In-vitro activity of HMR 3647 against Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and ß-haemolytic streptococci

M. Wootton, K. E. Bowker, A. Janowska, H. A. Holt and A. P. MacGowan*

Bristol Centre for Antimicrobial Research and Evaluation, Southmead Health Services NHS Trust and University of Bristol, Department of Medical Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in-vitro activity of HMR 3647 and seven comparators (azithromycin, clarithromycin, erythromycin A, roxithromycin, penicillin G, ciprofloxacin and levofloxacin) were tested against 207 Streptococcus pneumoniae and 200 ß-haemolytic streptococci. Ten comparators (azithromycin, clarithromycin, erythromycin A, roxithromycin, ampicillin, co-amoxiclav, cefuroxime, cefotaxime, ciprofloxacin and levofloxacin) were tested against 143 Haemophilus influenzae and 58 Moraxella catarrhalis. The MIC50 of HMR 3647 for S. pneumoniae was <= 0.008 mg/L, less than that for the macrolides or quinolones tested. Pneumococci with an erythromycin A MIC of 0.06 mg/L (n = 23) had an MIC50 of HMR 3647 <= 0.008 mg/L, whereas isolates with an erythromycin A MIC >=1 mg/L (n = 34) had an MIC50 of HMR 3647 of 0.03 mg/L, a four-fold increase. In contrast, the difference in macrolide MIC50s for the two groups was >=64-fold. The MIC50s for ß-haemolytic streptococci, classified by Lancefield group, were in the range 0.015 to 0.06 mg/L for HMR 3647. H. influenzae were categorized into three groups according to cefuroxime MIC: <1 mg/L (n = 72); 2–4 mg/L (n = 29); and >4 mg/L (n = 42). The MIC50 of HMR 3647 increased two-fold with increasing cefuroxime MICs; ß-lactam MICs increased much more markedly. The MIC50 of HMR 3647 for M. catarrhalis was 0.03 mg/L. HMR 3647 has good activity against respiratory tract pathogens but in-vitro susceptibility is affected by erythromycin A susceptibility in S. pneumoniae and ß-haemolytic streptococci.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Respiratory tract infections, a major cause of morbidity and mortality in the community and hospitals, are caused by a variety of pathogens. Causes of lower respiratory tract infections (LRTI), outside the atypical group of bacteria, are Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. ß-Haemolytic streptococci are often implicated in upper respiratory tract infections. Increasing incidence of resistance in these organisms has resulted in calls for changes in antimicrobial treatment. In the last decade macrolides such as clarithromycin and azithromycin have assumed an increasing role in the management of LRTI. Azithromycin displays increased antimicrobial activity against Gram-negative respiratory pathogens compared with erythromycin A, with the former being four times more active against H. influenzae and twice as active against M. catarrhalis. Clarithromycin has an MIC90 of <=0.25 mg/L for most erythromycin A-susceptible pathogens except H. influenzae.1,2,3,4 Ketolides, such as HMR 3647, display the same antibacterial spectrum as the macrolides but, in addition, show good activity against erythromycin A-resistant isolates among Gram-positive cocci such as S. pneumoniae and ß-haemolytic streptococci of Lancefield Group A.5 Compared with clarithromycin and azithromycin, ketolides are very stable in acidic media. In this study we tested HMR 3647, a range of macrolides including azithromycin, clarithromycin, erythromycin A and roxithromycin, and other relevant comparators against 608 respiratory isolates including erythromycin A-resistant S. pneumoniae and cefuroxime-resistant H. influenzae strains.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The isolates used in this study, 207 S. pneumoniae, 200 ß-haemolytic streptococci, 58 M. catarrhalis and 143 H, influenzae, were from the collection held at Southmead Hospital. They were isolated from clinical specimens between 1990 and 1998 and grouped according to species and erythromycin A or cefuroxime sensitivity. The groups were: ß-haemolytic streptococci, divided into Lancefield groups A (59), B (41), C (19), F (35), G (46), of which five were erythromycin A resistant; S pneumoniae (207), of which 173 were erythromycin A sensitive and 34 resistant (four isolates were also clindamycin resistant); H. influenzae (143), of which 42 were cefuroxime resistant and 101 sensitive. The control strains used were H. influenzae NCTC 11931, Straphylococcus aureus NCTC 9144 and Escherichia coli NCTC 10536. The antimicrobials used were all obtained from Hoechst Marion Roussel (Romainville-cedex, France). Agar dilution MICs were performed according to the BSAC method for sensitivity testing6 on DST agar (Unipath, Basingstoke, UK) with 5% lysed horse blood and, for H. influenzae, 10 mg/L of both nicotinamide adenine dinucleotide and L-histidine.7 The antimicrobials were incorporated into the medium in a log 2 dilution series from 0.008 to 128 mg/L. Inocula were prepared by diluting bacterial suspensions equivalent in turbidity to a McFarland 0.5 standard, resulting in approximately 104 cfu/spot when applied by multipoint inoculator (Denley Instruments, Billingshurst, UK). Plates were incubated in an atmosphere of 6% CO2 for 18 h. The MIC was defined as the lowest concentration of drug to inhibit macroscopically visible colonies. The breakpoints used to define resistance to HMR 3647 were those of the manufacturer. For other agents, those of the BSAC6 were used, and where none were available those of the Comité de l'Antibiogramme de la Société Francaise de Microbiologie were used.8 Breakpoints were: for streptococci HMR 3647 <=1 mg/L, azithromycin <=1 mg/L, clarithromycin <=0.5 mg/L, erythromycin <=0.5 mg/L and roxithromycin <=0.5 mg/L; for H. influenzae HMR 3647 <=2 mg/L, low and high breakpoints of azithromycin <=0.25 mg/L and<=4 mg/L, clarithromycin <=0.5 mg/L and <=16 mg/L, erythromycin <=0.5 mg/L and <=8 mg/L, roxithromycin <=0.5 mg/L and <=4 mg/L. For H. influenzae and M. catarrhalis, a cefotaxime breakpoint of <=1 mg/L was used. For S. pneumoniae, penicillin breakpoints were <=0.06 mg/L for sensitive, 0.12–1 mg/L for moderate and >=2 mg/L for resistant; and for cefotaxime <=0.5 mg/L for sensitive, 1–2 mg/L for moderate and >=4 mg/L for resistant were used. for ß-haemolytic streptococci, the breakpoint for penicillin was <=0.12 mg/L. For all other species the breakpoints were ciprofloxacin <=0.5 mg/L, levofloxacin <=2 mg/L, ampicillin <=1 mg/L and co-amoxiclav <=1 mg/L.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in-vitro activity of HMR 3647 against ß-haemolytic streptococci is shown in Table I. HMR 3647 inhibited all ß-haemolytic streptococci at <=0.25 mg/L. HMR 3647 had greater activity than clarithromycin, azithromycin, roxithromycin and erythromycin A against ß-haemolytic streptococci group A, with an HMR 3647 MIC90 of 0.03 mg/L compared with 0.12 of 0.5 mg/L for the other agents. With ß-haemolytic streptococci group B, HMR 3647 exhibited similar activity to clarithromycin and with group C streptococci it was more active than all macrolides, having an MIC90 of 0.06 mg/L compared with MIC90s in the range 1–8 mg/L for the other agents. For group F streptococci, the HMR 3647 MIC90 was four-fold lower than the next most active macrolide. When tested against erythromycin A-resistant ß-haemolytic streptococci, HMR 3647 had an MIC50 eight-fold lower than clarithromycin, the next most active macrolide. With S. pneumoniae, HMR 3647 was eight-fold more active than clarithromycin. HMR 3647 had an MIC90 of 0.12 mg/L compared with clarithromycin 8 mg/L for erythromycin A-resistant pneumococci and a HMR 3647 MIC90 of 0.03 mg/L compared with clarithromycin 0.12 mg/L for erythromycin A-sensitive isolates. For those strains exhibiting the MLSB resistance, i.e. clindamycin- and erythromycin A-resistant, MR 3647 was 32-fold more active than clarithromycin (Table I).


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Table I. In-vitro activity of HMR 3647 and comparators against a range of respiratory tract pathogens
 
For H. influenzae, HMR 3647, with an MIC90 of 4 mg/L, was more active than all the macrolides and cefuroxime, but not cefotaxime which had an MIC90 of 0.5 mg/L (Table I). Against cefuroxime-resistant H. influenzae, HMR 3647 had an MIC90 four times higher than axithromycin, while against cefuroxime-sensitive strains it was twice as active. HMR 3647 had a similar MIC90 to clarithromycin for M. catarrhalis (Table I).

S. pneumoniae were categorized into four groups on the basis of their erythromycin MIC (Table II). Strains with erythromycin A MICs <=0.12 mg/L had an HMR 3647 MIC50 of <=0.08 mg/L, while those with erythromycin A MICs >=0.25 mg/L had an HMR 3647 MIC50 of 0.03 mg/L. It was notable that strains with an erythromycin MIC >=1 mg/L had very markedly higher MIC50s of azithromycin (32 mg/L), clarithromycin (4 mg/L) and roxithromycin (32 mg/L) but not of HMR 3647 (0.03 mg/L).


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Table II. Susceptibility of S. pneumoniae subdivided on the basis of erythromycin A MIC
 
H. influenzae isolates were categorized according to cefuroxime susceptibility. The MIC90 values of ampicillin and co-amoxiclav increased >=16-fold as the cefuroxime MIC increased from <=1 mg/L to >4 mg/L. The effect on the MIC90 of cefotaxime was less clear, although the MIC50 increased from 0.015 to 0.12 mg/L. In addition, the MIC90 of HMR 3647 and macrolides increased four-fold as the cefuroxime MIC increased (Table III).


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Table III. Susceptibility of H. influenzae subdivided on the basis of cefuroxime MIC
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance in streptococci to macrolides is usually due to modification in the ribosomal target of the macrolides. However, resistance due to an active efflux of erythromycin A (phenotypically defined as erythromycin A resistant, clindamycin sensitive) was recently reported in Streptomyces pyogenes and S. pneumoniae, and is increasing in incidence.9,10,11 The macrolides clarithromycin, axithromycin and roxithromycin all seem to have greater antibacterial activity than erythromycin A against S. pneumoniae, but do not overcome MLSB resistance (phenotypically defined as erythromycin A and clindamycin resistant).12 In this study, MR 3647 demonstrated an eight-fold increase in MIC90 compared with clarithromycin, the most active macrolide against MLSB-resistant S. pneumoniae. HMR 3647 is a semi-synthetic 14-membered ring macrolide.13 It differs from erythromycin A in that it has a 3-keto group on the erythronolide ring instead of 9-cladinose, a sugar with supposed antibacterial activity.14,15 It has been noted that the L-cladinose sugar moiety is responsible for inducing MLSB resistance in staphylococci causing cross resistance among macrolides.7 HMR 3647 lacks this moiety and the results seen in this study confirm this view in streptococci. Increased activity of MR 3647 against erythromycin A-resistant, clindamycin-sensitive streptococci shows that even if the efflux mechanism of resistance is present, HMR 3647 is not greatly affected. Some cross resistance does seem to exist, however, between HMR 3647 and erythromycin A, but to a lesser degree. This data is consistent with other ketolide studies16,17 which suggests that the ketolides are not inducers of MLSB resistance. HMR 3647 is more active than all the comparator macrolides against ß-haemolytic streptococci.

Against H. influenzae that are cefuroxime susceptible, HMR 3647 had a lower MIC90 than the macrolides tested. These strains are more likely to represent the wild-type population than the cefuroxime-resistant strains, which occur at an incidence of <2%.18 As expected, cefuroxime susceptibility affects susceptibility to other ß-lactams19 but surprisingly also seems to be associated with effects on macrolide, ketolide and quinolone susceptibility. Cefuroxime resistance in H. influenzae is thought to be caused by an alteration in penicillin-binding proteins (PBPs) and, therefore, should have no effect on the activity of other agents, perhaps suggesting another mechanism of resistance in these strains. Some cefuroxime-resistant H. influenzae are ampicillin sensitive by MIC testing. Whether these strains would respond to ampicillin therapy is as yet unknown.

In conclusion, HMR 3647 has activity against a wide range of ß-haemolytic streptococci and S. pneumoniae, but has less activity against S. pneumoniae which are resistant to macrolides due to efflux or MLSB mechanisms. However, all these strains remain susceptible as MIC values are still well below the breakpoint. HMR 3647 is also equipotent or has superior potency to macrolides against cefuroxime-susceptible H. influenzae and M. catarrhalis. Surprisingly, ketolide, macrolide and quinolone activity declined with increasing cefuroxime MICs in H. influenzae, possibly suggesting a further mechanism of resistance in addition to PBP changes. It is likely that HMR 3647 will have useful clinical activity against S. pneumoniae and ß-haemolytic streptococci, irrespective of their macrolide susceptibility.


    Acknowledgments
 
We wish to thank Dr A. Bryskier of Hoechst Marion Roussel for his advice and financial support.


    Notes
 
* Corresponding author. Tel: +44-117-9595652; Fax: +44-117-9593154. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 26 October 1998; returned 23 February 1999; revised 12 March 1999; accepted 28 May 1999