Guidelines for the Testing and Reporting of Antimicrobial Susceptibilities of Streptococcus pneumoniae

Canadian External Quality Assessment
Advisory Group for Antibiotic Resistance

September 1998

ISBN 0-662-27499-7
© 1998

1.0 Introduction

These guidelines are produced under the auspices and authority of the Canadian External Quality Assessment - Advisory Group on Antibiotic Resistance (CEQA-AGAR). They represent a consensus of peer reviewed information and expert opinion on the most appropriate ways to test for and report antimicrobial susceptibility and resistance. Clinical situations and individual laboratory operations may necessitate some variations from these guidelines. These guidelines are strongly recommended for use by all laboratories providing information for national surveillance programs on antimicrobial resistance.

2.0 Preamble

Streptococcus pneumoniae historically was susceptible to penicillins and many other antimicrobial agents. A developing body of both laboratory and clinical evidence indicates that this is no longer true ^{1, 6, 7, 10}. Data from the literature indicates that in some countries as many as 40% of strains are intermediate or resistant to penicillin ^{2, 6, 8}. Along with penicillin, resistance has emerged to other agents, including cephalosporins, macrolides and co-trimoxazole ⁸. It is now essential that laboratories test strains of S. pneumoniae for resistance to these agents in defined circumstances.

3.0 Definitions

3.1 Penicillin-susceptible S. pneumoniae. Strains that have penicillin MICs of <= 0.06 mg/L

3.2 Penicillin-intermediate S. pneumoniae. Strains that have penicillin MICs of 0.1 - 1.0 mg/L

3.3 PRSP. (Penicillin-Resistant S. pneumoniae). Strains that have penicillin MICs of >= 2.0 mg/L.

3.4 DRSP (Drug-Resistant S. pneumoniae). Strains that are intermediate or resistant to penicillin and to at least one other commonly tested antimicrobial agent of a different class.

3.5 NCCLS (National Committee for Clinical Laboratory Standards). A U.S.A. based organization that sets guidelines for testing and reporting of antimicrobials for some species of bacteria. Where appropriate these guidelines will be utilized in the CEQA-AGAR documents.

3.6 Disk-diffusion. Refers to Kirby-Bauer method ³.

3.7 Broth microdilution. Refers to testing in micro-titre trays according to NCCLS M7 documents ⁴.

3.8 Gradient diffusion. Refers to such methods as E-Test^® that provide an MIC according to standard testing protocols on agar plates ⁹.

4.0 What to Test

The decision on what and when to test depends on the clinical situation, the antimicrobial formulary, and laboratory capabilities. These are some guidelines on what antimicrobial agents to test from specific sites.

4.1 All S. pneumoniae from sterile body sites should be tested for antimicrobial resistance. These include blood, cerebrospinal fluid, joint aspirates, pleural fluids, abscesses, and any other site in which there is clinical indication of a life-threatening illness. These strains should be tested against the following agents: penicillin (oxacillin - see below), cefotaxime and/or ceftriaxone, and vancomycin.

4.2 S. pneumoniae from non-sterile sites. These would include sputum, eye, ear, etc. Isolates from non-sterile sites may not require routine susceptibility testing. However, it may be useful to establish in one's own area on a periodic basis the degree of resistance of S. pneumoniae to various antimicrobials commonly used for treatment of infections caused by this organism including penicillin (oxacillin - see below), and other agents as clinically indicated:

1. macrolides,
2. cefuroxime, cefotaxime or ceftriaxone
3. co-trimoxazole
4. clindamycin
5. tetracycline

5.0 Methods

5.1 Screening for penicillin resistance with oxacillin ². All clinically important strains of S. pneumoniae should be tested by disk diffusion according to the most current NCCLS document (M2-A6) using a 1 µg oxacillin disk. The medium is Mueller-Hinton agar with 5% sheep blood incubated in 5% CO2 for 20-24 hr at 35^o C.

Note that penicillin disks are not reliable and can not be used to test for penicillin susceptibility.

5.2 Penicillin MIC Testing. Quantitative MIC testing using penicillin should be done by broth micro-dilution in cation adjusted Mueller-Hinton broth with 2 - 5% lysed horse blood incubated in ambient air at 35^o C for 20-24 hr.

Alternatively, a gradient diffusion method may be used. For this method use Mueller-Hinton agar with 5% sheep blood incubated in 5% CO2 for 20-24 hr.

5.3 Testing of other antimicrobials.

5.3.1 Macrolides. Erythromycin can be tested using disk diffusion. Erythromycin will predict susceptibility to clarithromycin and azithromycin.

5.3.2 Reliable disk diffusion criteria do not exist for testing S. pneumoniae against cefuroxime, ceftriaxone and cefotaxime. These should be tested by an MIC method.

6.0 Interpretation and Reporting

6.1 Interpretation

6.1.1 Penicillin susceptibility

Oxacillin zone diameter >= 20 mm. The isolate is susceptible to penicillin. Oxacillin zone diameter <= 19 mm. Susceptibility to penicillin must be confirmed by an MIC method. These strains may be resistant, intermediate or susceptible to penicillin.

6.1.2 Penicillin MIC Interpretation.

0.1 - 1.0 mg/L Intermediate

>= 2.0 mg/L Resistant

Note: All gradient diffusion MICs greater than 1.0 mg/L should be interpreted and reported as >= 2 mg/L.

6.1.3 Interpretation of Susceptibility to Other Antimicrobial Agents

Please refer to Tables 2G in NCCLS Informational Supplement M100-S8 ⁵ for specific zone diameter criteria (for M2-A6; Disk Diffusion) and MIC breakpoints (for M7-A4; MIC Testing) for interpretation of results to appropriate antimicrobial agents other than penicillin. Examples of these interpretive guidelines are:

Note: No strains with vancomycin zone diameters <= 17 or MICs >= 1 mg/L have been observed. If such strains are observed they should be submitted to a reference laboratory for confirmation before reporting. Additional breakpoint criteria are available for newer quinolones that have activity against S. pneumoniae. Some of these agents are not licensed in Canada. Laboratories should consult the NCCLS documents listed above and their local pharmacy and therapeutics guidelines before testing and reporting these agents.

6.2 Reporting

6.2.1 Oxacillin susceptible (>= 20 mm). These strains can reliably be considered as susceptible to penicillin and all beta-lactam antibiotics.

6.2.2 Oxacillin result <= 19 mm. The report should indicate that the strain may have reduced susceptibility to penicillin, and that this result will be confirmed.
Note: It has been observed that strains producing no zone of inhibition around an oxacillin disk have a very high probability of reduced susceptibility to penicillin.

6.2.3 Penicillin MIC >= 0.1 mg/L. The report should indicate that the strain has reduced susceptibility to penicillin, and that patients may not respond to therapy with this agent.

In addition, there is an increasing body of evidence that strains of S. pneumoniae that are either intermediate-resistant or fully-resistant to penicillin may be more resistant to cephalosporins including ceftriaxone and cefotaxime and to macrolides and co-trimoxazole.

6.2.4 For external/national surveillance reporting purposes, for isolates confirmed to be either intermediate or fully resistant to penicillin, report one isolate per patient.

7.0 Referral Strategies

Laboratories that do not have the capabilities for full MIC determination but who are capable and authorized to conduct screening tests should perform oxacillin screening tests on isolates from patients with life threatening infections. If the laboratory is unable to perform other tests, presumptively resistant isolates should be sent to a reference laboratory for full antimicrobial susceptibility testing.

8.0 Summary

It is incumbent on laboratories to be able to accurately test for the antibiotic resistance of S. pneumoniae as noted above and be able to inform physicians of problem isolates. Surveillance and ongoing evaluation of methods to detect resistance are also important in order to prevent the spread of resistant strains.

9.0 References

1. Simor, A.E., Louie, M., Low, D.E. 1996. Canadian national survey of prevalence of antimicrobial resistance among clinical isolates of Streptococcus pneumoniae. The Canadian bacterial surveillance network. Antimicrob. Agents Chemother. 40: 2190-2193.

2. Swenson, J.M., Hill, B.C. Thornsberry, C. 1986. Screening pneumococci for penicillin resistance. J. Clin. Microbiol. 24: 749-752.

3. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility tests. Seventh ed. Approved Standard. M2-A6. National Committee for Clinical Laboratory Standards, Wayne Pennsylvania. January 1997.

4. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard. M7-A4. National Committee for Clinical Laboratory Standards, Wayne Pennsylvania. January 1997.

5. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing; Eighth informational supplement. M100-S8. Vol. 17, No. 1. National Committee for Clinical Laboratory Standards, Wayne, Pennsylvania. January, 1998.

6. Butler, J.C., Hofmann, J., Cetron, M.S. et al. 1996. The continued emergence of drug-resistant Streptococcus pneumoniae in the United States: an update from the Centers for Disease Control and Prevention's pneumococcal surveillance system. J. Clin. Microbiol. 174: 986-993.

7. Morbidity and Mortality Weekly Report. 1996. Centers for Disease Control and Prevention. Defining the public health impact of drug-resistant S. pneumoniae: Report of a working group. MMWR 45: 1-20.

8. Bradley, J.S., Scheld, W.M. 1997. The challenge of penicillin resistant Streptococcus pneumoniae meningitis: current antibiotic therapy in the 1990s. Clin. Infect. Dis. 24: S213- S221.

9. Skulnik, M., Small, G.W., Lo, P., et al. 1995. Evaluation of accuracy and reproducibility of E-test for susceptibility testing of Streptococcus pneumoniae to penicillin, cefotaxime and ceftriaxone. J. Clin. Microbiol. 33: 2334-2337.

10. Lovgren, M., Spika, J.S., Talbot, J.A. 1998.Invasive Streptococcus pneumoniae infections: serotype distribution and antimicrobial resistance in Canada, 1992 - 1995. Canad. Med. Assoc. J. 158: 327-331.

[CEQA-AGAR]