number of Isolates for Determination of In Vitro Antibacterial 
Activity and for Correlation of MIC and Disk Diffusion Methods 
 
 
Bacteria or Bacterium  Number of Isolates 
Enterobacteriaceae   300 
Pseudomonas aeruginosa   100 
Acinetobacter spp.   100 
Staphylococcus spp.1, 2   100 
Enterococcus spp.1, 2   100 
Haemophilus influenzae and H. parainfluenzae   100 
Neisseria gonorrhoeae   100 
Streptococcus pneumoniae   100 (prefer > 300) 
Beta-hemolytic streptococci1   100 (prefer > 300) 
Alpha-hemolytic streptococci (other than S. pneumoniae)1   100 (prefer > 300) 
Other single species   100 
 
                                                 
1 Bacterial isolates in these genera or bacterial groups should be identified to species level (or, in some cases, to 
more specific groups (e.g., Streptococcus anginosus group)).  In these cases, the recommended number of isolates 
applies to the particular species or group. 
 
2 When a particular phenotype (e.g., methicillin-resistant Staphylococcus aureus, vancomycin-resistant 
Enterococcus faecalis) will be included in labeling, the recommended number of isolates applies to that phenotype. 
13  Contains Nonbinding Recommendations 
 
APPENDIX B:  
STUDY REPORTS OF SPECTRUM OF ACTIVITY AND RESISTANCE 
 
Study Reports of Spectrum of Activity 
 
We recommend that study reports of spectrum of activity include the following elements: 
 
  The name and location of each investigator conducting or contributing to the study. 
 
  The standardized methods for bacterial identification and antibacterial susceptibility test 
(AST) used to determine the activity of the antibacterial drug; details of the method and 
the performance characteristics of the assay in the actual laboratory where such testing is 
done should be included if a nonstandard method is used. 
 
  A description of all susceptibility testing quality control (QC) measures; all AST results 
should be accompanied by QC data. 
 
  The number of isolates tested in each laboratory, the specimen source, and the 
geographical region from which the isolates were obtained. 
 
  A description of the spectrum of activity by all regions and individual geographic regions. 
 
  The phenotypic and/or genotypic characterization of isolates relative to their resistance to 
other antibacterial drugs; the methodology and the criteria used to characterize isolates as 
resistant should be described. 
 
  The phenotypic and/or genotypic characterization of isolates relative to virulence 
characteristics. 
 
Results from studies evaluating antibacterial activity should be presented in tabular form under 
appropriate sections as described below: 
 
  Genera and species tested  species with unique mechanisms of resistance should be 
grouped separately; serotype, phenotype, and/or genotype should be included if known 
 
  Drug name 
 
  The minimum inhibitory concentration (MIC) range and the number of isolates tested 
 
  The values for the MIC required to inhibit growth of 50 percent of bacteria (MIC50) and 
the MIC required to inhibit growth of 90 percent of bacteria (MIC90)  
 
  Minimum bactericidal concentration (MBC) 
 
  MIC:MBC ratio for members of clinically relevant genera 
14  Contains Nonbinding Recommendations 
 
 
Study Reports of Resistance 
 
Results from studies evaluating resistance should be presented in tabular form under appropriate 
sections as described below: 
 
  Genera and species tested  species with unique mechanisms of resistance should be 
grouped separately; if serotype, phenotype, and/or genotype are known then that 
information should be included 
 
  Drug name 
 
  MIC range  for each group of organisms and the number of isolates tested in each 
laboratory 
 
  MIC50 and MIC90 values 
 
  MBC 
 
The complete study report should include stability and lot numbers of the drug used for 
microbiology testing.  In addition, reproducibility of test results should be submitted.  If the data 
are derived from a publication, a copy of the publication should be included in the submission. 
 
 
15  Contains Nonbinding Recommendations 
 
APPENDIX C:  
DATABASE FOR FINAL IN VITRO ANTIBACTERIAL  
SUSCEPTIBILITY TEST INTERPRETIVE CRITERIA 
 
We recommend a single electronic database for the clinical trial with the subject level data 
presented in columns.  Each column heading should identify the scope of information below it.  
For instance, a subject identification (ID) number can include a coding arrangement that 
differentiates the trial center as well as the individual subject.  Sponsors should discuss the 
format of the microbiology datasets with the FDA at the time of the pre-new drug application or 
pre-biologics license application meeting.  We recommend that a sponsor provide the following 
information in the database under appropriate columnar headings: 
 
  Clinical trial center number  
 
  Subject ID number 
 
  Treatment group 
 
  Sample source 
 
  Species of bacterial isolate 
 
  Indication 
 
  Subject-by-subject clinical evaluations including separate rows for each subject, the 
subjects status of microbiological eradication, and the subjects overall clinical response 
(e.g., cure, failure) 
 
  Antibacterial susceptibility test (AST) results by diffusion methods for the investigational 
drug and the comparator drug 
 
  AST results by dilution methods for the investigational drug and the comparator drug 
 
Sponsors should provide an interpretation of the data described below for the investigational 
drug and comparator drugs.  Because of possible geographic differences in antibiograms and the 
clonal nature of bacteria, sponsors should present data in both combined and separate formats 
(e.g., United States and non-United States in separate tables).  Where appropriate, we 
recommend that U.S. data be broken down into regions (e.g., Northeast, Southeast, Midwest, 
Northwest, Southwest) and that non-U.S. data be broken down by region (e.g., Asia, Europe, 
Africa) and within region by country (e.g., France, Germany). 
 
We recommend that a sponsor include the following in the database: 
 
1.  Minimum inhibitory concentration (MIC) values and subject microbiological responses 
for each baseline bacterium within each proposed indication; all subsets of bacteria 
demonstrating unique mechanisms of resistance (e.g., methicillin-resistant 
16  Contains Nonbinding Recommendations 
 
Staphylococcus aureus, beta-lactamase-positive Haemophilus influenzae) and virulence 
should be listed separately. 
 
2.  MIC values and subject clinical response for each baseline bacterium for each proposed 
indication; all subsets of bacteria demonstrating unique mechanisms of resistance and 
virulence should be listed separately. 
 
3.  Zone diameter values and subject microbiological responses for each bacterium for each 
proposed indication; all subsets of bacteria demonstrating unique mechanisms of 
resistance and virulence should be listed separately. 
 
4.  Zone diameter values and subject clinical responses for each bacterium for each proposed 
indication; all subsets of bacteria demonstrating unique mechanisms of resistance and 
virulence should be listed separately. 
 
5.  For each subset of bacteria requiring defined AST interpretive criteria, all individual 
isolates in the range of MICs from two dilutions below the susceptible and two dilutions 
above the resistant provisional AST interpretive criteria. 
 
6.  For each subset of bacteria requiring defined zone diameter AST interpretive criteria, all 
individual isolates in the range of zone diameters from 4 to 6 millimeters above the 
susceptible and 4 to 6 millimeters below the resistant provisional AST interpretive 
criteria. 
 
7.  By indication and bacteria relevant to the indication, all MICs for isolates associated with 
microbiological failures.  The bacteria should be identified to the species level. 
 
8.  By indication and bacteria relevant to the indication, all zone diameters for isolates 
associated with microbiological failures.  The bacteria should be identified to the species 
level. 
 
9.  For each bacterium (e.g., nonfastidious, fastidious, and anaerobic), the MIC value 
indicating the number and percent of isolates at that MIC associated with each 
microbiological response.  MIC values should be grouped by bacterial type. 
 
10. For each bacterium (e.g., nonfastidious, fastidious), the zone diameter indicating the 
number and percent of isolates at the zone diameter associated with each microbiological 
response.  Zone diameter information should be grouped by bacterial type. 
 
11. For each group of bacteria, a histogram showing the number of isolates at each MIC from 
clinical trials overlaying isolates from nonclinical studies.  Sponsors should present 
bacteria with characterized phenotypic resistance and virulence markers as a subset. 
 
12. For each group of bacteria, a histogram showing the number of isolates at each zone 
diameter from clinical trials overlaying isolates from nonclinical studies.  Sponsors 
17  Contains Nonbinding Recommendations 
 
should present bacteria with characterized phenotypic resistance and virulence markers as 
a subset. 
 
18  Contains Nonbinding Recommendations 
 
APPENDIX D:  
EXAMPLE FORMAT FOR SECTIONS OF LABELING  
THAT PERTAIN TO MICROBIOLOGY 
 
Recommended language for and organization of the microbiology information that is discussed 
in the CLINICAL PHARMACOLOGY section of labeling are provided below along with 
additional recommendations in italics.1  For additional information concerning this section of 
labeling, see the guidance for industry Clinical Pharmacology Labeling for Human Prescription 
Drug and Biological Products  Considerations, Content, and Format.2  Also included are 
recommended citations for the REFERENCES section of labeling.  We recommend a consistent 
formatting approach for headings and subheadings in the CLINICAL PHARMACOLOGY 
section and the other sections of labeling to help organize the information (e.g., underlining for 
headings and italics for subheadings).  We recommend the use of title case for headings and 
subheadings. 
 
12  CLINICAL PHARMACOLOGY 
 
12.1  Mechanism of Action 
X is an anti- (e.g., bacterial, as appropriate) drug [see Microbiology (12.4)].3 
 
Recommendation:  The antimicrobial mechanism of action should be described in subsection 
12.4 Microbiology.  
 
12.4  Microbiology 
 
Mechanism of Action 
 
Resistance 
 
Interaction With Other Antimicrobials 
 
[Heading Title] [Other relevant information to be determined on a case-to-case basis] 
 
Recommendation:  Additional relevant microbiological information that provides 
characterization of the antimicrobial drug should be placed under another appropriately 
named heading. 
 
                                                 
1 As provided for in the final rule Requirements on Content and Format of Labeling for Human Prescription Drug 
and Biological Products (71 FR 3922, January 24, 2006), the microbiology portion of labeling should be added as 
subsection 12.4 under the CLINICAL PHARMACOLOGY section. 
 
2 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the FDA 
Drugs guidance web page at 
https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.  
 
3 Note that this is an exception to the preferred presentation of cross-referencing in the Full Prescribing Information, 
which is to use the section heading followed by the numerical identifier. 
19  Contains Nonbinding Recommendations 
 
Antimicrobial Activity 
[Name of drug] has been shown to be active against most isolates of the following 
microorganisms, both in vitro and in clinical infections [see Indications and Usage (1)]: 
 
Recommendation:  Each organism in this list must be associated with an indication in the 
INDICATIONS AND USAGE section (e.g., acute bacterial skin and skin structure 
infections caused by susceptible isolates of Staphylococcus aureus) (21 CFR 
201.57(c)(2)(i)(C)). 
 
[Microorganisms should be listed under the following categories in alphabetical order] 
Aerobic bacteria 
Gram-positive bacteria 
Gram-negative bacteria 
Anaerobic bacteria 
Gram-positive bacteria 
Gram-negative bacteria 
Other microorganisms (as applicable) 
 
The following in vitro data are available, but their clinical significance is unknown.4  At least 
90 percent of the following bacteria exhibit an in vitro minimum inhibitory concentration 
(MIC) less than or equal to the susceptible breakpoint for [name of drug] against isolates of 
similar genus or organism group.  However, the efficacy of [name of drug] in treating clinical 
infections caused by these bacteria has not been established in adequate and well-controlled 
clinical trials. 
 
[Microorganisms should be listed under the following categories in alphabetical order] 
Aerobic bacteria 
Gram-positive bacteria 
Gram-negative bacteria 
Anaerobic bacteria 
Gram-positive bacteria 
Gram-negative bacteria 
Other microorganisms (as applicable) 
 
Recommendation:  For an organism to be included in the above list (i.e., the second 
list), the organism at a minimum should:  (1) be relevant to an indication granted in 
the labeling; and (2) have an MIC  below the concentration of the antimicrobial 
90
achievable in the plasma or at the infection site using the dosing regimen approved in 
the labeling as determined from in vitro testing of the targeted species or organism 
group.  See Appendix A for the recommended number and characteristics of test 
isolates.  See Appendix E for a summary of information for microorganisms to be 
included on the second list as well as additional information. 
 
                                                 
4 This statement must be included for the second list of bacteria; see 21 CFR 201.57(c)(13)(ii)(A). 
20  Contains Nonbinding Recommendations 
 
Susceptibility Testing 
For specific information regarding susceptibility test interpretive criteria, and associated test 
methods and quality control standards recognized by FDA for this drug, please see 
https://www.fda.gov/STIC. 
 
 
21  Contains Nonbinding Recommendations 
 
APPENDIX E:  
INFORMATION REGARDING THE SECOND BACTERIA LIST IN THE 
MICROBIOLOGY SUBSECTION OF LABELING 
 
 
Factors to Consider When Developing the Lists 
 
The following factors should be considered when developing the bacteria lists: 
 
  Certain bacteria are disease-specific and therefore can be properly placed only in the first 
list.  Examples of such pathogens are Mycobacterium tuberculosis, Bacillus anthracis, 
Mycobacterium leprae, Yersinia pestis, Neisseria meningitidis, Neisseria gonorrhoeae, 
and Brucella species. 
 
  There should be scientific evidence demonstrating that the bacteria are frequently 
associated with an indication for which approval is being sought. 
 
  Sponsors should support the species included in the second list with antibacterial 
susceptibility test (AST) results of recent clinical isolates (minimum inhibitory 
concentrations (MICs)) correlated with the achievable concentrations of the antibacterial 
drug using the recommended dosing regimen. 
 
  Bacteria included in the second list should have MIC  values less than or equal to the 
90
clinically relevant AST interpretive criteria established for the particular genera, species, 
or group of bacteria related to a specific indication or indications. 
 
Summary Information for the Second List 
 
The summary should include the following information for the bacteria on the second list: 
 
  A discussion of the relevance of the bacteria to a specific clinical indication 
 
  The frequency in which the bacteria is shown to cause disease in the general population 
 
  Relevant literature references and/or laboratory test data summary tabulations of 
susceptibility data (e.g., range, MIC , MIC  for relevant antibacterial drugs) and 
50 90
annotated supporting literature for the listed bacteria 
 
  In vitro susceptibility information for (e.g., MIC, MIC range, MIC , MIC ) of the 
50 90
bacteria proposed for the second list (see Appendix C for the suggested number of 
isolates and characteristics of pathogens that should be included for testing), 
accompanied by appropriate quality control (QC) data 
 
  A discussion of the methods used and their comparability to assess susceptibility as 
described in the supporting literature 
 
22  Contains Nonbinding Recommendations 
 
  Comparisons of U.S. and foreign data analyzed separately and together 
 
  Susceptibility data that are accompanied by the appropriate QC data 
 
23