In-vitro activities of 16 non-ß-lactam antibiotics against penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae

J. Verhaegen* and L. Verbist

Department of Microbiology, University Hospitals KU Leuven, B-3000 Leuven, Belgium


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs of 16 non-ß-lactams were determined by agar dilution for 283 penicillin-susceptible, 122 intermediate and 23 fully penicillin-resistant isolates of pneumococci. Penicillin-resistant pneumococci were more likely to be resistant to tetracyclines, macrolides and related compounds. In the latter group, quinupristin/dalfopristin was the only compound whose activity was not influenced by penicillin resistance. Among the fluoroquinolones, clinafloxacin and trovafloxacin showed excellent activity. Strains resistant to macrolides, tetracycline and penicillin were concentrated in serogroups 6, 9, 14, 19 and 23.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae remains an important bacterial pathogen in human infection. In Belgium the prevalence of penicillin resistance has increased significantly over the past 4 years from 2.3% in 1993 to 10% in 1997.

The in-vitro activities of 21 ß-lactam antibiotics against pneumococci isolated in Belgium during 1995 and 1996 were examined in a previous study. 1 Although penicillin-susceptible (pen-S) strains also exhibit non-ß-lactam resistance, penicillin-intermediate (pen-I) and penicillin- resistant (pen-R) pneumococci are more likely to be resistant to antibiotics other than ß-lactams. The aim of this study was to evaluate the activity of non-ß-lactam antibiotics against S. pneumoniae.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

Isolates cultured from blood or pleural fluid (75%), cerebrospinal fluid (7.5%), middle ear aspirates (14%) and from various other puncture sites (3.5%) were collected by more than 90 laboratories and sent to the national reference centre for S. pneumoniae. Capsular types were determined by phase-contrast microscopy, with sera from the Statens Seruminstitut (Copenhagen, Denmark).

All pen-I (122) and pen-R (23) isolates from 1995 and 1996 were included in this study, together with 283 pen-S isolates selected at random over the same period. Susceptibility to penicillin was defined according to NCCLS criteria as follows: susceptible, MIC <=0.06 mg/L; intermediate, MIC 0.12-1 mg/L; resistant, MIC >=2 mg/L. 2

Antimicrobial agents

Stock solutions of the following 16-non-ß-lactam antibiotics were prepared from powders with known potency supplied by representatives of the respective companies in Belgium: erythromycin and clarithromycin (Abbott, Louvain-la-Neuve, Belgium); azithromycin, doxycycline and trovafloxacin (Pfizer, Brussels, Belgium); dirithromycin, vancomycin and LY 333328 (Eli Lilly, Brussels, Belgium); tetracycline, ofloxacin and teicoplanin (Hoechst-Marion-Roussel, Brussels, Belgium); clindamycin (Pharmacia-Upjohn, Brussels, Belgium); quinupristin/dalfopristin and sparfloxacin (Rhone-Poulenc Rorer, Brussels, Belgium); ciprofloxacin (Bayer, Brussels, Belgium); and clinafloxacin (Parke Davis, Zaventem, Belgium).

Determination of MICs

MICs were determined by agar dilution in Mueller- Hinton agar (Difco, Detroit, MI, USA) supplemented with 5% defibrinated sheep blood. The antimicrobial agents were incorporated into the agar in two-fold serial dilutions (range 0.016- 8 mg/L). Suspensions with turbidity equivalent to that of a 0.5 McFarland standard were prepared from overnight blood agar cultures in sterile water and further diluted to obtain 10 7 cfu/mL. Plates were inoculated with a multipoint replicator delivering approximately 10 4 cfu/spot.

S. pneumoniae ATCC 49619 was used for quality control. The agar plates were incubated at 36°C for 18 h in 5% CO 2. The MIC was defined as the lowest concentration of the agent that produced complete inhibition.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Results of susceptibility testing are presented in Table I as the range of MICs and the geometric mean MIC. Since there were no significant differences in MIC distributions of non-ß-lactam antibiotics between pen-I and pen-R isolates, these two subgroups were put together (n = 145).


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Table I. Susceptibility of pneumococci to non-ß-lactam antibiotics
 
The MICs of doxycycline were two to three concentrations lower than those of tetracycline. Susceptibility to tetracycline (<=2 mg/L) was 75% in the pen-S isolates (n = 283) and 59% in the penicillin non-susceptible pen-I/pen-R isolates.

Among the macrolides and related antibiotics, clarithromycin showed the lowest MICs and dirithromycin the highest MICs. Clindamycin MICs paralleled those of the macrolides in most cases. Quinupristin/dalfopristin had the narrowest MIC distribution and was the only compound that was equally active against pen-I/pen-R isolates and pen-S isolates. The susceptibilities at NCCLS breakpoints against pen-S and pen-I/pen-R isolates were, respectively: erythromycin, 76% and 66%; clarithromycin, 78% and 66%; azithromycin, 76% and 66%; and clindamycin, 84% and 68%.

Among the fluoroquinolones, clinafloxacin and trovafloxacin had the lowest MICs, about four dilutions lower than those of ofloxacin and ciprofloxacin and one dilution lower than sparfloxacin. Based on NCCLS breakpoints, susceptibility to ofloxacin (<=2 mg/L) was 100% for the pen-S isolates and 99% for the pen-I/pen-R isolates.

All pneumococci were susceptible to the glycopeptides: teicoplanin showed the highest activity.

Table II shows the distributions of different susceptibility patterns over the most frequent serogroups. Serogroups 1, 3, 4, 5 and 7 contained only pen-S isolates (in 1995- 1996) and all but one isolate in this collection were susceptible to erythromycin. In contrast, serogoups with a higher percentage of pen-I/pen-R isolates (6, 9, 14, 19 and 23) showed a high percentage of erythromycin and tetracycline resistant isolates; furthermore, the majority of erythromycin- resistant isolates were also resistant to clindamycin.


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Table II. Distribution of susceptibility patterns over serogroups
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
S. pneumoniae no longer has predictable antibiotic susceptibility in Belgium, since isolates with increased resistance to penicillin and to other antibiotics have become more common. There is a need for antimicrobial agents that can be used for oral and parenteral therapy of infections caused by pen-R pneumococci. The aim of the current study was to examine the in-vitro activities of a wide range of non-ß- lactam antibiotics against pen-S and pen-I/pen-R isolates of S. pneumoniae.

Macrolides used to be good alternative agents against pneumococci. However, 23% of the pen-S and 34% of pen-I/pen-R S. pneumoniae isolates were also resistant to macrolides, limiting the potential usefulness of these agents. Erythromycin-resistant S. pneumoniae were uniformly cross-resistant to other macrolides and so erythromycin remains a good reference for susceptibility testing of all macrolides against S. pneumoniae. The relative activities of the different macrolides were very similar to those observed in other studies. 3,4

Based on in-vitro activity and known pharmacokinetics, clarithromycin is probably the best choice among the macrolides for treatment of infections caused by macrolide-susceptible S. pneumoniae. Resistance to macrolides in our S. pneumoniae isolates was mainly due to the constitutive MLS B type, since most macrolide-resistant strains were also highly resistant to clindamycin. Constitutive MLS B type resistance is presumably the result of altered rRNA, which blocks binding of macrolides, clindamycin and streptogramin B to their target site. 5 Acquisition of resistance to penicillin in pneumococci is thought to originate from horizontal transfer of genetic material from other bacterial species. It is not surprising that serotypes that are very competent in uptake of foreign DNA and become resistant to penicillin may possibly acquire constitutive MLS B resistance by the same route.

Ciprofloxacin and ofloxacin have been reported to be marginally active against pneumococci. 6 Consequently, the fluoroquinolones currently available should not be used as first-line treatment for pneumococcal infections. In contrast, the newer fluoroquinolones clinafloxacin and trovafloxacin may be promising therapeutic options in patient populations that are likely to be infected with multiply resistant pneumococci. 7

All glycopeptides had excellent activities against pneumococci with MICs <0.5 mg/L. The superior in-vitro activity of teicoplanin compared with that of vancomycin is well known. However, taking into account the higher degree of protein binding of teicoplanin, the two glycopeptides are probably similar in clinical efficacy. 8 The clinical usefulness of the new semisynthetic glycopeptide LY 333328 remains to be determined.


    Notes
 
* Correspondence address. Laboratory of Bacteriology, University Hospital St Rafaël, Kapucijnenvoer 36, B-3000 Leuven, Belgium. Tel: +32-16-33-21-50; Fax: +32-16-33-63-31. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Verhaegen, J. & Verbist, L. (1998). In-vitro activity of 21 ß-lactam antibiotics against penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 41, 381–5.[Abstract]

2 . National Committee for Clinical Laboratory Standards. (1998). Performance Standards for Antimicrobial Susceptibility Testing—Eighth Informational Supplement: Approved Standard M100-S8. NCCLS, Wayne, PA.

3 . Ednie, L. M., Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1996). Comparative activities of clarithromycin, erythromycin, and azithromycin against penicillin-susceptible and penicillin-resistant pneumococci. Antimicrobial Agents and Chemotherapy 40, 1950–2.[Abstract]

4 . Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1997). Susceptibility of penicillin-susceptible and -resistant pneumococci to dirithromycin compared with susceptibilities to erythromycin, azithromycin, clarithromycin, roxithromycin, and clindamycin. Antimicrobial Agents and Chemotherapy 41, 1867–70.[Abstract]

5 . Leclercq, R. & Courvalin, P. (1993). Resistance to macrolides, azalides and streptogramins. In The New Macrolides, Azalides, and Streptogramins: Pharmacology and Clinical Applications (Neu, H. C., Young, S. S. & Zinner, S. H., Eds), p. 33–40. Marcel Dekker, New York.

6 . Fuchs, P. C., Barry, A. L. & Brown, S. D. (1996). Tentative interpretive criteria for testing the susceptibility of Streptococcus pneumoniae to eight fluoroquinolones. Diagnostic Microbiology and Infectious Disease 26, 23–7.[ISI][Medline]

7 . Knudsen, J. D., Fuursted, K., Espersen, F. & Frimodt-Moller, N. (1997). Activities of vancomycin and teicoplanin against penicillin-resistant pneumococci in vitro and in vivo and correlation to pharmacokinetic parameters in the mouse peritonitis model. Antimicrobial Agents and Chemotherapy41 , 1910–5.[Abstract]

8 . Schwalbe, R. S., McIntosh, A. C., Qaiyumi, S., Johnson, J. A., Johnson, R. J., Furness, K. M.et al. (1996). In vitro activity of LY333328, an investigational glycopeptide antibiotic, against enterococci and staphylococci. Antimicrobial Agents and Chemotherapy 40, 2416–9.[Abstract]

Received 23 June 1998; returned 21 August 1998; revised 26 October 1998; accepted 24 November 1998