Prevalence of bacterial pathogens resistant to the available antibiotics has been increasing over the past several decades. This feature represents a major challenge for developing innovative therapeutic modalities, particularly for treatment of Gram-positive infections. Among these, methicillin resistant Staphylococcus aureus (MRSA) is an increasing common cause of nosocomial infections, causing severe morbidity and mortality worldwide, and accounting in some hospitals for more than 50% of all S. aureus diseases 1234. Methicillin resistance was firstly identified in 1961 among nosocomial isolates of S. aureus and, till the last 10 years, was mainly restricted to the nosocomial setting 5. However in the past decade new strains of MRSA in the community have been increasingly isolated reaching even 30% in some areas 67. Of particular concern is that methicillin resistance, particularly in nosocomial strains, is often associated with resistance to other antibiotics such as macrolides and fluoroquinolones, so that glycopeptides and the oxazolidinones constitute the only available therapeutic options, once an MRSA infection has been diagnosed.

In the last years new antimicrobials specifically targeted against Gram-positive or MRSA strains have been launched, but strains with decreased susceptibility to these antibiotics have been increasingly isolated and, unfortunately, development of new antimicrobial agents in the next future seems to be declining 8910. On the other hand, antimicrobial combination therapy should be useful in improving efficacy, providing broad-spectrum coverage and preventing the emergence of resistant mutants 11. Moreover, combination therapy is argued when empirical treatment of severe infections is needed, such as in severe pneumonia. As an etiological diagnosis with susceptibility results is almost never available to assist in the selection of the prompt therapy, the initial approach to treatment is largely empirical and is ordinarily guided by consensus guidelines. Moreover, early administration of appropriate empirical therapy for severe pneumonia has been demonstrated to significantly improve survival 12. For this reason, antimicrobial therapy should cover both Gram-positive and Gram-negative bacteria, until obtainment of laboratory results. Guidelines from the American Thoracic Society suggest to add an anti-MRSA agent for treatment of pneumonia when MRSA is suspected, so that double or triple antimicrobial combinations are often used 13.

Despite their wide use, antimicrobial activity of antibiotics' combinations, particularly when more than two drugs are associated, is only rarely assessed in in vitro assays.

This study has been addressed to rate the interaction between glycopeptides (vancomycin and teicoplanin) and a β-lactam (cefotaxime) or a fluoroquinolone (levofloxacin) and among glycopeptides, a fluoroquinolone (levofloxacin) and either an aminoglycoside (amikacin) or a β-lactam (cefepime, ceftazidime, imipenem, piperacillin/tazobactam), which are suggested for empirical combinations in the hospital setting when a MRSA etiology is suspected 13. Moreover, frequency of spontaneous mutations for single and combined antibiotics were also evaluated.

Antimicrobial combination therapy may be used to extend spectrum coverage, prevent the emergence of resistant mutants and gain synergy between antimicrobials 11. Combination therapy is often recommended for empirical treatment of bacterial infections in intensive care units, where monotherapy is not likely to cover all potential pathogens, and the emergence of resistance is a potential threat 20. In the last years, development of new antimicrobials targeted to treatment of MRSA infections, such as linezolid, has led to assess antibacterial activity of such drugs in combination with several antibacterials, particularly β-lactams 21222324. Also combination of vancomycin with aminoglycoside or β-lactams has been widely investigated, generally reporting synergy with β-lactams while no interactions of even antagonism were observed with aminoglycosides 25262728. Activity of combinations containing teicoplanin against S. aureus has been less investigated 2728. The present study has compared antimicrobial combinations containing teicoplanin or vancomycin for their antibacterial activity and ability to select for mutants able to grow in their presence in nosocomial MRSA. To our knowledge this is the first study evaluating the efficacy of triple combinations of a glycopeptide, a β-lactam and a fluoroquinolone against MRSA strains. Triple combinations with vancomycin have been recently indicated by American Thoracic Society and Infectious Diseases Society of America for initial empiric therapy for hospital acquired pneumonia, ventilator associated pneumonia and healthcare associated pneumonia in patients with potential MRSA etiology 13. These guidelines suggest the use of vancomycin or, as alternative, of linezolid, combined with one among antipseudomonal cephalosporins or carbapenems or β-lactam/β-lactamase inhibitor (i.e. piperacillin-tazobactam) and an antipseudomonal fluoroquinolone or an aminoglycoside. Teicoplanin is not included, since it has not been approved in the United States, however, in all countries where it has been approved, teicoplanin represents an alternative to vancomycin 13. In this study, data from the checkerboard assay indicates that teicoplanin with cefotaxime showed the highest rate of synergy among the tested combinations, followed by those with levofloxacin and ceftazidime or piperacillin/tazobactam. Ability of β-lactams in triple combinations to yield synergy was quite different for the various molecules, ranging from 20 to 80%.

Killing curves confirmed superior activity of teicoplanin plus cefotaxime, while smoothed differences among β-lactams. Since in in vitro experiments glycopeptides are slowly bactericidal, it may be hypothesized that their antistaphylococcal bactericidal activity could be increased by use of combinations 29. It is also evident that this ability was not a class effect since marked differences were found among β-lactams tested and between the two glycopeptides. Therefore it seems rather difficult to compare our data with those of other studies in which different β-lactams have been used.

Methicillin resistance is known to favor development of multi-drug resistance, including macrolides and fluoroquinolones. In fact, for the strains evaluated in this study, determination of frequency of mutation for each antibiotic in single was not always possible, since they were already resistant to the molecules tested and therefore able to grow at antibiotic concentrations equal to the resistant breakpoint. Data obtained indicate that glycopeptides in double or triple combinations were effective in preventing the emergence of mutants able to grow in presence of the antibiotics. No mutants were isolated in vitro after incubation with glycopeptides alone, thus correlating the low frequency of development of glycopeptides resistance observed in vivo. Different results have been reported for teicoplanin and vancomycin after serial exposure to these antibiotics by other authors, but the differences between the methods employed are likely the cause of this discrepancy 30313233. However, further studies on development of resistance possibly induced by the tested antibiotics in single and in combinations after serial passages are now in progress.

In summary, double and triple combinations of glycopeptides with levofloxacin and/or β-lactams were studied as far as synergy and frequency of mutations were concerned in a congruent number of MRSA strains. High rates of synergy were obtained for some combinations including teicoplanin, while combinations with both glycopeptides reduced frequency of mutations to not detectable levels. Although in vitro studies need to be confirmed by in vivo findings, the data obtained support the use of antimicrobial combinations including glycopeptides and β-lactams in initial empirical therapy of severe pneumonia, potentially sustained by MRSA and non fermenting gram-negative bacteria, limiting the development of mutants able to grow in presence of antibiotics.

LD received research funding from sanofi aventis.

LD participated in design and coordination of the study, interpretation of the data, and co-drafted the manuscript. EDV participated in study design, co-performed killing curves and frequency of mutation assays, analysis and interpretation of the data and co-drafted the manuscript. LN performed checkerboard assays, time kill curves and frequency of mutation assays and participated in analysis and interpretation of the data. MRG participated in revising the manuscript.

All authors read and approved the final manuscript.