Comparative activity of garenoxacin and other agents by susceptibility and time–kill testing against Staphylococcus aureus, Streptococcus pyogenes and respiratory pathogens

Silvana Noviello1, Filomena Ianniello2, Sebastiano Leone2 and Silvano Esposito2,*

1 Dottorato di Ricerca in Scienze Farmacologiche e Fisiopatologia Respiratoria XVII° ciclo, Seconda Università degli Studi di Napoli, Napoli; 2 Dipartimento di Medicina Pubblica Clinica e Preventiva, Sezione Malattie Infettive, Seconda Università degli Studi di Napoli, Via D. Cotugno, 1, 80135, Napoli, Italy

Received 12 June 2003; returned 6 July 2003; revised 25 July 2003; accepted 27 July 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Garenoxacin is a novel des-F(6)quinolone that has shown excellent antimicrobial activity against a wide range of clinically important microorganisms. In this study, its activity was examined, in comparison with that of other antimicrobial agents, by susceptibility and time–kill testing against Staphylococcus aureus, Streptococcus pyogenes and respiratory pathogens.

Methods: Overall, 200 bacterial strains were tested. The antimicrobial activity of garenoxacin was compared with that of ciprofloxacin, levofloxacin, moxifloxacin, amoxicillin, co-amoxiclav, cefuroxime, cefotaxime, ceftriaxone, imipenem, erythromycin and clarithromycin. In addition, the bactericidal activity of garenoxacin, moxifloxacin, levofloxacin and ciprofloxacin was evaluated by time–kill analysis against four strains each of staphylococci [two methicillin-susceptible (MSSA) and two methicillin-resistant (MRSA)], pneumococci (two penicillin-susceptible and two penicillin-resistant) and Streptococcus pyogenes (two erythromycin-susceptible and two erythromycin-resistant). Antibiotics were tested at concentrations 1–8 x MIC.

Results: MIC90 values of garenoxacin for the MSSA and MRSA strains were 0.03 and 2 mg/L, respectively. Among all the quinolones tested, garenoxacin yielded the lowest MIC values against all pneumococci (MIC90 0.12 mg/L) irrespective of macrolide resistance; the rank order of activity was garenoxacin> moxifloxacin>levofloxacin>ciprofloxacin. Excellent activity was shown also against Haemophilus influenzae (MIC90 <= 0.03 mg/L) and Moraxella catarrhalis (MIC90 <= 0.03 mg/L). Ninety percent of S. pyogenes were inhibited at garenoxacin concentrations equal to 0.25 mg/L, its activity not being influenced by macrolide susceptibility. Garenoxacin was rapidly bactericidal against staphylococci, producing a >= 3 log10 decrease in viable counts (cfu/mL) within 3 h at 4 x MIC, whereas a moderate, slower killing rate was observed versus streptococci.

Conclusions: This investigational des-F(6)quinolone represents a promising alternative for the treatment of respiratory tract infections.

Keywords: new fluoroquinolones, Gram-positive, bactericidal activity


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Quinolones are a class of antimicrobials that were first described by Lescher et al. in 19621 and since then, following a wide array of chemical and structural modifications, they have become very important therapeutic agents for the treatment of several bacterial infections. The weak activity of the first-generation fluoroquinolones against respiratory pathogens and some clinically important pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), as well as the increased importance of penicillin-resistant pneumococci, have led to the development of new fluoroquinolones with an enhanced activity against Gram-positive pathogens. Garenoxacin has exhibited excellent activity against a broad spectrum of clinically important bacteria, including some atypical pathogens, such as Mycobacterium, Mycoplasma and Chlamydia.

The aim of this study was to assess and confirm comparatively the antibacterial efficacy of garenoxacin with that of a representative panel of other antimicrobial agents, oral and parenteral, such as ciprofloxacin, levofloxacin, moxifloxacin, amoxicillin, co-amoxiclav, cefuroxime, cefotaxime, ceftriaxone, imipenem, erythromycin and clarithromycin. The activities of these drugs were tested against S. aureus, either methicillin-susceptible (MSSA) or -resistant (MRSA), respiratory pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis) and Streptococcus pyogenes.

This evaluation was performed by means of MIC determination and kinetic time–kill analysis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

A total of 200 strains [MSSA, n = 30 strains; MRSA, n = 30 strains; S. pneumoniae, n = 40 strains (35 penicillin-susceptible and five penicillin-resistant); H. influenzae, n = 30 strains; M. catarrhalis, n = 30 strains; S. pyogenes, n = 40 strains] were tested. Microorganisms had been isolated during January–July 2002 from patients affected by community-acquired respiratory tract infections. In all cases, the isolates tested in this study were recovered from consecutive unique patients. Following subcultures, the isolates were identified by conventional tests and criteria. All isolates were stored frozen at –70°C in Brain Heart Infusion broth (Oxoid, Basingstoke, UK) plus 20% glycerol, with two subcultures made before the organisms were tested.

Antimicrobial agents

Garenoxacin was kindly supplied as pure laboratory powder of known potency by Bristol Myers Squibb (Pharmaceutical Research Institute, Princeton, NJ, USA). Other antimicrobials were obtained from their respective manufacturers or alternatively from Sigma-Aldrich. All of them were stored at +4°C and protected from light and moisture. The different antibiotic dilutions were prepared in accordance with the NCCLS guidelines. Garenoxacin dilutions were prepared, according to the manufacturer’s instructions, in DMSO.2

MICs

MICs were determined by standard broth (staphylococci, streptococci and haemophili) or agar (moraxellae) dilution, according to NCCLS recommendations. Cation-adjusted Mueller–Hinton (CAMH) broth (Oxoid, Basingstoke, UK), supplemented with 5% lysed horse blood for streptococci, was employed for testing staphylococci, whereas Haemophilus Test Medium was used when H. influenzae strains were tested. To determine the antimicrobial susceptibility of M. catarrhalis strains by agar dilution, CAMH agar was utilized. The inoculum was ~5 x 105 cfu/mL. The inoculated trays and plates were incubated in ambient air at 35°C for 18–24 h. Testing of ß-lactams against staphylo- cocci was performed in CAMH broth supplemented with 2% NaCl. The supplemental NaCl was excluded when testing non-ß-lactam drugs.

The MIC was defined as the lowest antibiotic concentration that yielded no visible growth. The following standard quality control strains were included in each run of broth dilution MICs: S. aureus ATCC 29213 and S. pneumoniae ATCC 49619.

Time–kill studies

The bactericidal activity of garenoxacin was compared with that of other fluoroquinolones (moxifloxacin, levofloxacin and ciprofloxacin) against 12 strains [two MSSA, two MRSA, four pneumococci (two penicillin-susceptible and two penicillin-resistant), and four S. pyogenes, two erythromycin-susceptible and two erythromycin-resistant].

Time–kill studies were performed in flasks according to the method described by Eliopoulos & Moellering.3 Overnight bacterial cultures were adjusted to a turbidity equivalent to that of a 0.5 McFarland standard, and further diluted to yield a starting inoculum ranging between 5 x 105 and 5 x 106 cfu/mL. Cultures were incubated at 35°C in a shaking water bath for 1 h in order to achieve exponential growth. Subsequently, aliquots (25 mL) of inoculum were exposed to concentrations of the compounds at 1, 2, 4 and 8 x MIC. In each case, an antibiotic-free control was prepared and the same procedure applied. At 0, 3, 6, 12 and 24 h of incubation at 37°C in a shaking water bath, samples were removed from test and growth-control cultures and plated onto suitable agar plates in order to perform a count of viable bacteria. A count of 300 cfu/mL was the limit of quantification; plates showing less than 30 colonies were counted as an estimate only. Time–kill curves were constructed by plotting the number of surviving bacteria against time.

Antimicrobial activity was interpreted as bactericidal when a reduction of >=3 log10 cfu/mL (99.9% kill) occurred, compared with the initial inoculum at time 0. Experiments were performed in duplicate.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs

MIC values, expressed as MIC50 and MIC90 (mg/L) are depicted in Table 1. Garenoxacin proved to be highly active against MSSA, with an MIC90 of 0.03 mg/L, four-fold lower than ciprofloxacin but simi-lar to moxifloxacin. However, when tested against MRSA, this new fluoroquinolone showed an MIC90 six-fold higher than that assessed for MSSA. In vitro potency of garenoxacin against pneumococci was eight- to 16-fold superior to that of ciprofloxacin, and eight-fold superior to that of levofloxacin, all pneumococcal strains being inhibited by a concentration of 0.12 mg/L. MIC values of garenoxacin were similar for five strains of penicillin-resistant S. pneumoniae; these were also resistant to macrolides (data not shown in Table 1). Among the haemophilus strains (n = 30), the production of ß-lactamase was observed in six (20%). MIC values of garenoxacin for H. influenzae were very similar to those of the other fluoroquinolones considered. The high activity of garenoxacin was similar to that exhibited by the other fluoroquinolones against the M. catarrhalis isolates. Although garenoxacin proved to be highly active against S. pyogenes clinical isolates, the ß-lactams considered and the two macrolides, erythromycin and clarithromycin (at least for the erythromycin-susceptible strains), were more potent.


View this table:
[in this window]
[in a new window]
 
Table 1. MICs (mg/L) of all antimicrobial agents against all bacterial strains
 
Time–kill studies

MIC values of garenoxacin, moxifloxacin, levofloxacin and ciprofloxacin were the following: MSSA = 0.03, 0.06, 0.12, 0.12; MRSA = 0.25, 0.25, 0.5, 0.5; penicillin-susceptible and -resistant S. pneumoniae = 0.06, 0.12, 0.5, 1; erythromycin-susceptible and -resistant S. pyogenes = 0.06, 0.12, 1, 1. Time–kill results of the four antibiotics against the different microorganisms are reported in Table 2 (results averaged between two strains each). More than 99.9% of the initial inoculum of the four strains of S. aureus, either methicillin-susceptible or -resistant, was killed following 3 or 6 h exposure to concentrations of all the four fluoroquinolones tested at values equal to, or greater than, 4 x MIC. No re-growth was observed during the 24 h duration of the experiments. A moderate, slower killing rate was observed versus pneumococci and streptococci.


View this table:
[in this window]
[in a new window]
 
Table 2. Antibacterial performance in in vitro time–kill studies.
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Penicillin resistance in S. pneumoniae is increasing all over the world. This type of resistance has been described as being cross-related, to different extents, to other antimicrobial agents, such as third-generation cephalosporins, macrolides, tetracycline and co-trimoxazole. Using a mathematical transmission model, McCormick et al.4 analysed temporal trends in the proportions of singly and dually resistant organisms and found that pneumococcal strains resistant to both penicillin and erythromycin are increasing faster than strains singly resistant to either. The authors predicted that by 2004, in the absence of a vaccine, 41% of pneumococci at the Centers for Disease Control and Prevention Active Bacterial Core surveillance sites, taken together, will be dually resistant, with 5% resistant to penicillin only and 5% resistant to erythromycin only.4

Therefore, the efficacy of first-line ß-lactams has been affected by pneumococcal resistance to penicillin and ß-lactamase production in H. influenzae and M. catarrhalis. However, whereas some antimicrobial classes are still useful in the treatment of infections sustained by haemophili and moraxellae ß-lactamase-producers, penicillin-resistant pneumococci require a reappraisal for the usage of fluoroquinolones in the treatment of respiratory tract infections since fluoroquinolone activity is not influenced at all by penicillin resistance.

Older generation fluoroquinolones yield only a moderate in vitro antimicrobial activity against pneumococci, with MIC values clustering around the breakpoints. The MICs we obtained in our study were similar to those reported by others.5,6 Garenoxacin showed excellent in vitro activity against all the microorganisms tested, with MIC90 values ranging between <=0.03 and 2 mg/L for all tested strains; its activity was affected neither by penicillin susceptibility in pneumococci nor by erythromycin resistance in S. pyogenes, and it was rapidly bactericidal against staphylococci.

The development of ‘respiratory quinolones’ has raised a great deal of interest and they are likely to be used even more widely for the treatment of respiratory tract infections, as emergence of resistance in respiratory pathogens to common empirical antimicrobial agents is still increasing. Nevertheless, as recently underlined by Dalhoff & Schmitz,7 all the new fluoroquinolones exhibit a similar good activity against the most important respiratory tract pathogens with few exceptions, gemifloxacin, sitafloxacin and garenoxacin being one-to-two dilution steps more active than moxifloxacin against S. pneumoniae. In this scenario, it will be their pharmacokinetic and pharmacodynamic properties that will represent clinically important differentiators and determinants of overall activity and efficacy. In addition, emergence of resistant subpopulations following exposure to antibiotics has to be taken into account: this is functionally related to drug concentrations achievable in vivo and to the susceptibility profile of bacterial pathogens. Drugs with the most favourable properties should be used as first-line agents in order to preserve the potential of fluoroquinolones, and, most importantly, to provide the patient with an optimally effective regimen. However, considering the ability of some microorganisms, such as S. pneumoniae, to develop multidrug resistance, careful monitoring of microbial susceptibility to all antimicrobial agents is mandatory.


    Footnotes
 
* Corresponding author. Tel: +39-081-566-62-18; Fax: +39-081-566-62-03; E-mail: silvanoesposito{at}libero.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Lescher, G. Y., Troelich, C. J. & Brundage, R. P. (1962). 1,8-Naphtyridine derivatives, a new class of chemotherapy agents. Journal of Medicinal Chemistry 5, 1063–5.

2 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Third Edition: Approved Standard M7-A4. NCCLS, Villanova, PA, USA.

3 . Eliopoulos, B. M. & Moellering, R. C. (1996). In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., Ed.), pp. 104–5. William & Wilkins Co., Baltimore, MD, USA.

4 . McCormick, A. W., Whitney, C. G., Farley, M. M. et al. (2003). Geographic diversity and temporal trends of antimicrobial resistance in Streptococcus pneumoniae in the United States. Nature Medicine 9, 424–30.[CrossRef][ISI]

5 . Biedenbach, D. J., Jones R. N., Pfaller, M. A. et al. (2001). Activity of BMS284756 against 2,681 recent clinical isolates of Haemophilus influenzae and Moraxella catarrhalis: report from The SENTRY Antimicrobial Surveillance Program (2000) in Europe, Canada and the United States. Diagnostic Microbiology and Infectious Disease 39, 245–50.[CrossRef][ISI]

6 . Pankuch, G. A., Nagai, K. & Davies, T. A. (2002). Antipneumococcal activity of BMS284756 compared to those of six other agents. Antimicrobial Agents and Chemotherapy 46, 251–4.

7 . Dalhoff, A. & Schmitz, F. J. (2003). In vitro antibacterial activity and pharmacodynamics of new quinolones. European Journal of Clinical Microbiology and Infectious Disease 22, 203–21.[ISI][Medline]





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
52/5/869    most recent
dkg429v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Disclaimer
Request Permissions
Google Scholar
Articles by Noviello, S.
Articles by Esposito, S.
PubMed
PubMed Citation
Articles by Noviello, S.
Articles by Esposito, S.