Haemophilus Species and Moraxella catarrhalisUsing ... - Europe PMC

JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1994, p. 2786-2790

Vol. 32, No. 11

0095-1137/94/$04.00+0 Copyright ©D 1994, American Society for Microbiology

Disk Diffusion versus Broth Microdilution Susceptibility Testing of Haemophilus Species and Moraxella catarrhalis Using Seven Oral Antimicrobial Agents: Application of Updated Susceptibility Guidelines of the National Committee for Clinical Laboratory Standards PAMELA C. KIBSEY,t ROBERT P. RENNIE,* AND JOYCE E. RUSHTON

Department of Medical Microbiology and Infectious Diseases, University of Alberta Hospitals, Edmonton, Alberta, Canada T6G 2J2 Received 13 April 1994/Returned for modification 23 June 1994/Accepted 25 August 1994

Susceptibility testing of Haemophilus species and Moraxella catarrhalis is medium and inoculum dependent. Seven oral agents, ampicillin, amoxicillin-clavulanic acid, cefaclor, loracarbef, cefuroxime-axetil, cefixime, and erythromycin, were tested against 400 ,1-lactamase-positive and -negative clinically significant respiratory strains of Haemophilus species and 100 strains of M. catarrhalis. Sources of the strains included teaching and regional hospitals and a private laboratory. All strains were tested by broth microdilution and disk diffusion in haemophilus test medium for Haemophilus species and Mueller-Hinton broth and agar for M. catarrhalis. Appropriate National Committee for Clinical Laboratory Standards (NCCLS) standards were followed. For Haemophilus species, by disk diffusion and broth microdilution, respectively, 27 and 27% of strains were resistant to ampicillin, 37 and 5% were resistant to erythromycin, 3 and 0.5% were resistant to cefaclor, 2 and 0.5% were resistant to loracarbef, and 0% were resistant to cefuroxime-axetil, cefixime, and amoxicillinclavulanic acid. P-Lactamase-negative ampicillin-resistant strains were not observed. Of M. catarrhalis strains, 56% were resistant to ampicillin by disk diffusion and 95% were resistant by broth microdilution. This species was susceptible to all other agents tested by either method. The disagreements between disk diffusion results and MICs for cefaclor, ampicillin, cefuroxime, and loracarbef that occurred with use of the 1990 NCCLS tables were resolved when the 1992 NCCLS tables were used.

Haemophilus influenzae is a well-documented pathogen of the respiratory tract. More recently, Moraxella catarrhalis has been associated with symptoms of respiratory infections and is now considered a potential pathogen in acute exacerbation of chronic obstructive pulmonary disease in adults and in otitis media in children (4). Treatment with oral antimicrobial agents is important in order to keep patients infected with these organisms out of hospital (9). Haemophilus species and M. catarrhalis produce 1-lactamases, which should by now have obviated the use of less-expensive antibiotics like ampicillin as the first-line treatment when the infection is of bacterial origin and clearly shown to be caused by one of these 3-lactamaseproducing strains. Most studies have shown that 15 to 30% of Haemophilus strains and up to 85% of M. catarrhalis strains are now resistant to ampicillin by means of 1-lactamase production (1, 3, 7, 11, 13-15, 26). It is therefore important to have available other oral agents which retain their efficacy in spite of the presence of 3-lactamase-producing strains of these bacteria. It is also important to periodically conduct surveys of susceptibility of recent clinical isolates to ensure that these agents retain their activity against Haemophilus and Moraxella species (22, 23, 25).

There have been a variety of methods and interpretive criteria used to define the susceptibilities of Haemophilus and Moraxella species to 1-lactam antibiotics (1, 2, 6). Recently, haemophilus test medium (HTM) was endorsed by the National Committee for Clinical Laboratory Standards (NCCLS) (document M7-A2) as the standard medium for susceptibility testing of Haemophilus species (15, 21). New criteria for zone diameter interpretation and MIC breakpoints in effect until December 1992 produced false intermediate rates of 9.6 to 14%, which were not relevant clinically, for many laboratories (1, 10, 17). When Jorgensen's original criteria for ampicillin (susceptible at zone diameters .22 mm) were incorporated (NCCLS standard M100-S4 [M2-A4], December 1992), the false intermediate rate for disk diffusion with ampicillin on HTM dropped (5, 10, 12, 20). The present study was undertaken to identify the development of resistance to currently available oral antimicrobial agents among recent clinical strains of Haemophilus species and M catarrhalis isolated from respiratory infections in Alberta, Canada. The study was done with current NCCLS standards regarding media and incubation and with the latest published zone size criteria for penicillins and cephalosporins with Haemophilus species and other fastidious organisms.

* Corresponding author. Mailing address: Microbiology and Public Health, University of Alberta Hospitals, WMC 2B3.08, 8440 112th St., Edmonton, Alberta, Canada T6G 2J2. Phone: (403) 492-4461. Fax: (403) 492-3864. t Present address: Department of Laboratory Medicine, Grey Nun's Hospital, Edmonton, Alberta, Canada.

(Results of this research were presented in part at the 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, Calif., October 1992 [21a], and at the Conjoint Meeting on Infectious Diseases, Toronto, Ontario, Canada, December 1992 [15a].) 2786

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VOL. 32, 1994

MATERLILS AND METHODS Bacterial strains. Four hundred recent clinical strains of H.

influenzae and Haemophilusparainfluenzae were obtained from a tertiary care teaching hospital (University of Alberta Hospitals), from colleagues at three large referral hospitals in southern, central, and northern Alberta (D. Church at Alberta Children's Hospital, T. Louie at Calgary General Hospital, and D. McNaughton at Red Deer Regional Hospital), and from a private laboratory with a large community referral base in the province (J. Nigrin at T. A. Kasper and Associates). There were 224 strains confirmed as H. influenzae, 13 as H. parain-

fluenzae, and 163 as Haemophilus strains not identified to the

species level. One hundred strains of M. catarrhalis from the University of Alberta Hospitals laboratory were included in the study. Of the 400 strains of Haemophilus species, 380 (95%) were isolated from expectorated sputum or auger suction specimens; the remainder were isolated from the eyes, from the ears, from sinus aspirates, or from the nose. All of the M.

catarrhalis strains were isolated from sputum. None of the isolates were recovered from surveillance cultures, and all were considered as probable causes of infection. Strains were collected in a prospective manner over 1 year and were frozen in double-strength skim milk at -70°C. Strains of Haemophilus were identified according to conventional methods (16). M. catarrhalis strains were identified by positive reactions for indophenyl oxidase, butyrase, and DNase. For the study, strains were subcultured twice on chocolate-blood agar medium to ensure their viability and purity before testing. Antimicrobial agents. The following oral antimicrobial agents were tested: ampicillin (Ayerst), amoxicillin-clavulanic acid (Smith Kline Beecham), cefaclor (Eli Lilly), cefuroxime (Glaxo), cefixime (Lederle Cyanamid), loracarbef (Eli Lilly), and erythromycin (Eli Lilly). Stock solutions from assayed powders were prepared according to the manufacturers' instructions, from information in the literature (24), and from Table 4 of NCCLS standard M7-A2 (21). Antibiotic disks were stored at -70°C until required for use. The drugs were used at the following ranges of dilutions: ampicillin and loracarbef, 0.12 to 16 ,ug/ml; amoxicillin-clavulanic acid, 1/0.5 to 32/16 ,ug/ml; cefaclor, cefuroxime-axetil, and cefixime, 0.5 to 32 ,ug/ml; and erythromycin, 0.25 to 8 ,ug/ml. Test methods. Two sources of HTM were used for disk diffusion tests with Haemophilus species. An in-house HTM was prepared according to the recipe of Jorgensen et al. (15): Mueller-Hinton agar (BBL, Cockeysville, Md.), 38 g/liter; hematin (Sigma, St. Louis, Mo.), 30 ml of a 0.05-g/liter (wt/vol) solution made in 0.01 N NaOH; yeast extract (Unipath, Nepean, Ontario, Canada), 5 g/liter; P-NAD, 3.0 ml of a 5-g/liter (wt/vol) solution made in distilled water (filter sterilized and added after autoclaving); and distilled water to 1,000 ml. The final pH of this medium was 7.2 to 7.4. A commercially prepared HTM was purchased from Prepared Media Laboratories, Richmond, British Columbia, Canada. The broth microdilution medium was prepared in-house with cation-adjusted Mueller-Hinton broth (BBL) as the base instead of Mueller-Hinton agar, but other ingredients were the same as in the agar formulation. Strains of M. catarrhalis were tested on Mueller-Hinton agar and in the same broth medium (7, 8). The inoculum for both Haemophilus species and M catarrhalis in the disk diffusion method was 1 x 108 to 4 x 108 CFU/ml as described in NCCLS standard M2-A4 (18); for the broth microdilution method, the inoculum for the organisms was ca. S x 105 CFU/ml as described in NCCLS standard M7-A2 (19). The number of organisms in each inoculum was confirmed by limited-dilution colony counts based on a McFarland standard

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optical density of 0.5. Suspensions of organisms for inoculation were prepared directly from overnight cultures on chocolate medium. Inocula were diluted in sterile saline and adjusted to the appropriate optical density with a nephelometer to ensure that the same optical density was achieved each time the tests were done. Ninety-six-well blank, sterile microdilution plates (Sceptor; BBL) were inoculated with 100-,u1 aliquots of HTM containing twofold dilutions of antibiotics. Agar plates were incubated after inoculation for 16 to 18 h at 35°C in 5% C02, and microdilution plates were incubated for 24 h at 35°C in air. Zone sizes were measured with digital calipers and were recorded to the nearest 0.1 mm. The MIC was determined for each agent from duplicate observations as the concentration in the first well showing no visible growth. P-Lactamase activity was determined for each strain by a nitrocefin disk method

(Cefinase; BBL). Control strains of Haemophilus influenzae ATCC 49247 and 49766 (for H. influenzae) and Eschenchia coli ATCC 25922 and 35218 (for M. catarrhalis) were used with each batch of media and antibiotics tested.

RESULTS AND DISCUSSION The strains of Haemophilus tested in this study were recent clinical isolates from four hospital laboratories and one private laboratory in Alberta. They represented a cross section of significant isolates from respiratory tract infections in both inpatients and outpatients. Repeat isolates from individual patients were excluded from the study. Data available on strains received from the other hospitals and laboratories suggested that up to 30% of H. influenzae strains were ,B-lactamase positive and that of these strains, 80% were also resistant to cefaclor, 5% were resistant to cefuroxime, 16% were resistant to trimethoprim-sulfamethoxazole, and 0% were resistant to amoxicillin-clavulanic acid. This apparent high percentage of resistance to cefaclor was thought to be due to use of earlier zone size limits for susceptibility and to arbitrary interpretation of MIC criteria for susceptibility breakpoints other than those published by the NCCLS. The criteria used to determine breakpoints for disk diffusion and broth microdilution susceptibility tests for Haemophilus species and M. catarrhalis are given in Table 1. Susceptibility interpretations were first based on Tables 2 and 2A in NCCLS approved standards M2-A4 (18) and M7-A2 (19) and were then reevaluated by use of the updated Tables 2A for standards M2 and M7 published in the NCCLS fourth informational supplement M100-S4 (5, 20, 21) when they became available. The zone diameter and breakpoint criteria for M. catarrhalis were measured according to Tables 2 in these documents (for organisms other than Haemophilus and Neisseria species), since there were no specific criteria for Moraxella species and it was considered desirable to have some measure of comparison of susceptibility with other organisms. Our criteria for erythromycin with Haemophilus species were based on reference 15, again to permit some measure of comparison since there is no guideline from NCCLS. The most important changes in NCCLS documents which affected zone diameter breakpoints were for ampicillin and cefaclor with Haemophilus species. For ampicillin, the zone diameter breakpoint for susceptibility was reduced from .25 mm to .22 mm, and that for resistance was reduced from .21 mm to .18 mm; for cefaclor, the zone diameter breakpoint for susceptibility was reduced from .24 mm to .20 mm, and for resistance it was reduced from .18 mm to .16 mm. These changes have clarified a large number of intermediate results that were not

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J. CLIN. MICROBIOL.

KIBSEY ET AL.

TABLE 1. Criteria used for zone diameter interpretive standards for disk diffusion and equivalent MIC breakpoints for broth microdilution tests for Haemophilus speciesa and M. catarrhalis' Criterion for indicated categoryc by: Antibiotic

Organism

Disk diffusion zone of inhibition (mm)

MIC

(pug/ml)

R

I

S

R

I

S

Haemophilus spp. M. catarrhalis

s18 s19

19-21

.22

.4 .0.5

2

220

s1 s0.25


l19 l13

220

14-17

.18

.8/4 .32/16

16/8

.4/2 s8/4


l16 l14

17-19

.20

.32

15-17

.18

.32

16 16

s8 s8

Cefuroxime


.20 l14

21-23 15-22

.24 .23

.16 .32

8 8-16

s4 s4

Cefixime


l15

16-18

'-21 .19

.4

2

l1 l1

Haemophilus spp.

s15 l14

16-18 15-17

.19

.8

.18

.8

Ampicillin

Amoxicillin-clavulanic acid Cefaclor

Loracarbef'

M. catarrhalis

Erythromycin


.13

14-17

l13

14-22

.18

.8

.23

s0.5

1-4

.8

.0.5

1-4

a The outcome criteria used for Haemophilus species were derived from Tables 2A of NCCLS standards M2-A4 and M7-A2 (20, 21) and from reference 15 (for erythromycin). b The outcome criteria used for M. catarrhalis were derived from Tables 2 of NCCLS standards M2-A4 and M7-A2 (18, 19) for organisms other than Haemophilus and Neisseria species. cR, resistant; I, intermediate; S, susceptible. d Intermediate (16 ,g/ml) and resistant (32 ,g/ml) categories were added for loracarbef with Haemophilus species in 1993 NCCLS document M7-A3.

in agreement with the equivalent MIC breakpoints for susceptibility and resistance. As shown in Table 2, with the exception of ampicillin and erythromycin, by broth microdilution MIC almost all strains of Haemophilus species and M. catarrhalis were susceptible to the

oral agents tested at concentrations achievable with oral doses of the antibiotics. Variability related to methodology is shown in Table 3. Only minor differences were observed in results for Haemophilus species with the in-house and commercial HTMs used for disk diffusion. In particular, only four (1.2%) results

TABLE 2. Cumulative percentage of strains inhibited by broth microdilution method Antibiotic

Ampicillin


Amoxicillin-clavulanic acida Cefaclor

Cefuroxime

Cefixime Loracarbef

50.25

0.5

1

2

4

8

16

-32

63 5

68 5

74 5

74 13

76 29

83 84

91 100

100

99

100

Haemophilus spp.

NT"

83 100

100

NT

NT NT

98

M. catarrhalis


NT NT

13 12

28 40

57 75

82 95

98 100


NT NT

72 15

93 59

99 86

100 99

100

Haemophilus spp.

NT

99

100

NT

94 99

99

M. catarrhalis

100

12 4

32 8

67 39

85 76

95 91

99 99

3 100

10

27

65

95

100


Erythromycin

Cumulative % of strains inhibited at indicated concn (,ug/ml) of antibiotic

Organism


100 100

a For amoxicillin-clavulanic acid, the concentration identified for the cumulative percent inhibited is the concentration of amoxicillin. The concentration of clavulanic acid is one-half that of amoxicillin. bNT, not tested.

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VOL. 32, 1994

TABLE 3. Comparison of errors between disk diffusion (in-house and commercial HTMs) and broth microdilution

MIC for 328 strains of Haemophilus species Antibiotica

No. (%) of errorsb vs broth microdilution MIC with: In-house HTM Commercial HTM

Total

Ampicillin 1 (0.5) Amoxicillin-clavulanic acid 0 Cefaclor 23 (7) Cefuroxime 15 (5) 0 Cefixime 24 (7) Loracarbef

Very major

Total

Very major

0 0 2

5 (1.0) 0 24 (7) 13 (5)

1 0 1 0

0 0 2

0 26 (7)

0 1

a Greater than 50% discordance was observed with erythromycin, and no tabulation was performed for this drug. All errors were minor. Most strains had intermediate MICs and were variable (either sensitive, intermediate, or resistant) by disk diffusion. There was no correlation between ,-lactamase activity and discordant results by these methods for any of the cephalosporin antibiotics or for erythromycin. b Very major errors were defined as ratings of susceptible by the disk method and resistant by broth microdilution MIC. Total errors for each combination of medium and antibiotic also include major and minor effors.

were considered very major errors when in-house HTM was used, and three (0.9%) results were considered similarly erroneous when commercial HTM was used for disk diffusion tests. There were no very major errors when strains of M. catarrhalis were tested. A comparison of disk diffusion data for both in-house and commercial HTMs for H. influenzae with ampicillin and cefaclor is shown in Table 4. There were no differences in the two HTMs, only one difference for cefaclor, and two differences for ampicillin with respect to strains with intermediate zone sizes according to the latest NCCLS criteria (20). Favorable resolution of clinically irrelevant intermediate zones for ampicillin and cefaclor is illustrated in Table 4. The intermediate category for ampicillin for Haemophilus species decreased from 5 to 1% (in-house) and 5 to 2% (commercial), and that for cefaclor decreased from 25 to 1% (in-house) and 23 to 2% (commercial) under the 1992 NCCLS guidelines. This study was undertaken to identify potential problems in test methodologies for strains of Haemophilus species and M. catarrhalis that should normally be expected to be susceptible to oral antibiotics commonly used for treatment of communityassociated respiratory infections. Failure to apply standardization of criteria or methods for testing these combinations of TABLE 4. Comparison of disk diffusion susceptibilities of

ampicillin and cefaclor for H. influenzae strains with HTMs from different sources

Antibiotic Antibiotic

Ampicillin

Medium preparation

In-house Commercial

Cefaclor

In-house Commercial

NCCLS protocola 1990 1992 1990 1992 1990 1992 1990 1992

No. (%) of strains in categoryb S I R

128 (61) 11 (5) 140 (63) 2 (1) 124 (57) 11 (5) 136 (62) 4 (2) 158 (72) 54 (25) 211 (96) 3 (1) 156 (74) 49 (23) 209 (96) 4 (2)

82 (37) 80 (36) 84 (38) 80 (36) 7 (3) 6 (3) 6 (3) 4 (2)

a 1990, reference 18; 1992, reference 20. b S, susceptible; I, intermediate; R, resistant (under relevant NCCLS criterion).

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organisms and antimicrobial agents may lead to apparent increases in numbers of resistant isolates. In fact, these organisms are still susceptible to the usual concentrations of these agents that can be achieved with oral dosing regimens. The results of this investigation suggest that two major factors may account for potential problems in interpretation. Using carefully standardized media and methodologies, we have shown (Table 3) that in-house and commercially prepared HTMs perform equally well. Second, the previous NCCLS guidelines for ampicillin and cefaclor interpreted in laboratories that use a disk diffusion method with either in-house or commercial HTM tend to overestimate the percentage of resistant isolates for these two antibiotics. The magnitude of this increase in our study would have been significant (Table 4). Approximately 28% of strains of Haemophilus would have been considered intermediate or resistant to cefaclor if the earlier zone sizes and only disk diffusion had been used. The most recent guidelines adopted by the NCCLS (21) significantly improve the correlation between disk diffusion and broth microdilution for ampicillin and cefaclor, and also for loracarbef. Between 2 and 3% of strains would be considered intermediate or resistant to these oral agents if tested by disk diffusion (Table 4), but only 0.25 to 0.5% would be considered resistant if tested by broth microdilution (Table 3). If 20% or more of strains were resistant in vitro, as suggested by the earlier susceptibility breakpoint criteria, it would certainly be expected that the resolution of symptoms and bacteriological cures when documented would not be as good as the results of clinical studies would indicate (9). A reassuring finding was that the susceptibility to cefaclor is virtually unchanged since last surveyed in Canada (26). ,-Lactamase-negative, ampicillin-resistant strains are still rare (1). However, the latest zone size criteria for both ampicillin and cefaclor would not be expected to alter the percentage of intermediate or resistant ,B-lactamase-negative, ampicillin-resistant strains, since their zone sizes in the presence of these antibiotics are already in the resistant category (1). We included erythromycin in our study to collect zone diameter and MIC data on local strains to convince physicians of the in vitro and in vivo discrepancy with this agent against Haemophilus species. While the MIC for 90% of the strains tested is near 4 ug/ml, the achievable levels in blood are only 0.7 to 1.5 ,ug/ml, making this antibiotic inappropriate for treatment of Haemophilus infections, and therefore with justification it is not included in the NCCLS tables for Haemophilus species. Our conclusions support the change of interpretive criteria published by the NCCLS Subcommittee on Antimicrobial Susceptibility Testing in December 1992 (5, 20) and suggest that laboratories should carefully monitor their susceptibility results to look for significant and rapid changes in resistance to antibiotics being tested. When susceptibility surveys are being performed, broth microdilution should be considered the "gold standard." Oral agents such as cefaclor, cefuroxime-axetil, cefixime, amoxicillin-clavulanic acid, and loracarbef should now be considered empirically for treating respiratory infections with Haemophilus or Moraxella species. The continued persistence of high percentages of P-lactamase-producing strains of H. influenzae and M. catarrhalis should prompt the consideration of alternative oral agents when appropriate cultures clearly show that these strains are responsible for the infectious process. When susceptibility patterns show significant deviation from this kind of surveillance data, the results should be corroborated by additional testing. Careful standardization of methodologies is necessary to ensure accurate results when

KIBSEY ET AL.

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testing fastidious organisms against certain antimicrobial agents. ACKNOWLEDGMENTS We thank the participating laboratories for supplying the strains used in this investigation. This study was supported by a grant in aid from Eli Lilly Canada, Inc. REFERENCES 1. Barry, A. L., P. C. Fuchs, and M. A. Pfaller. 1993. Susceptibilities of P-lactamase-producing and -nonproducing ampicillin-resistant strains of Haemophilus influenzae to ceftibuten, cefaclor, cefuroxime, cefixime, cefotaxime, and amoxicillin-clavulanic acid. Antimicrob. Agents Chemother. 37:14-18. 2. Barry, A. L, J. H. Jorgensen, and D. J. Hardy. 1991. Reproducibility of disc susceptibility tests with Haemophilus influenzae. J. Antimicrob. Chemother. 27:295-301. 3. Bergeron, M. G., P. Simard, and P. Provencher. 1987. Influence of growth medium and supplement on growth of Haemophilus influenzae and on antibacterial activity of several antibiotics. J. Clin.

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