Vol. 28, No. 3

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1990, p. 430-434 0095-1137/90/030430-05$02.00/0 Copyright © 1990, American Society for Microbiology

Use of Haemophilus Test Medium for Broth Microdilution Antimicrobial Susceptibility Testing of Streptococcus pneumoniae Department

J. H. JORGENSEN,* L. A. MAHER, AND A. W. HOWELL The University of Texas Health Science Center, San Antonio, Texas 78284

of Pathology,

Received 8 September 1989/Accepted 11 November 1989

A recently described medium (Haemophilus test medium [HTM]) for antimicrobial susceptibility testing of Haemophilus influenzae was evaluated in this study for broth microdilution testing of Streptococcus pneumoniae. A total of 137 clinical isolates was tested against 11 antimicrobial agents, using Mueller-Hinton broth supplemented with 3% lysed horse blood in parallel with HTM. Inocula of 5 105 CFU/ml and incubation for 20 to 24 h were used with both media. All isolates of S. pneumoniae produced acceptable growth in both media, and MICs determined in HTM agreed closely with those determined in lysed horse blood. Drugs which provided a MIC within 1 log2 concentration difference in both media included penicillin (100%), ampicillin (98.0%), amoxicillin-clavulanate (100%), ampicillin-sulbactam (100%), cephalexin (98.9%), cefaclor (96.8%), cefuroxime (99.0%), chloramphenicol (96.2%), tetracycline (96.2%), and erythromycin (100%). HTM MICs with trimethoprim-sulfamethoxazole were 1 to 2 log2 concentration increments higher in 92.0% of isolates than MICs determined in Iysed horse blood. Based on the results of this study, HTM appears to represent a promising alternative medium for broth microdilution susceptibility testing of S. pneumoniae. X

or more of the study antimicrobial agents. Control strains used with both susceptibility test media included Escherichia coli ATCC 25922, E. coli ATCC 35218, and Enterococcusfaecalis ATCC 29212. Reference microdilution susceptibility tests. Broth microdilution susceptibility tests were performed on each isolate by the method recommended by the NCCLS (12), which incorporates use of cation-supplemented Mueller-Hinton broth (Difco, Detroit, Mich.) with 3% LHB. LHB was prepared at 50% by aseptically adding equal volumes of sterile distilled water to defibrinated horse blood (Remel, Lenexa, Kans.) and then performing five freeze-thaw cycles, i.e., completely freezing the 50% blood at -20°C and then completely thawing the blood at room temperature. The lysed blood was then clarified by centrifugation at 12,000 x g for 20 min. The supernatant was aseptically decanted (now 50% LHB) and added to cation-supplemented Mueller-Hinton broth to yield a final concentration of 3% LHB. Twofold concentration increments of the antimicrobial agents were dispensed in 100-,ul amounts in plastic 96-well microdilution trays. A suspension of colonies from an overnight blood agar plate culture was adjusted to the turbidity of a 0.5 McFarland standard. This suspension was further diluted to achieve a final inoculum of 5 x 105 CFU/ml in the microdilution wells. Following incubation at 35°C for 20 to 24 h in ambient air, MIC endpoints were interpreted in the usual manner. HTM microdilution tests. Broth microdilution tests were also performed with HTM (8), which consisted of MuellerHinton broth base (Difco) supplemented with 15 ,ug of bovine hematin (Sigma Chemical Co., St. Louis, Mo.), 5 mg of yeast extract (Scott Laboratories, Fiskeville, R.I.), and 15 ,ug of ,-NAD (Sigma) per ml. A 30-ml amount of hematin stock solution and 5 g of yeast extract were added to 1 liter of dissolved Mueller-Hinton broth base before autoclaving. A fresh hematin stock solution was prepared on the day of use by dissolving 50 mg of the bovine hematin in 100 ml of 0.01 N NaOH with gentle heat and stirring for 20 to 30 min until the powder was completely dissolved. After autoclaving and cooling, 3 ml of P-NAD stock solution was added aseptically. The NAD stock solution was made by dissolving

The increasing prevalence of relative penicillin resistance in Streptococcus pneumoniae (3, 4, 9, 15), along with recognition of resistance to chloramphenicol, tetracycline, and erythromycin (2, 3, 5, 9, 17), places new emphasis on the need for routine antimicrobial susceptibility testing of this organism when isolated from serious infections. The oxacillin disk diffusion test, which many clinical laboratories now use, cannot differentiate frank penicillin resistance from relative resistance (7, 14). A penicillin MIC test must be performed to distinguish between these two resistance categories. However, false susceptibility to penicillin has been reported when laboratories have attempted to adapt certain commercial microdilution MIC systems for testing of S.

pneumoniae (7, 16). The medium currently recommended by the National Committee for Clinical Laboratory Standards (NCCLS) for broth microdilution testing of pneumococci is MuellerHinton broth supplemented with 2 to 5% lysed horse blood (LHB) (13). While pneumococci grow reliably in this medium, the horse blood lysate is not available commercially and is difficult for most clinical laboratories to prepare. The purpose of this investigation was to determine whether an optically clear, relatively simple medium developed recently for testing Haemophilus species (Haemophilus test medium [HTM]) (8) could be used for broth microdilution testing of S. pneumoniae.

MATERIALS AND METHODS Test strains. A group of 100 S. pneumoniae isolates was selected randomly from a larger collection of 487 clinical isolates recovered during a recent U.S. national surveillance study of antimicrobial resistance in respiratory isolates of S. pneumoniae (J. H. Jorgensen, L. A. Maher, A. W. Howell, J. S. Redding, and G. V. Doern, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 1286, 1989). Thirty-seven additional clinical isolates from the same collection were selected for testing because they demonstrated resistance or diminished susceptibility to one *

Corresponding author. 430

S. PNEUMONIAE SUSCEPTIBILITY TESTING

VOL. 28, 1990

the agar surface (for induction of the enzyme). Growth was taken from around the inhibition zone margin and suspended in sterile 0.9% saline to match the turbidity of a no. 1 McFarland standard. A CAT test disk and a control disk, placed in separate glass test tubes, were each overlaid with 0.2 ml of the growth suspension. After incubation for 30 min at 35°C, the color of the eluate in the tube containing the test disk was compared with that in the tube containing the control disk. The test was considered positive when the yellow in the test disk tube was more intense than the color in the control disk tube.

TABLE 1. Agreement between MICs performed with LHB and HTM on S. pneumoniae isolates No. of strains tested

Drug

Penicillin Ampicillin

Amoxicillin-clavulanate Ampicillin-sulbactam Cephalexin Cefaclor Cefuroxime Chloramphenicol Tetracycline Erythromycin Trimethoprim-sulfamethoxazole

112 99 98 98 94 93 100 106 104 94 112

No. of HTM MICs within the given log2 concn increments of LHB MICs

-2

-1

Same

+1

2

45 8 43

64 83 53 83 55 52 72 55 47 60 4

3 6 2 4 16 2 5 14 1 27 45

il 1 3 2 4

22 36 22 33 52 7

+2

+3

1 2

RESULTS

All 137 S. pneumoniae clinical isolates tested in this study produced acceptable growth characteristics in both LHB and HTM, although the density of growth generally appeared somewhat heavier in LHB. Interpretation of growth endpoints was quite simple in the optically clear HTM broth. MIC endpoints in the HTM broth were sharp, with either clear growth buttons or no visible turbidity (at the MICs). MICs determined by use of the two media agreed closely with the majority of antimicrobial agents tested (Table 1). MICs were the same or agreed within 1 log2 concentration increment with penicillin (100%), ampicillin (98.0%), amoxicillin-clavulanate (100%), ampicillin-sulbactam (100%), cephalexin (98.9%), cefaclor (96.8%), cefuroxime (99.0%), chloramphenicol (96.2%), tetracycline (96.2%), and erythromycin (100%). However, MICs determined in HTM tended to be 1 concentration increment lower than when determined in LHB with penicillin, amoxicillin-clavulanate, cefaclor, cefuroxime, and tetracycline (Table 1). This trend combined with the fact that a number of penicillin MICs were clustered near the interpretive breakpoints (Fig. 1) led to 8.9% minor interpretive errors with that drug. Trimethoprim-sulfamethoxazole MICs were 1 to 2 log2 concentration increments higher with 92% of isolates in HTM as compared with the parallel LHB determinations

5

58

50 mg of P-NAD in 10 ml of distilled water followed by filter sterilization using a 0.22-,um-pore size membrane filter. Cations (Ca2" and Mg2+ [12]) were added after chilling the HTM broth. Since trimethoprim-sulfamethoxazole tests were to be performed, thymidine phosphorylase (Burroughs Wellcome Co., Research Triangle Park, N.C.) was also added aseptically at a final concentration of 0.2 IU/ml to the sterilized and cooled HTM broth. Antimicrobial agent-containing HTM broth was dispensed into plastic 96-well microdilution trays as described above. Inoculum preparation and incubation conditions were as stated above. CAT tests. Strains demonstrating elevated chloramphenicol MICs were tested for the presence of chloramphenicol acetyltransferase (CAT), using a commercial paper disk method (Remel). Modifications of the manufacturer's method were made as described by Matthews et al. (11). Specifically, strains were grown overnight on a sheep blood agar plate with inclusion of a 30-,ug chloramphenicol disk on

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HTM Microdilution MIC (j±g/mi) FIG. 1. Comparison of penicillin MICs (n criteria (14).

=

112) determined

in LHB and in HTM. Dotted

lines indicate current NCCLS interpretive

J. CLIN. MICROBIOL. JORGENSEN ET AL.J.CI.MROO.

432

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and

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HTM Microdilution MIC Comparison of trimethoprim-sulfamethoxazole MICs (n= NCCLS interpretive criteria (14). FIG.

I

1

~ ~

32

16

>32

(,~g/mI)

118) determined in LHB and in HTM. Dotted

lines

indicate current

Fig. 2). Similarly, erythromycin MICs tended to higher when determined in

be 1 concentration increment HTM.

HTM

correlated

well with MICs determined in LHB and with the

production

Chloramphenicol MICs determined

in

of CAT (Fig. 3). Ail six CAT-producing strains had chloramphenicol MICs of .:16 ptg/ml when tested in HTM. Induction of the CAT enzyme with a 30-p.g chloramphenicol disk

was

essential for

a

correct CAT test result, in that ail six

negative when tested without induction. Tetracycline-resistant strains were also readily detected by MIC testing in HTM (Fig. 4). MIC testing with HTM provided accurate categorization of susceptible and resistant strains according to current NCCLS interpretive guidelines (Table 2) (13). No very major or major errors of interpretation occurred with penicillin, erythromycin, tetracycline, or trimethoprim-sulfamethoxazole. Error rates were not calculated for ampicillin, the cephalosporins, and P-lactamase inhibitor combinations since specific interpretive guidelines for pneumococci have not been described by the NCCLS for those drugs. Error rates calculated for chloramphenicol used the interpretive breakpoints suggested by Matthews et al. (11). Tests of five separate lots of HTM and five lots of LHB provided MIC results consistently within the NCCLS control ranges (for unsupplemented Mueller-Hinton broth) with ail three of the quality control strains. strains

were

Fl=CAT+

32

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Strains

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DISCUSSION

The oxacillin disk diffusion test has been shown to be

quite reliable for screening pneumococci for susceptibility to penicillin (7). However, the disk test cannot make the

8

4

HTM Microdilution MIC FIG. mined

3.

in

and

in

HTM.

Dotted

criteria proposed by Matthews et

ai.

lines

(11).

32

(Q.£g/mi)

Comparison of chloramphenicol MICs (n LHB

16

=

indicate

106) deter-

interpretive

VOL. 1990 VOL. PNEUMONIAE SUSCEPTIBILITY TESTING 1990 ~~~~~~~S. 28,28,

433

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3216

1

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-

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w

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23

40

0.5 -

t-

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4

0.25-~

13

3

2

0.125-

2

0.06 I-

1

1 0.06

0.25

0.125

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2

0.5

32

64

128

HTM Microdilution MIC Q.£g/mI) FIG. 4. criteria

Comparison of tetracycline (14).

important distinction penicillin (7, 14).

To

necessary

either the broth

or

=

104) determined in LHB and in HTM. Dotted lines indicate

between frank and relative resistance

to

resistance, it is

MICs (n

classify accurately the level of to determine the penicillin MIC by

the agar dilution method. Pneumococci

may also be resistant to chloramphenicol (by CAT production), tetracycline, trimethoprim-sulfamethoxazole, erythromycin, or rifampin (2, 3, 5, 9, 17). Therefore, it may now be important to determine routinely the susceptibility of pneumococcal isolates causing serious infections to several other agents in addition to penicillin.

The NCCLS recommends

lution

or

use

current

NCCLS interpretive

of LHB for broth microdi-

sheep blood supplementation of Mueller-Hinton

agar for agar dilution tests of

streptococci (including

pneu-

mococci; 12, 13). However, relatively few laboratories routinely perform agar dilution MICs, and LHB has not been

supplement is laborious to properly, it makes interpretation of microdilution growth endpoints very difficult. Significant very major errors have been reported with certain available

commercially.

The LHB

prepare, and when not clarified

commercial microdilution

or

automated

susceptibility

test

they have been used to test pneumococci (7, 16). However, one recent study (1) has suggested that whole defibrinated sheep blood may be used as a growth supplement in commercial microdilution trays, with the growth endpoints detected by the greening of the medium which occurs in the presence of pneumococcal growth. HTM has been recommended recently by the NCCLS as the medium of choice for susceptibility testing of Haemophilus spp. (13, 14). While HTM contains growth supplements tailored to the specific growth requirements of H. influenza, this study has shown that it also provides adequate nutritive properties for testing of S. pneumoniae clinical isolates. Ail of the strains examined in this study grew well in HTM

methods when

TABLE 2. Recognition of S. pneumoniae clinical isolates with resistance MICs in HTM as compared with MICs in LHB Drug and resistance

Nso.aof slae

No. of isolates with resistance MIC in HTM

19

10

i

i

6

6

No

MIC (~tg/m1) in LHBI

f

Penicillin

RRO0.1-1 R :-2

Chloramphenicol R.::-8 Tetracycline 1 =8 R .-16

O i il10

micrbdilution tests. were quite comparable to MICs pneumococci tested in LHB. While there were no very major or major errors in this data set, there were a number of minor errors with penicillin and trimethoprim-sulfamethoxazole based on the current NCCLS breakpoints because of slightly lower or' higher MICs respectivelyl) in HTM. The definition of relative penicillin resistance of pneumococci as a MIC of 0.1 toi F.g/ml was proposed as a working definition

MICs determined in HTM

for

Erythromycin

i

R.~-8

Trimethoprim-sulfamethoxazole R:.-4 RR, Relative resistance; R,

a

22

resistance; I. intermediate.

22

434

JORGENSEN ET AL.

which allowed separation of such strains from those that appeared to possess a normal level of susceptibility to penicillin (5, 6). Because penicillin MICs in HTM are sometimes 1 concentration increment lower than those in LHB, relative resistance to penicillin could be defined as MICs of 0.06 to 1 ,ug/ml for tests performed in HTM. This change would be quite consistent with the recent report that pneumococci with penicillin MICs of .0.06 ,ug/ml possess various alterations of their penicillin-binding proteins (10). Trimethoprim-sulfamethoxazole MICs for S. pneumoniae are generally 1 to 2 log2 concentration increments higher in HTM than when determined in LHB. An analogous situation has been seen with trimethoprim-sulfamethoxazole MICs for H. influenza when tested in these two media (8). Since categorization of resistant isolates is not affected by the shift in MICs for susceptible strains when tested in HTM (Fig. 2), it is probably unnecessary to alter the trimethoprim-sulfamethoxazole breakpoints to adjust for these slight medium differences. HTM has been shown in this study to represent a promising alternative medium for determining broth microdilution MICs of a variety of antimicrobial agents with S. pneumoniae clinical isolates. Because HTM is simpler to prepare than LHB, and because HTM can be obtained commercially in prepared form, it may represent a practical choice for routine determination of broth dilution MICs for both H. influenza and S. pneumoniae in clinical laboratories. ACKNOWLEDGMENTS This study was supported in part by a grant from Glaxo Inc., Research Triangle Park, N.C. We gratefully acknowledge the invaluable contributions of the participant laboratories which contributed the strains used in this study. LITERATURE CITED 1. D'Amato, R. F., J. M. Swenson, G. A. McKinley, L. Hochstein, A. A. Wallman, D. J. Cleri, A. J. Mastellone, L. Fredericks, L. Gonzalez, D. H. Pincus, and C. Thornsberry. 1987. Quantitative antimicrobial susceptibility test for Streptococcus pneumoniae using inoculum supplemented with whole defibrinated sheep blood. J. Clin. Microbiol. 25:1753-1756. 2. Dang-Van, A., G. Tiraby, J. F. Acar, W. V. Shaw, and D. H. Bouanchaud. 1978. Chloramphenicol resistance in Streptococcus pneumoniae: enzymatic acetylation and possible plasmid

linkage. Antimicrob. Agents Chemother. 13:577-583. 3. Istre, G. R., J. T. Humphreys, K. D. Albrecht, C. Thornsberry, J. M. Swenson, and R. S. Hopkins. 1983. Chloramphenicol and penicillin resistance in pneumococci isolated from blood and

cerebrospinal fluid: a prevalence study in metropolitan Denver. J. Clin. Microbiol. 17:472-475. 4. Jackson, M. A., S. Shelton, J. D. Nelson, and G. H. McCracken. 1984. Relatively penicillin-resistant pneumococcal infections in

J. CLIN. MICROBIOL.

pediatric patients. Pediatr. Infect. Dis. 3:129-132. 5. Jacobs, M. R., H. J. Koornhof, R. M. Robins-Browne, C. M. Stevenson, Z. A. Vermaak, I. Freiman, G. B. Miller, M. A. Whitcomb, M. Isaacson, J. I. Ward, and R. Austrian. 1978.

Emergence of multiply resistant pneumococci. N. Engl. J. Med. 299:735-740. 6. Jacobs, M. R., Y. Mithal, R. M. Robins-Browne, M. N. Gaspar, and H. J. Koornhof. 1979. Antimicrobial susceptibility testing of pneumococci: determination of Kirby-Bauer breakpoints for penicillin G, erythromycin, clindamycin, tetracycline, chloramphenicol, and rifampin. Antimicrob. Agents Chemother. 16: 190-197. 7. Jones, R. N., D. C. Edson, and the CAP Microbiology Resource

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9.

10. 11.

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15.

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Committee. 1983. Special topics in antimicrobial susceptibility testing: test accuracy against methicillin-resistant Staphylococcus aureus, pneumococci, and the sensitivity of P-lactamase methods. Am. J. Clin. Pathol. 80(Suppl.):609-614. Jorgensen, J. H., J. S. Redding, L. A. Maher, and A. W. HoweIl. 1987. Improved medium for antimicrobial susceptibility testing of Haemophilus influenza. J. Clin. Microbiol. 25:2105-2113. Klugman, K. P., and H. J. Koornhof. 1988. Drug resistance patterns and serogroups or serotypes of pneumococcal isolates from cerebrospinal fluid or blood, 1979-1986. J. Infect. Dis. 158:956-964. Markiewicz, Z., and A. Tomasz. 1989. Variation in penicillinbinding protein patterns of penicillin-resistant clinical isolates of pneumococci. J. Clin. Microbiol. 27:405-410. Matthews, H. W., C. N. Baker, and C. Thornsberry. 1988. Relationship between in vitro susceptibility test results for chloramphenicol and production of chloramphenicol acetyltransferase by Haemophilus influenza, Streptococcus pneumoniae, and Aerococcus species. J. Clin. Microbiol. 26:2387-2390. National Committee for Clinical Laboratory Standards. 1985. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A. National Committee for Clinical Laboratory Standards, Villanova, Pa. National Committee for Clinical Laboratory Standards. 1988. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Tentative standard M7-T2. National Committee for Clinical Laboratory Standards, Villanova, Pa. National Committee for Clinical Laboratory Standards. 1988. Performance standards for antimicrobial disk susceptibility tests. Tentative standard M2-T4. National Committee for Clinical Laboratory Standards, Villanova, Pa. Saah, A. J., J. P. Mallonee, M. M. Tarpay, C. Thornsberry, M. A. Roberts, and E. R. Rhodes. 1980. Relative resistance to penicillin in the pneumococcus. A prevalence and case-controlled study. J. Am. Med. Assoc. 243:1824-1827. Shanholtzer, C. J., and L. R. Peterson. 1986. False susceptible penicillin G MICs for Streptococcus pneumoniae with a commercial microdilution system. Am. J. Clin. Pathol. 85:626-629. Simberkoff, M. S., M. Lukaszewski, A. Cross, M. At-Ibrahim, A. L. Baltch, R. P. Smith, P. J. Geiseler, J. Nadler, and A. S. Richmond. 1986. Antibiotic-resistant isolates of Streptococcus pneumoniae from clinical specimens: a cluster of serotype 19A organisms in Brooklyn, New York. J. Infect. Dis. 153:78-82.