In vitro activity of LY333328 (oritavancin) against Gram-positive aerobic cocci and synergy with ciprofloxacin against enterococci

S. Noviello, F. Ianniello and S. Esposito,*

Clinica Malattie Infettive, Seconda Universita' di Napoli, Via Cotugno 1, 80135 Napoli, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gram-positive cocci are a major cause of nosocomial bacteraemias and are often resistant to most antibiotics. The emergence of enterococci with reduced susceptibility to vancomycin has created an urgent need for novel antibiotics to combat infections associated with these bacteria. In this study the in vitro activity of LY333328 (oritavancin), a semi-synthetic glycopeptide, was evaluated. LY333328 was effective against all strains of staphylococci, streptococci and enterococci tested. A combination of LY333328 and ciprofloxacin was additive for 50% of Enterococcus faecium strains resistant to both vancomycin and ciprofloxacin, and for 100% of vancomycin-susceptible and either ciprofloxacin-susceptible or -resistant strains.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Over the last few decades, Gram-positive cocci including enterococci have become important nosocomial pathogens.1 The incidence of Gram-positive cocci with single or multiple resistance to antimicrobial agents such as methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant Streptococcus pneumoniae (PRSP) has increased in both community and nosocomial settings.

Combination antimicrobial therapy for serious enterococcal infections is employed in clinical practice for a variety of reasons, the major one being synergy. The most appropriate treatment for serious enterococcal infections such as endocarditis is based on combinations of a penicillin or a glycopeptide and an aminoglycoside.2 The increasing frequency of enterococcal strains exhibiting single or multiple resistance traits such as ß-lactamase production, aminoglycoside resistance and, more recently, the emergence of glycopeptide resistance renders such treatment inappropriate.1 If ß-lactams are ineffective, an alternative treatment consists of the synergic vancomycin– aminoglycoside combination. However, the recent widespread use of vancomycin has resulted in an increased prevalence of enterococci resistant to glycopeptides (23.9% in USA). Strains of staphylococci with intermediate susceptibility to vancomycin have also been reported.1 The emergence of high-level aminoglycoside resistance also highlights the need for new combinations of antibiotics.

LY333328 is a new semi-synthetic glycopeptide derived from the N-alkylation of LY264826, a naturally occurring, vancomycin-like drug. In previous studies it has been shown that this new glycopeptide is highly active against Gram-positive bacteria.35 The present study was carried out to evaluate: (i) the in vitro activity of LY333328 in comparison with other currently available glycopeptides against 637 isolates; and (ii) the potential for in vitro synergy between LY333328 and ciprofloxacin, which, because of its moderate activity against enterococci, can be considered as a good alternative to aminoglycosides against 30 enterococcal strains with different patterns of drug resistance.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial agents

Antimicrobial agents were kindly supplied as pure powders by their respective manufacturers (LY333328 and vancomycin by Eli Lilly and Co., Indianapolis, IN, USA; ciprofloxacin by Bayer, Milan, Italy; teicoplanin by Aventis, Milan, Italy). Antimicrobial stock solutions were prepared in accordance with NCCLS guidelines.6 LY333328 was dissolved in water and diluted in cation-adjusted Mueller–Hinton broth (CAMHB) in accordance with the manufacturer's instructions (Eli Lilly and Co.).

Clinical isolates

The in vitro activity of LY333328 was compared with vancomycin and teicoplanin against 637 isolates, which included: 220 S. aureus [180 methicillin-susceptible (MSSA), 40 MRSA], 216 S. pneumoniae [194 penicillin-susceptible (PSSP), 22 PRSP; 12 strains characterized by intermediate resistance and 10 by high resistance)], 156 Streptococcus pyogenes strains, 20 Enterococcus faecium isolates [10 vancomycin-resistant (VRE), 10 vancomycin-susceptible (VSE)] and 25 Enterococcus faecalis strains.

A collection of 30 clinical isolates of E. faecium and E. faecalis, selected on the basis of different susceptibility patterns to vancomycin (VAN) and ciprofloxacin (CIP), was used in the combination drug studies. These were VANr CIPr E. faecium (n = 10), VANs CIPr E. faecium (n = 10) and VANs CIPs E. faecalis (n = 10).

Susceptibility tests

The MIC, defined as the lowest concentration of antibiotic that yielded no visible growth, and the minimal bactericidal concentration (MBC), defined as the lowest antibiotic concentration that resulted in <=0.1% survival in the subculture, were determined by a broth microdilution method in CAMHB according to NCCLS recommendations.6 Lysed horse blood (5%) was added when streptococci other than enterococci were tested. Plates were incubated at 35°C for 18–24 h for MIC testing, and MBC evaluation was carried out after MIC reading was performed by incubating the plates for a further 24–48 h.

Bacterial resistance to vancomycin and teicoplanin was indicated by an MIC >= 16 mg/L. A projected breakpoint of >=4 mg/L was adopted for LY333328. Since no NCCLS breakpoints have yet been determined for LY333328, the selection of strains was performed on the basis of susceptibility or resistance to vancomycin.

Assessment of synergy

MICs of each antibiotic were determined by the broth microdilution method; synergy was assessed by a microdilution chequerboard method using CAMHB and repeated twice for each experiment. Fractional inhibitory concentration indexes (>=FIC) were interpreted as follows: synergy <=0.5; additivity = 1.0; antagonism >=2, in accordance with the criteria of Krogstad & Moellering.7


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
S. aureus ATCC 29213 and E. faecalis ATCC 29212 were used as quality control strains. MICs obtained for these strains were within the ranges proposed by NCCLS for all the antimicrobials tested.

The in vitro activity of LY333328, vancomycin and teicoplanin is summarized in Table IGo. No difference was observed between methicillin-susceptible and -resistant staphylococcal strains. All streptococcal strains, irrespective of penicillin resistance or susceptibility, were highly susceptible to LY333328.


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Table I. In vitro comparative activity (mg/L) of LY333328, vancomycin and teicoplanin against Gram-positive cocci
 
The median of MICs and the MIC ranges for the different antimicrobial agents against the different enterococcal strains are shown in Table IIGo. The susceptibility to LY333328 was similar for ciprofloxacin-resistant and vancomycin-resistant and -susceptible strains of enterococci.


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Table II. MICs of vancomycin, ciprofloxacin and LY333328 against 30 enterococcal isolates
 
Table IIIGo shows the combined effects of LY333328 and ciprofloxacin against the 30 enterococcal isolates in vitro. The antimicrobial drug combination resulted in synergy for 50% of E. faecium strains resistant to both vancomycin and ciprofloxacin; additivity was observed for all the other strains. There was no antagonistic effect observed with any strain.


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Table III. In vitro interactions between LY333328 and ciprofloxacin against 30 enterococcal strains
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Vancomycin-resistant enterococci have become a major cause of nosocomial infections. Infections caused by S. aureus with reduced susceptibility to vancomycin and teicoplanin have also been reported recently.8,9 One approach to the problem of antimicrobial resistance is the modification of currently known agents to overcome resistance. In the present study LY333328, a semisynthetic glycopeptide derived from a naturally occurring vancomycin-like drug, displayed good in vitro activity against all the Gram-positive microorganisms tested, including those that are resistant to currently available glycopeptides.

LY333328 was highly effective against strains of both MSSA and MRSA, penicillin-susceptible and PRSP, VSE and VRE E. faecium and vancomycin-susceptible E. faecalis. The MIC90s obtained are consistent with previous studies in which the inhibitory and bactericidal activities of LY333328 against Gram-positive pathogens were evaluated.3 The MIC50s for LY333328 were similar for all strains of staphylococci, streptococci and enterococci studied. Biavasco et al.4 have shown that LY333328 was highly active against staphylococci and enterococci and its effects did not vary substantially between the vancomycin-susceptible and -resistant enterococci. LY333328 was effective against vancomycin-susceptible E. faecium and E. faecalis. Thus, the present results complement and extend the previous studies suggesting that LY333328 could be an effective therapeutic alternative to glycopeptides in current use.

In the present study the synergic or additive effects of combined LY333328 and ciprofloxacin on enterococci were also demonstrated by the chequerboard method.

The results of 50% synergy in vancomycin-resistant/ ciprofloxacin-resistant strains of E. faecium and 100% additivity in vancomycin-susceptible/ciprofloxacin-resistant strains of E. faecium and vancomycin-susceptible/ ciprofloxacin-susceptible strains of E. faecalis indicate that this combination could result in a therapeutic advantage for treatment of infections sustained by enterococci with decreased susceptibility to vancomycin. These results are also consistent with those reported by Unal et al.,10 who observed a similar synergy between ciprofloxacin and vancomycin in six E. faecium strains that were resistant to both antimicrobial agents. While they hypothesized that such interactions may be related to a diminished induction of the proteins that mediate resistance for doubly resistant E. faecium, no explanation was given for the absence of synergy in isolates susceptible to vancomycin but resistant to ciprofloxacin.10

This new glycopeptide compound overcomes the resistance expressed by some enterococcal strains towards the currently available glycopeptides. Results of this study may be of clinical importance for the treatment of those infections caused by enterococci with high-level aminoglycoside resistance as well as vancomycin resistance, but more studies are necessary to confirm these results and to evaluate the clinical efficacy.


    Notes
 
* Corresponding author. Tel: +39-81-5666218; Fax: +39-81-5666203; E-mail: silvanoesposito{at}libero.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Kaye, K. S., Fraimow, H. S. & Abrutyn, E. (2000). Pathogens resistant to antimicrobial agents. Infectious Disease Clinics of North America 14, 293–319.[ISI][Medline]

2 . Sanford, J. P., Gilbert, D. N., Moellering, R. C. & Sande, M. A. (2000). The Sanford Guide to Antimicrobial Therapy, 30th edn. Antimicrobial Therapy, Inc., USA.

3 . Jones, R. N., Barrett, M. S. & Erwin, M. E. (1997). In vitro activity and spectrum of LY333328, a novel glycopeptide derivative. Antimicrobial Agents and Chemotherapy 41, 488–93.[Abstract]

4 . Biavasco, F., Vignaroli, C., Lupidi, R., Manso, E., Facinelli, B. & Varaldo, P. E. (1997). In vitro antibacterial activity of LY333328, a new semi-synthetic glycopeptide. Antimicrobial Agents and Chemotherapy 41, 2165–72.[Abstract]

5 . Garcìa-Garrote, F., Cercenado, E., Alcalà, L. & Bouza, E. (1998). In vitro activity of the new glycopeptide LY333328 against multiply resistant Gram-positive clinical isolates. Antimicrobial Agents and Chemotherapy 42, 2452–5.[Abstract/Free Full Text]

6 . National Committee for Clinical Laboratory Standards. (1999). Standard Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Anaerobically. NCCLS, Villanova, PA.

7 . Krogstad, D. J. & Moellering, R. C. (1996). Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 4th edn, (Lorian, V., Ed.), pp. 330–95. Williams and Wilkins, Baltimore, MD.

8 . Perl, T. M. (1999). The threat of vancomycin resistance. American Journal of Medicine 106, 26S–37S.[Medline]

9 . Paterson, D. L. (1999). Reduced susceptibility of Staphylococcus aureus to vancomycin—a review of current knowledge. Communicable Diseases Intelligence 23, 69–73.[Medline]

10 . Unal, S., Flokowitsch, J., Mullen, D. L., Preston, D. A. & Nicas, T. I. (1993). In-vitro synergy and mechanism of interaction between vancomycin and ciprofloxacin against enterococcal isolates. Journal of Antimicrobial Chemotherapy 31, 711–23.[Abstract]

Received 18 December 2000; returned 10 March 2001; revised 23 April 2001; accepted 30 April 2001