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Antimicrobial Chemotherapy

Chemotherapy Research and Practice

Chemotherapy Research and Practice


Copyright © 2011 Hindawi Publishing Corporation. All rights reserved.

This is a focus issue published in volume 2011 of “Chemotherapy Research and Practice.” All articles are open access articles distributedunder the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, pro-vided the original work is properly cited.

Editorial Board

H. Akaza, JapanEmilio Bajetta, ItalySandro Barni, ItalyJoseph R. Bertino, USAEnzo Bonmassar, ItalyP. D. Bonomi, USAMichael H. Cynamon, USAN. Frimodt-Moller, DenmarkNaoya Fujita, JapanVassilis Georgoulias, GreeceM. R. Hammerschlag, USAKiyohiko Hatake, JapanPo-Ren Hsueh, TaiwanKatsuki Ito, Japan

Hiromichi Iwasaki, JapanElias Jabbour, USABarton A. Kamen, USAM. Kaneko, JapanMasaru Kubota, JapanCharles F. Levenback, USAVito Lorusso, ItalyN. H. Mulder, The NetherlandsRyuzo Ohno, JapanG. J. Peters, The NetherlandsPiero Picci, ItalySpyros Pournaras, GreecePaolo Pronzato, ItalyTadeusz Robak, Poland

Ethan Rubinstein, CanadaNagahiro Saijo, JapanW. Scheithauer, AustriaH. J. Schmoll, GermanyS. Seeber, GermanyMark S. Soloway, USAKazuo Tamura, JapanUmberto Tirelli, ItalyHaruaki Tomioka, JapanAthanassios Tsakris, GreecePaul M. Tulkens, BelgiumJ. B. Vermorken, BelgiumTakuya Watanabe, Japan

Contents

Factors Affecting the Cost Effectiveness of Antibiotics, Steven SimoensVolume 2011, Article ID 249867, 6 pages

Antibacterial and Antioxidant Properties of the Methanolic Extract of the Stem Bark of Pteleopsishylodendron (Combretaceae), Aristide Laurel Mokale Kognou, Rosalie Annie Ngono Ngane, Jules RogerKuiate, Martin Luther Koanga Mogtomo, Alembert Tchinda Tiabou, Raymond Simplice Mouokeu,Lucie Biyiti, and Paul Henri Amvam ZolloVolume 2011, Article ID 218750, 7 pages

Susceptibility of Bifidobacteria of Animal Origin to Selected Antimicrobial Agents, Sigrid Mayrhofer,Christiane Mair, Wolfgang Kneifel, and Konrad J. DomigVolume 2011, Article ID 989520, 6 pages

Cardiac Conduction Safety during Coadministration of Artemether-Lumefantrine andLopinavir/Ritonavir in HIV-Infected Ugandan Adults, Pauline Byakika-Kibwika, Mohammed Lamorde,Peter Lwabi, Wilson B. Nyakoojo, Violet Okaba-Kayom, Harriet Mayanja-Kizza, Marta Boffito,Elly Katabira, David Back, Saye Khoo, and Concepta MerryVolume 2011, Article ID 393976, 4 pages

Antibiotic Combinations with Daptomycin for Treatment of Staphylococcus aureus Infections,Kristina Nadrah and Franc StrleVolume 2011, Article ID 619321, 10 pages

Activity of Antimicrobial Peptides and Conventional Antibiotics against Superantigen PositiveStaphylococcus aureus Isolated from the Patients with Neoplastic and Inflammatory Erythrodermia,Wioletta Baranska-Rybak, Oscar Cirioni, Malgorzata Dawgul, Malgorzata Sokolowska-Wojdylo,Lukasz Naumiuk, Aneta Szczerkowska-Dobosz, Roman Nowicki, Jadwiga Roszkiewicz,and Wojciech KamyszVolume 2011, Article ID 270932, 6 pages

Hindawi Publishing CorporationChemotherapy Research and PracticeVolume 2011, Article ID 249867, 6 pagesdoi:10.1155/2011/249867

Review Article

Factors Affecting the Cost Effectiveness of Antibiotics

Steven Simoens

Research Centre for Pharmaceutical Care and Pharmaco-Economics, Katholieke Universiteit Leuven,Onderwijs en Navorsing 2, P.O. Box 521, Herestraat 49, 3000 Leuven, Belgium

Correspondence should be addressed to Steven Simoens, [email protected]

Received 29 October 2010; Accepted 5 January 2011

Academic Editor: Enzo Bonmassar

Copyright © 2011 Steven Simoens. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In an era of spiraling health care costs and limited resources, policy makers and health care payers are concerned about thecost effectiveness of antibiotics. The aim of this study is to draw on published economic evaluations with a view to identify andillustrate the factors affecting the cost effectiveness of antibiotic treatment of bacterial infections. The findings indicate that thecost effectiveness of antibiotics is influenced by factors relating to the characteristics and the use of antibiotics (i.e., diagnosis,comparative costs and comparative effectiveness, resistance, patient compliance with treatment, and treatment failure) and byexternal factors (i.e., funding source, clinical pharmacy interventions, and guideline implementation interventions). Physiciansneed to take into account these factors when prescribing an antibiotic and assess whether a specific antibiotic treatment addssufficient value to justify its costs.

1. Introduction

Antibiotics have made a significant contribution to improv-ing the health of patients suffering from bacterial infections.For instance, antibiotics are commonly used in the treatmentof lower respiratory tract infections. The scientific literatureand international guidelines recommend antibiotic therapyin patients with acute exacerbations of chronic obstruc-tive pulmonary disease (COPD) and community-acquiredpneumonia (CAP) [1–3]. Also, antibiotics appear effectivein improving cure rates and decreasing duration of acutesinusitis in patients who have a microbiological diagnosisof bacterial infection or severe disease [4]. In fact, theadded value of antibiotics for therapeutic and prophylacticpurposes is so persuasive that many older antibiotics neverunderwent controlled clinical trials [5].

In an era of spiraling health care costs and limitedresources, policy makers and health care payers are also con-cerned about the cost effectiveness of antibiotics. Economicevaluation is a technique that assesses the cost effectiveness ofantibiotics by exploring whether antibiotic treatment makesa sufficient contribution to health to justify its costs. Aneconomic evaluation is defined as a comparative analysis ofat least two health technologies in terms of both their costsand outcomes [6].

Information about the cost effectiveness of antibiotictreatment of bacterial infections can be used for decision-making purposes by a variety of stakeholders [7]. Policymakers can use economic evaluation to inform the allocationof scarce health care resources. Health care payers inan increasing number of countries apply evidence aboutcost effectiveness to inform drug pricing/reimbursementdecisions (see Table 1). Antibiotics that provide better costeffectiveness are rewarded by means of a more favourableprice/reimbursement. Health care professionals can rely oneconomic evaluation to shed light on alternative approachesto treat bacterial infections. Finally, pharmaceutical com-panies can employ techniques of economic evaluation todemonstrate the cost effectiveness of their antibiotics.

A number of economic evaluations assessing the costeffectiveness of antibiotic treatment of bacterial infectionshave been published in the literature. The aim of this studyis to identify and discuss the factors that affect the costeffectiveness of antibiotics.

2. Materials and Methods

The literature review did not focus on presenting evidenceabout the cost effectiveness of antibiotics but rather drewon published economic evaluations with a view to identify

2 Chemotherapy Research and Practice

Table 1: Use of economic evaluation in drug pricing/reimbursement.

Country Organisation Implementation date

Australia Pharmaceutical Benefits Advisory Committee 1993

Belgium Medicine Reimbursement Committee 2002

England and Wales National Institute for Health and Clinical Excellence 1999

France High Health Authority 2008

Germany Institute for Quality and Efficiency in Health Care 2007

Netherlands Health Care Insurance Board 1999

New Zealand Pharmaceutical Management Agency 1993

Scotland Scottish Medicines Consortium 2002

Sweden Dental and Pharmaceutical Benefits Agency 2002

Taiwan Centre for Drug Evaluation 2008

and illustrate the factors affecting the cost effectiveness ofantibiotic treatment of bacterial infections. As such, theliterature review of economic evaluations was not systematic.

Economic evaluations were identified by searchingPubMed, Centre for Reviews and Dissemination databases(Database of Abstracts of Reviews of Effects, NationalHealth Service Economic Evaluation Database, and HealthTechnology Assessments Database), Cochrane Database ofSystematic Reviews, and EconLit up to September 2010.Additionally, the bibliography of included studies waschecked for other relevant studies. Search terms related tomultiple infection types and antibiotic classes and included“pharmaco-economics,” “economic evaluation,” “cost effec-tiveness,” “cost minimisation,” “cost utility,” and “cost bene-fit” alone and in combination with each other.

The review focused on studies published between 1995and 2010. Earlier studies were considered to be of limitedpractical relevance due to likely changes over time inantibiotic treatment modalities and in the organisation andfinancing of health care systems. Both original economicevaluations and literature reviews of economic evaluationswere included.

3. Results

The cost effectiveness of antibiotic treatment of bacterialinfections is influenced by factors relating to the character-istics and the use of antibiotics (i.e., diagnosis, compara-tive costs and comparative effectiveness, resistance, patientcompliance with treatment, and treatment failure) and byexternal factors (i.e., funding source, clinical pharmacyinterventions, and guideline implementation interventions)(see Figure 1).

3.1. Diagnosis. Diagnosing a bacterial infection is rendereddifficult by the fact that the diagnosis is generally based onpatients’ self-reported clinical symptoms. This is exemplifiedwith the case of COPD exacerbations. The diagnosis ofa COPD exacerbation is complex because exacerbationsare heterogeneous and there is debate about the definitionof an exacerbation. Furthermore, in practice, high-qualitysputum specimens are not always available [8]. This implies

that exacerbations are not always identified as such andappropriate treatment is not always administered. In fact,there is evidence that up to 50% of exacerbations arenot identified by a health care professional when using asymptom-based definition [9].

With respect to the identification of the bacterial aeti-ology, a Spanish economic evaluation showed that themost valuable treatment strategy for CAP depended on thebacterial pathogen involved and the physician needed toadapt the antibiotic treatment strategy to the cause [10].The authors concluded that amoxicillin 1 g for treating CAPwas more effective and less expensive than moxifloxacin,telithromycin, or clarithromycin if the physician was able todiscriminate clinically the bacterial aetiology. If the physicianneeded to initiate empirical treatment in the absence ofinformation about the causative pathogen and the antibioticsusceptibility pattern of the isolated organism, moxifloxacinbecame the most valuable option. However, the model oftreatment pathways in this study was necessarily simplistic,and future modelling work in this domain would benefitfrom better and more recent data on resistance.

Viruses can be mistaken for microbial pathogens andmay be treated empirically with antibiotics. For instance, twoeconomic evaluations using the same study design exploredthe cost effectiveness of moxifloxacin in the treatmentof CAP in different countries [11, 12]. Viruses were notconsidered in the base case analysis, and results indicatedthat moxifloxacin was more effective and less expensivethan alternative antibiotics. A sensitivity analysis consideredviruses with respect to the prevalence of pathogens; thestudy assumed a normalized frequency distribution of 20%for viruses, 54% for S. pneumoniae, 8% for H. influenza,and 18% for atypical pathogens. Antibiotic treatment ofpathogens including viruses reduced health care costs, therate of first-line clinical failure, and the hospitalization ratebut did not change the overall conclusions about the costeffectiveness of moxifloxacin. As these economic evaluationswere carried out from the perspective of the third-partypayer, the analyses considered health care costs only and didnot include costs due to productivity loss. The inclusion ofindirect costs would result in an even better cost effectivenessfor treatment with moxifloxacin.

Chemotherapy Research and Practice 3

Characteristics and useof antibiotics

Diagnosis

Comparative costs

Comparative effectiveness

Resistance

Patient compliance

Treatment failure

Cost-effectivenessof antibiotics

External factors

Funding source

Clinical pharmacy

Guideline implementation

Figure 1: Factors affecting the cost effectiveness of antibiotics.

3.2. Comparative Costs and Comparative Effectiveness. Thecomparative costs and comparative effectiveness of antibi-otics play a key role in determining the cost effectiveness ofantibiotic treatment of bacterial infections.

A study carried out an economic evaluation of the useof teicoplanin and vancomycin in the treatment of intensivecare unit patients with catheter-related infections [13].Comparative trials of teicoplanin and vancomycin reportedno significant differences in their efficacy [14, 15] and, hence,the authors conducted a cost minimisation analysis. In acost minimisation analysis, only costs are analysed and theleast costly treatment approach is chosen because outcomesare known to be equal between approaches. This studyelicited data about resource use based on a Delphi panelof nine experts rather than actually observing resource usein patients. Mean treatment costs per patient amounted to1,272 C with teicoplanin and 1,041 C with vancomycin. Thehigher treatment cost with teicoplanin mainly originatedfrom higher drug acquisition costs. Treatment costs ofteicoplanin and vancomycin turned out to be sensitive tochanges in drug unit costs and unit costs of serum levelmonitoring tests.

A literature review of antibiotic treatment of COPD exac-erbations focused on the comparative costs and the compar-ative effectiveness of first-generation antibiotics (aminopeni-cillins, macrolides, and tetracyclines) and second-generationantibiotics (e.g., fluoroquinolones) [16]. Fluoroquinolonesgenerally had higher acquisition costs than first-generationantibiotics. Traditionally, studies suggested that second-generation macrolides and fluoroquinolones are equallyeffective as first-generation antibiotics [17]. If this is thecase, the cost effectiveness of antibiotic treatment can bedetermined by means of a cost minimisation analysis.However, this literature was limited by the fact that mosttrials were powered to demonstrate equivalence rather thanclinical superiority, had enrolled small samples that are notalways representative of the patient population, and didnot control for concomitant therapy or for comorbidities.Also, more recent evidence suggested that management ofCOPD exacerbations with moxifloxacin or gemifloxacin isassociated with a shorter time to resolution of symptoms,a lower hospitalisation rate, and a prolonged exacerbation-free interval, thereby generating clinical benefits as well ascost savings [18, 19]. In general, there is a need for economic

evaluations to determine the cost effectiveness of treatingCOPD exacerbations by comparing the comparative costs ofantibiotics with their comparative effectiveness.

3.3. Resistance. When antibiotics first became available,changes in the susceptibility of pathogens were of littleconcern. However, inappropriate use of antibiotics, (human-to-human) clonal spread of multidrug-resistant strains, andthe presence of comorbidities have all contributed to therise in resistance over the years. Resistance to antibioticscan have a substantial impact on outcomes and costs oftreatment. For instance, there is evidence that CAP patientswith pneumococcal resistance may be at greater risk ofpoor outcomes [20]. Also, if first-line treatment fails due toresistance, additional costs are incurred due to the need forsecond-line treatment or hospitalization, or both.

Using evidence from four economic evaluations ofantibiotic treatment of mild-to-moderate CAP in Belgium,Canada, France, Spain, and the United States [10–12, 21], itis possible to examine the impact of resistance on the costeffectiveness of antibiotics. The studies employed a similarstudy design; decision-analytic models evaluated the costeffectiveness of oral antibiotics from the third-party payerperspective, with first-line treatment being initiated in thecommunity and failure resulting in second-line treatment inthe community or hospitalization. The first-line interventionwas moxifloxacin in each study. Comparator treatments werebeta-lactams (e.g., coamoxiclav, cefuroxime), macrolides(e.g., clarithromycin, azithromycin), or tetracyclines (e.g.,doxycycline). Effectiveness was assessed in terms of the rate offirst-line clinical failures, of second-line treatments required,of hospitalizations required, and of mortality.

The impact of resistance on the cost effectiveness ofantibiotics was investigated in two ways. First, sensitivityanalyses examined the impact of various resistance rates forS. pneumoniae and H. influenzae on the cost effectivenessof antibiotics. Second, results on cost effectiveness can becompared between economic evaluations and thus betweencountries with different levels of resistance; Germany hasa low level of resistance in CAP pathogens [22]; Belgium,Canada, and the United States have an intermediate levelof resistance [23–25]; France and Spain have a high level ofresistance [22]. However, it should be noted that factors otherthan resistance may explain differences in results between


these economic evaluations (e.g., costs of care, treatmentprotocols).

The sensitivity analyses and the comparison betweencountries indicated that varying levels of resistance in CAPpathogens and multidrug resistance in S. pneumoniae isolatesaffected costs and clinical outcomes of antibiotic treatment[10–12]. However, conclusions did not change; treatment ofCAP with moxifloxacin was more effective and less expensivethan other antibiotic treatment strategies in Belgium, France,Germany, Spain, and the United States. At the moment,worldwide resistance of CAP pathogens to moxifloxacin islow [26] but continued vigilance with regard to the evolutionof resistance and its impact on the cost effectiveness ofmoxifloxacin and of other antibiotics is indicated.

In Canada, the sensitivity analysis showed that a 50%increase in fluoroquinolone resistance would decrease thecost effectiveness of moxifloxacin treatment as comparedwith azithromycin to CAN$ 101.47 per first-line clinicalfailure avoided [21]. Canada has faced a steady increase inmacrolide resistance in S. pneumoniae over time [23], andfurther increases in macrolide resistance rates cannot beruled out. Increases in macrolide resistance would improvethe cost effectiveness of treatment with moxifloxacin.

3.4. Patient Compliance. The cost effectiveness of antibiotictreatment also depends on patient compliance, with com-pliance being affected by the frequency of dosing, durationof treatment, adverse events, ease of administering drugs,ease of packaging, and price [27]. An economic evaluationof antibiotic treatment quantified patient compliance; ratesof compliance defined as an intake of at least 80% of theprescribed dose varied between 76% and 83% [28]. Variousstrategies to enhance patient compliance with antibiotictreatment have been proposed such as patient education,once-daily dosing schedules, a convenient and acceptableform of medication, easy-to-open packaging, and the choiceof an antibiotic with few side effects [27].

3.5. Treatment Failure. Resistance to antibiotics and patientcompliance may influence the cost effectiveness of antibioticsbecause they may lead to treatment failure and further antibi-otic treatment or hospitalisation. For instance, a literaturereview of the distribution of health care costs of COPDexacerbations found that hospitalization costs accounted formore than 45% of health care costs and drugs costs madeup between 6% and 21% of costs [16]. As hospitalizationis generally indicative of treatment failure, these estimateshighlight the cost effectiveness that can be attained frommore effective antibiotics that allow patients to be managedin primary care and that prevent treatment failure andhospitalization. In other words, if a new antibiotic wouldhave a lower failure rate than alternative treatments, it wouldbe likely to be cost effective, even if it is more expensive thanother antibiotics.

Treatment failure may be caused by a number of hostfactors. The literature suggests that frequency of exacerba-tions, presence of comorbidities, impairment in lung func-tion, need for more aggressive bronchodilator therapy, andprevious hospitalization predict treatment failure [29, 30].

The ability to identify patients at a higher risk of failingtreatment can aid clinicians in their choice of antibiotic. Thisimplies that it may be advisable to identify patient subgroupsin which treatment with a specific antibiotic provides the bestcost effectiveness and should be recommended by guidelines.

3.6. Funding Source. A recent study extracted the cost effec-tiveness of antibiotics from economic evaluations includedin the Tufts-New England Center Cost Effectiveness AnalysisRegistry through September 2009 [31]. The analysis included85 observations on the cost effectiveness of antibioticsderived from 23 economic evaluations. Economic eval-uations related to infectious diseases (58% of studies),respiratory diseases (13%), cardiovascular diseases (9%),critical care (4%), endocrine disorders (4%), genitor-urinarydiseases (4%), musculoskeletal and rheumatologic diseases(4%), and sensory organ diseases (4%). The results indicatedthat the median incremental cost effectiveness ratio of antibi-otics was 748 C per quality-adjusted life year. Specifically,38.8% of antibiotics were more effective and less costly thanthe comparator; 45.9% of antibiotics improved effectivenessbut also increased costs; 15.3% of antibiotics were lesseffective and more costly than the comparator.

The cost effectiveness of antibiotics derived from analysesfunded by industry tended to be better than the cost effective-ness derived from analyses funded from other sources (e.g.,government, foundations). However, the limited number ofobservations implied that it was not possible to statisticallytest for this association. Also, there were too few observationsto explore whether there was an association between themethodological quality of economic evaluations and thefunding source. The possible association between cost effec-tiveness and funding source may have several explanations;industry influences the design of economic evaluations witha view to improving the cost effectiveness of their products;as costs of research and development are high, industrymarkets those antibiotics that are cost effective only; industrysponsors economic evaluations of antibiotics that are likelyto be cost effective only; researchers conduct and journaleditors publish those economic evaluations that support thecost effectiveness of antibiotics. In response to the possiblemanipulation of studies, professional societies and healthcare payers are increasingly issuing guidelines for the conductand reporting of economic evaluations.

3.7. Clinical Pharmacy. During the last decades, clinicalpharmacy services have developed around the world [32].Even if there exists no consensus concerning the term“clinical pharmacy,” clinical pharmacy can be defined asthe contribution of pharmacists and their assistants to drugtherapy as a part of the total care supplied to patients, incooperation with physicians and nursing staff, with a viewto optimizing the cost effectiveness, the effectiveness, and thesafety of drug therapy.

A literature review examined the cost effectiveness ofclinical pharmacy interventions focusing on the manage-ment of antibiotic therapy in a hospital setting [33]. Extract-ing evidence from six economic evaluations, the authorsconcluded that clinical pharmacy interventions relating to


antibiotic therapy can lower costs of hospital care withoutadversely affecting clinical outcomes. Lower costs arose froma decrease in drug costs (e.g., due to switch from intravenousto oral drugs), lower pharmacy costs, and a decrease in lengthof stay. However, economic evaluations of clinical pharmacyinterventions suffered from a number of methodologicallimitations relating to the absence of a control group withoutclinical pharmacy interventions; limited scope of costs andoutcomes; focus on direct healthcare costs only; exclusion ofpharmacist employment cost; use of intermediate outcomemeasures; exclusion of health benefits; absence of incremen-tal analysis.

3.8. Guideline Implementation. Numerous guidelines havebeen published governing appropriate antibiotic treatmentof bacterial infections. Interventions surrounding the imple-mentation of guidelines may have an impact on health careprofessional compliance with guidelines and, hence, mayinfluence the cost effectiveness of antibiotics.

A literature review evaluated the cost effectiveness ofantibiotic treatment consistent with guidelines for patientswith CAP [34]. This literature indicated that antibiotictreatment consistent with guidelines reduced length of stay,decreased costs, and reduced the mortality rate. How-ever, existing studies suffered from methodological limita-tions, and high-quality economic evaluations examining theimpact of guideline implementation interventions on thecost effectiveness of antibiotic treatment are needed.

4. Conclusions

This study has identified and discussed the factors that affectthe cost effectiveness of antibiotics. The findings indicate thatthe cost effectiveness of antibiotics is influenced by factorsrelating to the characteristics and the use of antibiotics (i.e.,diagnosis, comparative costs and comparative effectiveness,resistance, patient compliance with treatment, and treatmentfailure) and by external factors (i.e., funding source, clini-cal pharmacy interventions, and guideline implementationinterventions). Physicians need to take into account thesefactors when prescribing an antibiotic and assess whether aspecific antibiotic treatment adds sufficient value to justifyits costs. Finally, it should be noted that cost effectivenessis only one of the factors and not necessarily the mostimportant factor informing the choice of physicians betweenantibiotics. Other factors that need to be taken into accountinclude, for example, route of administration, patient profile,and the occurrence of adverse events.

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Research Article

Antibacterial and Antioxidant Propertiesof the Methanolic Extract of the Stem Bark ofPteleopsis hylodendron (Combretaceae)

Aristide Laurel Mokale Kognou,1 Rosalie Annie Ngono Ngane,2 Jules Roger Kuiate,1

Martin Luther Koanga Mogtomo,2 Alembert Tchinda Tiabou,3

Raymond Simplice Mouokeu,1 Lucie Biyiti,4 and Paul Henri Amvam Zollo4

1 Laboratory of Microbiology and Antimicrobial Substances, University of Dschang, P.O. Box 67 Dschang, Cameroon2 Department of Biochemistry, University of Douala, P.O. Box 24157 Douala, Cameroon3 Laboratory of Phytochemistry, Institute of Medical Research and Medicinal Plants Study,Ministry of Scientific Research and Innovation, P.O. Box 6163, Yaounde, Cameroon

4 Department of Biochemistry, University of Yaounde I, P.O. Box 812 Yaounde, Cameroon

Correspondence should be addressed to Rosalie Annie Ngono Ngane, [email protected]

Received 27 October 2010; Accepted 17 January 2011

Academic Editor: Athanassios Tsakris

Copyright © 2011 Aristide Laurel Mokale Kognou et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Pteleopsis hylodendron (Combretaceae) is used in Cameroon and West Africa folk medicine for the treatment of various microbialinfections (measles, chickenpox, and sexually transmitted diseases). The antibacterial properties of the methanolic extract andfractions from stem bark of Pteleopsis hylodendron were tested against three Gram-positive bacteria and eight Gram-negativebacteria using Agar-well diffusion and Broth microdilution methods. Antioxidant activities of the crude extract and fractions wereinvestigated by DPPH radical scavenging activity and β-carotene-linoleic acid assays. The methanolic extract and some fractionsexhibited antibacterial activities that varied between the bacterial species (ID = 0.00–25.00 mm; MIC = 781–12500 μg/mL and0.24–1000 μg/mL). The activity of the crude extract is, however, very weak compared to the reference antibiotics (MIC = 0.125–128 μg/mL). Two fractions (FE and FF) showed significant activity (MIC = 0.97 μg/mL) while S. aureus ATCC 25922 was almostresistant to all the tested fractions. In addition, the crude extract and some fractions showed good antioxidant potential withinhibition values ranging from 17.53 to 98.79%. These results provide promising baseline information for the potential use of thisplant as well as some of the fractions in the treatment of infectious diseases and oxidative stress.

1. Introduction

Since the successive introduction of various antibiotics intotherapeutics, the sensitivity of pathogenic microorganismschanged a lot so that the proportion of antibiotically resistantstrains is currently important [1], what involves an increasein seriousness of infectious diseases as gastroenteritis (GE)which are a problem of public health on a worldwide scalebut especially in Africa [2]. Diarrhea, its main characteristicis a major cause of morbidity and mortality among childrenin developing countries. According to the World HealthOrganization (WHO), there are more than 2 million deaths

per year [3]. Moreover, therapy with synthetic antibioticsis not always possible because of their high cost as well astoxicity due to their extended use. To overcome this problem,people in developing countries use preparations obtainedfrom plants following folk tradition for their primary healthcare because of low cost with little or no undesirable sideeffects [4]. The plants represent a potential and almostinexhaustible source of new anti-infective compounds [5]and many of them are used to treat GE effectively [6].

Pteleopsis hylodendron Mildbr. belongs to the familyCombretaceae commonly found in the forest regions of Westand Central Africa. The genus Pteleopsis is represented in


Africa by ten species but only P. hylodendron is found inCameroon [7]. The aqueous decoction of the stem barkof P. hylodendron is used to treat measles, chickenpox,sexually transmitted diseases, GE, female sterility, liver andkidney disorders, as well as dropsy [8]. Phytochemical,antimicrobial, toxicity and antioxidant works have beenpreviously reported of this plant [9, 10]. In the same logic,we have analysed the stem bark of Pteleopsis hylodendron andreport here the antibacterial activity on pathogenic bacteriaof the gastro-intestinal tract and antioxidant activity of thecrude extract and fractions.

2. Material and Methods

2.1. Plant Material. The stem bark of P. hylodendron wascollected in February 2009 at Mbeyengue I, Centre region ofCameroon. Identification was done at the National Herbar-ium in Yaounde, Cameroon, where a voucher specimen (No1309/SFRK) has been deposited.

2.2. Microorganisms. Five bacterial strains and six isolatesknown to be pathogenic of the gastro-intestinal tract wereused in this work. These included three Gram+ bacteria(Enterococcus faecalis ATCC 10541, Staphylococcus aureusATCC 25922 and Staphylococcus aureus) and eight Gram−

bacteria (Escherichia coli ATCC 11775, Escherichia coli,Proteus mirabilis, Pseudomonas aeruginosa ATCC 27853,Salmonella paratyphi A, Salmonella paratyphi B, Salmonellatyphi ATCC 6539, and Shigella flexeneri). The bacterialisolates were obtained from Centre Pasteur of Yaounde,Cameroon, while the reference strains were obtained fromAmerican Type Culture Collection (ATCC). The bacterialstrains and isolates were grown at 35◦C and maintained onnutrient agar. The bacterial cell suspension was prepared at1.5 × 108 colony forming units per mL (CFU/mL) followingthe McFarland 0.5 turbidity standard.

2.3. Extraction and Fractionation. The air-dried and pow-dered stem bark of Pteleopsis hylodendron (2.5 kg) wasextracted with MeOH (8 L, 72 h) at room temperatureto obtain a crude extract (590 g) after evaporation undervacuum. A portion of this extract (100 g) was subjectedto silica gel column chromatography (Ø8 cm × L30 cm)eluted successively with pure hexane, hexane-EtOAc (90 : 10–30 : 70), pure EtOAc, EtOAc-MeOH (95 : 5–80 : 20) and pureMeOH. Forty-six fractions of 500 mL each were collected andcombined based on their TLC profile into ten major fractionsA–J (A: 2-3, B: 4–6, C: 7–13, D: 14–16, E: 17–21, F: 22–28, G:29–35, H: 36-37, I: 38–44, J: 45–46).

2.4. Antibacterial Assays

2.4.1. Agar-Well Diffusion Method. Diameters of inhibitionzones (ID) were determined using Mueller Hinton Agar(MHA) by the well diffusion method [11]. The bacterialsuspension (100 μL) was homogeneously seeded onto Petridishes containing sterile molten MHA (20 mL). The sterile6 mm diameter wells were impregnated (50 μL) with dif-ferent concentrations of plant extract (10, 5, and 2.5 mg–200, 100, and 50 μg/mL). The dishes were kept for 1 h at

room temperature for the diffusion of the extract. Subse-quently, dishes were incubated at 35◦C for 24 h. Antibiotics(Amoxicillin, Ciprofloxacin and Gentamicin) were used aspositive control (10 μg–200 μg/mL) and 10% aqueous DMSOwas used as negative control. Results were evaluated bymeasuring the inhibition zones around each well. The assaywas done in triplicate and the mean diameters recorded asinhibition zones. We considered that an extract is activewhen ID was up to 20 mm, and then the strain is knownas sensitive; moderately active when ID was between 10 and20 mm, and then the strain is known as moderate; little ornot active when ID was between 0 and 10 mm, and then thestrain is known as little sensitive or resistant [12, 13].

2.4.2. Broth Microdilution Method. Minimum inhibitoryconcentrations (MICs) were determined using Mueller Hin-ton Broth (MHB) by microdilution method [14]. A two-fold serial dilution of the crude extract (12.50–0.024 mg/mL)and fractions (1000–1.953μg/mL and 500–0.242μg/mL).A negative control (10%, v/v aqueous DMSO, mediumand inoculum) and positive control (10%, v/v aqueousDMSO, medium, inoculum and water-soluble antibiotics)were included. Each well of 96-well sterile microtitre platereceived 100 μL of MHB, 100 μL of test substances and100 μL of the bacterial inoculum (1.5 × 108 CFU/mL). Theplates were covered and incubated at 35◦C for 24 h. As anindicator of bacterial growth, 50 μL p-iodonitrotetrazoliumviolet (INT) dissolved in water was added to the wells andincubated at 35◦C for 30 min. MIC values are recorded asthe lowest concentration of the substance that completelyinhibited bacterial growth that is, the solution in thewell remained clear after incubation with INT. Minimumbactericidal concentrations (MBCs) were determined byplating 10 μL from each negative well and from the positivegrowth control on Mueller Hinton Agar. MBCs were definedas the lowest concentration yielding negative subcultures.The experiments were performed in triplicate. Amoxicillin,ciprofloxacin and gentamicin at the concentration rangingbetween 128 and 0.062 μg/mL served as positive control.

2.5. Antioxidant Assay

2.5.1. DPPH Assay. The free radical scavenging activity of theextract and fractions on the stable radical DPPH wereestimated by the method of Mensor et al. [15]. 1.5 mL of amethanol solution of sample test at different concentrations(10, 50, 100, 500, and 1000 μg/mL) was mixed with a 0.3 mMDPPH methanol solution and kept for 30 min at roomtemperature. The decrease in the solution absorbance, dueto proton donating of substances was measured at 517 nm.L-Ascorbic acid was used as positive control. The percentageof DPPH radical scavenging activity was calculated using thefollowing formula:

DPPH radical scavenging activity (%)

=⎡⎣(

Acontrol −Asample test

)

Acontrol

⎤⎦ × 100.

(1)


2.5.2. β-Carotene-Linoleic Acid Assay. In this assay antioxi-dant capacity is determined by measuring the inhibition ofthe volatile organic compounds and the conjugated dienehydroperoxides arising from linoleic acid oxidation [16].A stock solution of β-carotene-linoleic acid mixture wasprepared as follows: 1.5 mg β-carotene was dissolved in 3 mLof chloroform, and 75 μL linoleic acid and 600 mg tween 40were added. Chloroform was completely evaporated using avacuum evaporator. Then 150 mL distilled water saturatedwith oxygen was added with a vigorous shaking. 1340 μL ofthis reaction mixture was dispersed to test tubes and 160 μLextract (20 mg/mL) were added, and emulsion system wasincubated at 55◦C for 105 min. Same procedure was repeatedwith L-Ascorbic acid used as a standard control and a blank.After this incubation period absorbance of the mixtures weremeasured at 492 nm. Antioxidative capacity of the extractwas compared with those of ascorbic acid and blank.

2.6. Phytochemical Screening. Chemical tests were carriedout on the methanolic extract and fractions using standardprocedures to identify the constituents (alkaloids, antho-cyanins, anthraquinones, coumarins, flavonoids, glycosides,phenols, polyphenols, saponins, tannins, triterpenes, andsterols) as described by Brunetton [17].

2.7. Statistical Analysis. Data were expressed as mean ±standard deviation. Statistical analysis was carried out usingthe Waller-Duncan’s test. The 12.0 SPSS Windows softwarewas used for this analysis. Differences were consideredsignificant at P < .05.

3. Results

3.1. Extraction and Fractionation. The % yield of methanolicextract of P. hylodendron was 15.96%. FI (43.08%) and FJ

(24.54%) were the most abundant.

3.2. Phytochemical Screening. Phytochemical screening re-vealed the presence of medicinally active constituents. Thedifferences in the composition between crude extract andfractions and between fractions were noted. Except FA, allother substances contained at least one chemical group. Alka-loids, anthocyanins, anthraquinones, flavonoids, glycosides,phenols, polyphenols, saponins, and tannins were present incrude extract while coumarins, sterols, and triterpenes wereabsent. FB, FC, and FD had a similar chemical composition(alkaloids). It is the same for FG and FH (alkaloids, antho-cyanins, anthraquinones, flavonoids, phenols, polyphenols,and tannins); FI and FJ (flavonoids, glycosides, phenols,polyphenols, saponins, and tannins).

3.3. Antibacterial Activity. The results of the antibacterialactivity by the Agar-well diffusion method are presentedin Table 1. At the three concentrations of the methanolicextract tested, ID ranged from 0.00 to 25.00 mm for allthe bacteria 15.00–25.00 mm for the isolates 0.00–22.00 mmfor the Gram−, and 10.87–25.00 mm for the Gram+. S.aureus was the most sensitive (ID = 20.00–25.00 mm) while

S. aureus ATCC 25922 and E. coli ATCC 11775 were the leastsensitive (ID = 11.00–15.00 mm and 10.00–14.75 mm resp.).No activity was recorded at 2.5 mg against P. aeruginosaATCC 27853. However these values are weak compared withthose of the reference antibiotics (ID = 12–40 mm).

In view of the results obtained by diffusion method, MICand MBC values of the crude extract and fractions wereestablished and the results are shown in Tables 2 and 3.

All the bacteria tested were inhibited by the methanolicextract (Table 2) with MIC ranging from 781–12500 μg/mLfor all the bacteria, isolates, and Gram−; 781–3125 μg/mLfor the strains and Gram+. S. paratyphi A was the leastsensitive (MIC = 12500 μg/mL). P. aeruginosa ATCC 27853,P. mirabilis and S. aureus were the most sensitive (MIC =781 μg/mL). The important activity on S. aureus confirmsthe best activity obtained in solid medium; which revealedthis germ as one of the most susceptible. Antibiotics exerteda higher inhibitory effect on bacterial (MIC = 0.125–128 μg/mL) than the methanolic extract.

The fractionation of the methanolic extract showed aninactivity of FA, FB, FC, and FD on all the bacteria tested(Table 3). On the contrary, FE and FF saw their activityincreasing significantly. Indeed, on five of the eleven bacteria(E. coli, P. mirabilis, S. paratyphi B, E. faecalis ATCC 10541, S.aureus), MIC was 0.97 μg/mL, making the substances moreactive than reference antibiotics. FG had a fairly good activity(MIC = 0.24 μg/mL) on some bacteria (P. aeruginosa ATCC27853, E. faecalis ATCC 10541, S. aureus) whereas FH, FI

and FJ were slightly active (MIC = 125–1000μg/mL). S.aureus ATCC 25922 was almost resistant to all the fractions(MIC > 1000 μg/mL). The MBC/MIC ratio activity for allthe bacteria tested varied between one (1) and eight (8) forthe crude extract and between one (1) and twenty (20) forthe fractions. According to Marmonier [18], plant extractand fractions exerted two types of activities: a bacteriostatic(MBC/MIC ≥ 4) and bactericidal activity (MBC/MIC ≤4). Methanolic extract and fractions of P. hylodendron werebactericidal on at least 63% and 27% of the bacteriarespectively.

3.4. Antioxidant Activity. The antioxidant activity of themethanolic extract and fractions was assessed by the DPPHand β-carotene-linoleic acid assays. The results are presentedin Tables 4 and 5. Activity increased in a concentration-dependant manner compared to L-ascorbic acid (positiveantioxidant control). At the concentrations of 500 and1000 μg/mL the methanolic extract, FE, FF, FG, FH, FI, andFJ showed a similar activity to that one of L-ascorbic acid.FH was the most antioxidant fraction (94.05–98.79%) whileFA was the least antioxidant (1.86–21.26%).

4. Discussion and Conclusion

Phytochemical screening of the methanolic extract andfractions of P. hylodendron revealed the presence alkaloids,anthocyanins, anthraquinones, flavonoids, glycosides, phe-nols, polyphenols, saponins and tannins. Other investigators[19, 20] have reported the presence of these components in


Table 1: ID (mm) of the methanolic extract of P. hylodendron.

BacteriaCrude extract (mg/mL) Reference antibiotic (μg/mL)

P. hylodendron Amoxicillin Ciprofloxacin Gentamicin

200 100 50 200

Gram− bacteria

E. coli ATCC 11775 14.75± 0.17f 12.25± 0.17h 10.00± 0.00g 19.00± 0.26d 28.25± 0.17f 27.00± 0.26f

E. coli 21.75± 0.13b 19.00± 0.00c 16.75± 0.17c 40.00± 0.00a 27.75± 0.17f 26.00± 0.00h

P. aeruginosa ATCC 27853 18.62± 0.27d 13.75± 0.17f 0.00± 0.00h 14.50± 0.42g 32.50± 0.17d 30.00± 0.00d

P. mirabilis 20.12± 0.12c 18.00± 0.00d 16.00± 0.00d 16.00± 0.00f 33.00± 0.00c 33.25± 0.17a

S. flexneri 21.75± 0.17b 19.75± 0.17b 17.50± 0.18b 20.00± 0.00c 36.50± 0.42a 32.00± 0.00b

S. paratyphi A 20.25± 0.17c 17.00± 0.00e 15.00± 0.00e 12.00± 0.00h 30.00± 0.00e 28.00± 0.00c

S. paratyphi B 19.75± 0.17c 17.62± 0.12d 15.12± 0.12e 14.25± 0.17g 35.00± 0.00b 28.25± 0.17e

S. typhi ATCC 6539 22.00± 0.00b 20.00± 0.00b 16.87± 0.12c 12.00± 0.00h 32.33± 0.25d 32.00± 0.00b

Gram+ bacteria

E. faecalis ATCC 10541 16.00± 0.00e 13.25± 0.17g 10.87± 0.12f 18.00± 0.00e 32.00± 0.00d 31.00± 0.00c

S. aureus ATCC 25922 15.00± 0.00f 13.75± 0.17f 11.00± 0.00f 20.75± 0.17c 35.50± 0.18c 30.25± 0.17cd

S. aureus 25.00± 0.00a 22.00± 0.00a 20.00± 0.00a 35.00± 0.00b 26.00± 0.00h 26.75± 0.24fh

a,b,c,d,e,f,g,hIn the same column, values carrying different letters in superscript are significantly different at P ≤ .05 (Waller Duncan test).Diameters of inhibition zones (ID).

Table 2: MIC and MBC (μg/mL) of the methanolic extract of P. hylodendron.

Crude extract Reference antibiotics

Bacteria P. hylodendron Amoxicillin Ciprofloxacin Gentamicin

MIC MBC MBC/MIC MIC MBC MBC/MIC MIC MBC MBC/MIC MIC MBC MBC/MIC

Gram− bacteria

E. coli ATCC 11775 3125 12500 4 128 — — 4 4 1 16 128 8

E. coli 1562 — — 1 1 1 8 8 1 1 1 1

P. aeruginosa ATCC 27853 781 781 1 128 — — 1 16 16 8 16 2

P. mirabilis 781 6250 8 1 8 8 1 1 1 2 16 8

S. flexneri 3125 — — 32 64 2 0.25 1 4 0.25 0.25 1

S. paratyphi A 12500 — — — — — 0.125 0.5 4 2 2 1

S. paratyphi B 1562 6250 4 1 8 8 0.5 2 4 2 16 8

S. typhi ATCC 6539 1562 6250 4 — — — 0.25 2 8 32 128 4

Gram+ bacteria

E. faecalis ATCC 10541 1562 6250 4 1 1 1 4 16 4 1 1 1

S. aureus ATCC 25922 3125 6250 2 — — — 8 8 1 4 16 4

S. aureus 781 1562 2 1 — — 8 8 1 0.25 0.25 1

—: 12500 μg/mL for the extract and >128μg/mL for the reference antibiotics.

the Combretaceae family to which belongs the studied plant.However, Ngounou et al. [21] and Atta-Ur-Rahman et al.[22] working on the stem bark of P. hylodendron collectedfrom East region of Cameroon revealed the presence oftriterpenes which were absent in our sample. This differencecan be attributed to the difference in the geographical region,soil composition, and age of the plant [17].

The antimicrobial activities of Pteleopsis species werereported [19, 23]. Generally, the methanolic extract andsome fractions of the stem bark of P. hylodendron showedvariable antibacterial activities dose-dependant on the elevenbacterial strains and isolates tested. These broad spectraof action could be related to their chemical components[24]. Among these compounds, tannins induce an important

antimicrobial activity because they have an ability to inacti-vate microbial adhesions, enzymes, cell envelope transportproteins, and so forth, [25]. Due to their ability to bindto proteins and metals, tannins also inhibit the growth ofmicroorganisms through substrate and metal ion depriva-tion [26]. However, differences in chemical compositionrecorded between the crude extract and some fractions mayexplain their different degree of antimicrobial properties.Also, the amount of the active components in the crudeextract may be diluted and fractionation may have increasedtheir concentrations, thus the activities in the fractions [27].Moreover, the differences in susceptibility may be explainedby the differences in cell wall composition and/or geneticcontent of plasmids that can be easily transferred among


Table 3: MIC and MBC (μg/mL) of the fractions from chromatography separation of P. hylodendron.

Bacteria ParametersFractions Reference antibiotics

FA FB FC FD FE FF FG FH FI FJ Amox Cipro GentaGram− bacteria

E. coli ATCC 11775MIC — — — — 500 — 62.5 500 1000 1000 128 4 16MBC — — — — — — — — — — — 4 128

MBC/MIC — — — — — — — — — — — 1 8

E. coliMIC — — — — 0.97 0.97 62.5 500 1000 1000 1 8 1MBC — — — — — — 250 — — — 1 8 1

MBC/MIC — — — — — — 4 — — — 1 8 1

P. aeruginosa ATCC 27853MIC — — — — — — 0.24 1000 1000 1000 128 1 8MBC — — — — — — 250 — — — — 16 16

MBC/MIC — — — — — — 20 — — — — 16 2

P. mirabilisMIC — — — — 0.97 0.97 — 250 500 1000 1 1 2MBC — — — — 125 62.5 — — — — 8 1 16

MBC/MIC — — — — 14 12 — — — — 8 1 8

S. flexneriMIC — — — — — — — 250 500 1000 32 0.25 0.25MBC — — — — — — — — — — 64 1 0.25

MBC/MIC — — — — — — — — — — 2 4 1

S. paratyphi AMIC — — — — — — 500 1000 1000 — — 0.125 2MBC — — — — — — — — — — — 0.5 2

MBC/MIC — — — — — — — — — — — 4 1

S. paratyphi BMIC — — — — 0.97 0.97 500 500 500 1000 1 0.5 2MBC — — — — — — — — — — 8 2 16

MBC/MIC — — — — — — — — — — 8 4 8

S. typhi ATCC 6539MIC — — — — — — 500 1000 — — — 0.25 32MBC — — — — — — — — — — — 2 128

MBC/MIC — — — — — — — — — — — 8 4Gram+ bacteria

E. faecalis ATCC 10541MIC — — — — 0.97 0.97 0.24 500 1000 1000 1 4 1MBC — — — — 0.97 250 0.24 — — — 1 16 1

MBC/MIC — — — — 1 16 1 — — — 1 4 1

S. aureus ATCC 25922MIC — — — — — — — 500 — — — 8 4MBC — — — — — — — — — — — 8 16

MBC/MIC — — — — — — — — — — — 1 4

S. aureusMIC — — — — 0.97 0.97 0.24 500 125 250 1 8 0.25MBC — — — — — — 0.97 — — — — 8 0.25

MBC/MIC — — — — — — 4 — — — — 1 1

F: fraction; Amox: amoxicillin; Cipro: ciprofloxacin; Genta: gentamicin.—: >1000 μg/mL for the fractions, and >128 μg/mL for the reference antibiotic.

strains [28]. MIC values obtained from the extract by micro-dilution method revealed that S. aureus is the most sensitive.It was reported [29] that S. aureus is one of the mostsusceptible bacteria to the plant extracts. These values alsoshowed that the Gram− and Gram+ bacteria had a compara-ble susceptibility. This may suggest that the mode of actionof the extract was not related to the cell wall composition. S.aureus ATCC 25922 which was inhibited completely by themethanolic extract at 3125 μg/mL, was almost resistant to allthe fractions. This may suggest that this microbe required

high concentrations of the substance tested and synergiceffect of chemical compounds as extract. FA, FB, FC, and FD

containing only alkaloids did not show any inhibitory effecton the bacteria tested. This may suggest these compoundswhich also present in the methanolic extract do not havea detectable antibacterial activity. However, alkaloids werereported to possess antibacterial activities [30]. FE andFF, most active had a comparable chemical compositionthat FG. Differences in activity between these fractionscould be related to the absence of anthocyanins in FF and


Table 4: Antioxidant potential of the crude extract and fractions of P. hylodendron and L- ascorbic acid in DPPH assay.

% inhibition concentration (μg/mL)Substances tested 1000 500 100 50 10

FA 21.26± 0.24f 19.69± 0.17i 11.83± 0.47ef 05.28± 0.26j 1.86± 0.17m

FB 22.40± 0.94f 18.80± 0.10j 09.19± 0.20f 08.83± 0.34i 04.20± 0.17l

FC 42.58± 0.50e 28.16± 0.29h 12.31± 0.41e 10.87± 0.38h 08.65± 0.00k

FD 67.98± 0.72d 52.13± 0.10g 20.24± 0.45d 17.83± 0.28g 10.81± 0.37j

FE 95.73± 0.26bc 94.95± 0.00d 94.05± 0.14a 91.77± 0.33cd 26.30± 0.20h

FF 95.37± 0.10bc 94.65± 0.10d 93.75± 0.10a 93.33± 0.14ab 83.42± 0.48d

FG 97.83± 0.37ab 95.55± 0.17c 94.65± 0.17a 92.79± 0.29bc 91.89± 0.28b

FH 98.79± 0.33ab 96.21± 0.13b 94.47± 0.26a 94.29± 0.26a 94.05± 0.20a

FI 95.49± 0.14bc 94.83± 0.17d 93.93± 0.33a 92.67± 0.38bc 70.08± 0.64f

FJ 95.85± 0.00bc 95.13± 0.14cd 93.57± 0.30a 93.21± 0.17ab 74.23± 0.14e

PH 95.13± 0.14bc 93.87± 0.28e 93.39± 0.10a 91.35± 0.14d 89.42± 0.26c

ASC 100.00± 0.00a 100.00± 0.00a 87.36± 0.00b 53.23± 0.00e 29.42± 0.00g

F: fraction; PH: methanolic extract of P. hylodendron; ASC: L-ascorbic acid.a,b,c,d,e,f,g,h,i,j,k,l,mIn the same column, values carrying different letters in superscript are significantly different at P ≤ .05 (Waller Duncan test).

Table 5: Antioxidant potential of the crude extract of P. hylodendron and L-ascorbic acid in β-carotene-linoleic acid assay.

% inhibition concentration (μg/mL)Substances tested 1000 500 100 50 10

PH 38.03± 0.25b 31.83± 0.16a 31.80± 0.00a 30.47± 0.08a 27.73± 0.08a

ASC 56.48± 0.05a 27.73± 0.02b 20.20± 0.00b 19.86± 0.00b 17.81± 0.02b

PH: methanolic extract of P. hylodendron; ASC: L-ascorbic acid.a,bIn the same column, values carrying different letters in superscript are significantly different at P ≤ .05 (Waller Duncan test).

anthraquinones in FE. Generally, it is difficult at the sightof results of the phytochemical screening to attribute theactivities recorded to a chemical compounds group.

As L-ascorbic acid, the methanolic extract and somefractions showed great antioxidant potentials. This partic-ularly high activity could be attributed to the presenceof phenolic compounds [31]. The antioxidant activity ofphenolic compounds is mainly due to their redox properties,which can play an important role in neutralizing freeradicals, quenching singlet and triplet oxygen species, ordecomposing peroxides [32]. Numerous studies have sug-gested flavonoids, anthraquinones, anthocyanins and tan-nins [33, 34] for antioxidant activity. Previous phytochemicalinvestigations on this plant have reported the presence ofellagic acid derivatives as antioxidant source [22].

These results provide promising baseline informationfor the potential use of this plant as well as some of thefractions in the treatment of GE and oxidative stress. FE andFF by their high antibacterial activity could be the base ofdevelopment of new antibacterial agents with broad spectra.Their purification and pharmacological and toxicity studiesare essential.

Acknowledgment

This work was supported by AIRES-Sud, a programme fromthe French Ministry of Foreign and European Affairs imple-mented by the “Institut de Recherche pour le Developpement(IRD-DSF)”.

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Research Article

Susceptibility of Bifidobacteria of Animal Origin toSelected Antimicrobial Agents

Sigrid Mayrhofer, Christiane Mair, Wolfgang Kneifel, and Konrad J. Domig

Department of Food Sciences and Technology, Institute of Food Sciences, BOKU-University of Natural Resources and Life Sciences,Muthgasse 18, 1190 Vienna, Austria

Correspondence should be addressed to Konrad J. Domig, [email protected]

Received 30 November 2010; Accepted 28 January 2011

Academic Editor: Spyros Pournaras

Copyright © 2011 Sigrid Mayrhofer et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Strains of the genus Bifidobacterium are frequently used as probiotics, for which the absence of acquired antimicrobial resistancehas become an important safety criterion. This clarifies the need for antibiotic susceptibility data for bifidobacteria. Based on arecently published standard for antimicrobial susceptibility testing of bifidobacteria with broth microdilution method, the rangeof susceptibility to selected antibiotics in 117 animal bifidobacterial strains was examined. Narrow unimodal MIC distributionseither situated at the low-end (chloramphenicol, linezolid, and quinupristin/dalfopristin) or high-end (kanamycin, neomycin)concentration range could be detected. In contrast, the MIC distribution of trimethoprim was multimodal. Data derived fromthis study can be used as a basis for reviewing or verifying present microbiological breakpoints suggested by regulatory agencies toassess the safety of these micro-organisms intended for the use in probiotics.

1. Introduction

Probiotics are generally defined as “live micro-organismswhich confer a health benefit on the host when administeredin adequate amounts” [1]. There is considerable interest inprobiotics for a variety of medical conditions, and millionsof people around the world consume probiotic medicationsor foods daily for perceived health benefits [2]. Next tolactobacilli, members of the genus Bifidobacterium (B.) arefrequently incorporated in probiotic products [3]. Althoughthey have generally been regarded as safe (GRAS), thereare theoretical concerns regarding their safety [2]. Theseconcerns include the potential for transmigration and conse-quently the occurrence of diseases [2]. Hence, bifidobacteriahave already been isolated from various clinical samplesand reported as potential pathogens [4, 5]. Additionally,the potential transfer of antibiotic resistance genes fromprobiotic bacteria to commensals or potential pathogenswithin the gastrointestinal flora is taken very seriously [6, 7].

Thus, microorganisms used as probiotics for humans oradditives in animal nutrition should not contain transfer-able antimicrobial resistance determinants [8–10]. Hence,the Panel on Additives and Products or Substances Usedin Animal Feed (FEEDAP) of the European Food SafetyAuthority (EFSA) has defined criteria for the assessmentof antimicrobial resistance of bacterial strains used as feedadditives [11]. According to this panel, all bacteria intendedfor use as feed additives in Europe must be examined toensure the susceptibility of the component strains to arelevant range of antibiotics [11]. Additionally, EFSA hasproposed the use of the Qualified Presumption of Safety(QPS) status as a safety assessment tool for microorganismsadded to food and feed. Within the QPS approach, whichis a system similar in concept and purpose to the GRASdefinition used in the USA, but modified to take accountof different regulatory practices in Europe, the absence ofacquired antibiotic resistance traits has to be confirmed forall strains of species with QPS status [12, 13].


In contrast to bacteria with clinical significance, standardprocedures and breakpoints have been poorly validatedfor antimicrobial susceptibility testing of non pathogenicbacteria [14]. Because of missing standardized protocols andsusceptibility data for lactic acid bacteria and bifidobacteria,risk assessment of these industrially important bacteria hasbeen complicated in the past. The development of the lacticacid bacteria susceptibility test medium [15] proved to be afirst major step forward to establish a standardized methodfor lactic acid bacteria and bifidobacteria. Meanwhile, thismedium has been frequently applied for antimicrobialsusceptibility testing of bifidobacteria [16–20]. Based on theuse of this medium, standard operating procedures forantimicrobial susceptibility testing of bifidobacteria havebeen proposed [21] and were recently made available as ISO10932/IDF 233 standard [22].

The occurrence of antimicrobial resistance in animalstrains of B. animalis, B. pseudolongum, and B. thermo-philum to seven antibiotics (i.e., ampicillin, clindamycin,erythromycin, gentamicin, streptomycin, tetracycline, andvancomycin) has been already determined [16, 17]. As upto 13 antibiotics were proposed by EFSA at the time ofinvestigation [23], the susceptibility of strains of theseBifidobacterium species to the remaining antimicrobialagents chloramphenicol, kanamycin, linezolid, neomycin,quinupristin/dalfopristin, and trimethoprim was examinedwithin this study. The obtained data may serve as basisfor the definition of microbiological breakpoints for bifi-dobacteria. Furthermore, data could be used to eradicateof bifidobacteria from infections, although little is knownabout their pathogenic potential [4]. Comparing antimicro-bial susceptibility data of human and animal strains, whichwere tested with the same medium, resistances were moreprevalent in strains of animal origin [24]. The developmentof bacterial resistance in livestock may be favored due tothe use of antibiotics throughout whole periods of life andwidely used prophylaxis with medicated feeding stuff at lowdoses [25]. Thus, the spread of antimicrobial resistancesin bifidobacteria could be better predicted by investigatinganimal strains.


2.1. Bacterial Strains and Growth Conditions. A total of 112bifidobacteria of animal origin, isolated during the EUproject BIFID (CT-2000-00805) [26], were included in thisstudy belonging to the species: B. animalis (n = 8), B. pseu-dolongum (n = 33), and B. thermophilum (n = 71). Theidentification of the isolates at strain and species level waspreviously described by Matto et al. [16] and Mayrhoferet al. [17]. The following BCCM/LMG microbial collectionstrains (University Gent, Belgium) were additionally tested asreference microorganisms: B. pseudolongum subsp. globosumLMG 11569 (ATCC 25865), B. pseudolongum subsp. globo-sum LMG 11614 (ATCC 25864), B. pseudolongum subsp.pseudolongum LMG 11594, B. thermophilum LMG 21813(ATCC 25525; type strain), and B. thermophilum LMG 11574(ATCC 25866).

Bacteria were maintained at −80◦C, resuscitated in MRSbroth (Oxoid, Hampshire, UK) supplemented with 0.5 g/litercysteine-HCl (AppliChem, Darmstadt, Germany), and sub-cultured on the Lactic acid bacteria Susceptibility testMedium for bifidobacteria (LSM-C). This medium consistsof 90% Isosensitest broth (Oxoid), 10% MRS broth (Oxoid),and 1.5% Agar Bacteriological (Oxoid) supplemented with0.3 g/liter cysteine-HCl (LSM-C) [15]. All incubations wereperformed in an anaerobic cabinet (80% N2, 10% CO2 and10% H2; Scholzen Technik, Switzerland) at 37◦C.

2.2. Antimicrobial Susceptibility Testing. The minimum in-hibitory concentrations (MICs) of the antimicrobial agentschloramphenicol, kanamycin, linezolid, neomycin, quin-upristin/dalfopristin, and trimethoprim were determined bybroth microdilution according to the ISO 10932/IDF 233standard [22] with minor modifications. With the exceptionof quinupristin/dalfopristin (Sanalog, Kist, Germany) andlinezolid (Pfizer, New York, USA), all antibiotics originatedfrom Sigma-Aldrich (Daint Louis, Missouri, USA). Allantibiotics except for chloramphenicol and trimethoprimwere dissolved in water for preparing stock solutions of1280 µg/mL. To dissolve chloramphenicol 95% ethanol wasneeded, whereas 0.05 M HCl was required for trimethoprim.These solvents were used in volumes as low as possible,and water was finally added to receive the desired volumeof the stock solution. Subsequently, stock solutions werediluted in LSM-C broth to obtain solutions with preliminaryconcentrations in the range of 0.25–256 µg/mL. Of these,50 µl were dispensed in the wells of the microtiter plates.

Bacterial inocula were prepared by suspending coloniesfrom 48 h incubated LSM-C medium to 5 mL 0.85% NaClsolution. Subsequently, inocula were adjusted to McFarlandstandard 1 and diluted 1 : 500 in LSM-C broth for inoc-ulation of microdilution plates by adding 50 µl of dilutedinoculum to each well. This resulted in a final antibioticconcentration of 0.12–128 µg/mL.

After incubating plates under anaerobic conditions at37◦C for 48 hours, the MIC value was read as the lowestconcentration of an antimicrobial agent in which visiblegrowth was inhibited.

The accuracy of susceptibility testing was monitored byparallel use of the quality control strain Enterococcus faecalisATCC 29212.

3. Results and Discussion

The antimicrobial susceptibility of 8 B. animalis, 36 B.pseudolongum, and 73 B. thermophilum strains, including fivereference strains, to chloramphenicol, kanamycin, linezolid,neomycin, quinupristin/dalfopristin, and trimethoprim issummarized in Table 1. MICs (µg/mL) are reported in termsof the MIC range, MIC50 (MIC that inhibited 50% of thetested strains), and MIC90 (MIC that inhibited 90% of thetested strains). Accordingly, no marked difference in theMIC distributions between the different species was observedfor chloramphenicol, kanamycin, linezolid, neomycin, andquinupristin/dalfopristin. For these antimicrobial agents


Table 1: Susceptibility of 8 B. animalis, 36. B. pseudolongum, and 73 B. thermophilum strains to selected antimicrobial agents as determinedby the broth microdilution method using LSM-C medium.

Antibiotic SpeciesMIC (µg/mL)

MIC range MIC50 MIC90

Chloramphenicol B. animalis 2–4 2 4

B. pseudolongum 1-2 1 2

B. thermophilum 0.5–2 1 2

Kanamycin B. animalis >128 >128 >128

B. pseudolongum ≥128 >128 >128

B. thermophilum ≥128 >128 >128

Linezolid B. animalis 0.5–2 1 2

B. pseudolongum 0.5–2 1 1

B. thermophilum 0.5–1 0.5 1

Neomycin B. animalis 32–>128 64 >128

B. pseudolongum 16–>128 64 >128

B. thermophilum 16–>128 128 >128

Quinupristin/ B. animalis ≤0.12 ≤0.12 ≤0.12

Dalfopristin B. pseudolongum ≤0.12–0.25 ≤0.12 0.25

B. thermophilum ≤0.12–0.25 ≤0.12 0.25

Trimethoprim B. animalis ≤0.12 ≤0.12 ≤0.12

B. pseudolongum ≤0.12–32 ≤0.12 4

B. thermophilum 0.5–>128 8 128

MIC50 and MIC90: MICs (µg/mL) that inhibited 50% and 90% of the number of strains tested, respectively.

narrow unimodal MIC distributions either at the low-endconcentration range (i.e., for chloramphenicol, linezolid, andquinupristin/dalfopristin) or at the high-end concentrationrange (i.e., for kanamycin and neomycin) were determined(Figures 1(a)–1(e)). A unimodal distribution describes apopulation, which is either uniformly susceptible (i.e., forchloramphenicol, linezolid, and quinupristin/dalfopristin)or resistant (i.e., for kanamycin and neomycin) [27, 28].Next to a unimodal distribution MIC values, obtained bysusceptibility testing of a defined population of strains,can also follow a bimodal or multimodal distribution [29].For trimethoprim a multimodal distribution with threedifferent subpopulations was identified: one with very lowMICs (≤0.12 µg/mL), another one with higher MICs (0.5–16 µg/mL), and a last one with high MICs (32–>128µg/mL)(Figure 1(f)). While B. animalis strains generally had lowertrimethoprim MICs (≤0.12 µg/mL), the highest MICs weredetected for B. thermophilum strains (0.5–>128µg/mL).The MIC values of B. pseudolongum strains were between≤0.12 µg/mL and 32 µg/mL.

In order to allow the interpretation of antimicrobialsusceptibility profiles of bifidobacteria used in food andfeed applications, microbiological breakpoints are neededfor categorizing susceptible or resistant strains. Especiallybimodal distributions of MIC values play an importantrole in determining the microbiological breakpoint. Nev-ertheless, also unimodal and multimodal distributions cansupport the definition of microbiological breakpoints todistinguish strains with acquired resistance from the native,

susceptible population. Beside this, MICs for determiningmicrobiological breakpoints are only meaningful when themethods and conditions of the test are known [30]. It is forthis reason that the development of an international standardreference method for determining MICs for bifidobacteriawas recently proposed [22]. Thus, data obtained in thisstudy, following the newest method developments con-cerning antimicrobial susceptibility testing of bifidobacteria,can be used as a basis for reviewing or verifying presentmicrobiological breakpoints for bifidobacteria to assess thesafety of microorganisms intended for use in food and feedapplications.

According to the literature, bifidobacteria are usuallysensitive to chloramphenicol. Normal MICs to this antimi-crobial agent using various test techniques and media havebeen reported to range between 0.5 and 8 µg/mL [31–37].Only once in the literature, five strains belonging to thespecies B. infantis, B. longum, and B. suis as well as oneBifidobacterium sp. isolate with higher MICs up to 64 µg/mLwere detected by Kheadr et al. [38]. However, these authorsconcluded that the strains may still be sensitive. Thus,strains with acquired resistance have not been detected untilnow. Using the recommended LSM-C medium [22] andthe same [20] or similar conditions [19] for antimicrobialsusceptibility testing, a chloramphenicol MIC ≤2 µg/mL forone B. thermophilum strain [19] or MICs between 1 and2 µg/mL for ten B. longum strains [20] could be observed.As only a small number of strains were tested within theabove-mentioned studies, the widening of the MIC distri-bution from 0.5 to 4 µg/mL (Figure 1(a)) by investigating


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Figure 1: Distribution of minimum inhibitory concentrations (MICs) for (a) chloramphenicol, (b) kanamycin, (c) linezolid, (d) neomycin,(e) quinupristin/dalfopristin, and (f) trimethorpim in 8 B. animalis, 36. B. pseudolongum, and 73 B. thermophilum strains as determinedwith the microdilution broth method using LSM-C medium.

more than 100 strains in this study is obvious. Applyingthe recommended breakpoint of EFSA [11], all strains ofthe unimodal distribution are categorized as susceptible,approving the recent EFSA microbiological breakpoint forchloramphenicol.

Most bifidobacteria have been reported as resistant toaminoglycosides because of the lack of cytochrome-mediateddrug transport system, resulting in a failure of the drugto reach its target [39]. The resistance of the tested strainsto gentamicin and streptomycin has already been reportedbefore [16, 17]. Correspondingly, all strains were resistantto kanamycin (128–>128µg/mL, Figure 1(b)) and neomycin(16–>128µg/mL, Figure 1(d)). The particular resistance ofbifidobacteria to kanamycin is well known, and MICsbetween 64 and >1024 µg/mL were described for thisantimicrobial agent, whereas for neomycin MICs lay between16 and >1024 µg/mL testing a large number of speciessuch as B. bifidum, B. breve, B. catenulatum, B. infantis, B.longum, B. suis, B. thermophilum, and others [32, 35–37].Using the LSM-C medium, the antimicrobial susceptibilityto kanamycin was only tested by Kushiro et al. [20] andKlose et al. [19]. Whereas Kushiro et al. [20] received MICsbetween 128 and 512 µg/mL for ten B. longum strains, thetested B. thermophilum strain of Klose et al. [19] displayedan MIC value of 64 µg/mL. The lower MIC value detected

by Klose et al. may be due to other testing conditions (i.e.,24 hours instead of 48 hours of incubation), underlining theimportance of controlled and standardized conditions forsusceptibility testing [40]. While breakpoints for gentamicinand streptomycin are indicated in the EFSA document [11],none is required for kanamycin or specified for neomycin.

Low level, unimodal MIC distributions between 0.5and 1 µg/mL have been reported for linezolid and differentbifidobacterial species, suggesting all bifidobacteria are sus-ceptible to this antibiotic [37, 41]. This is in good accordancewith our results, since all tested strains were inhibited bya linezolid concentration lower than 4 µg/mL (Figure 1(c)).Ten B. longum strains, also tested using the same mediumand conditions, displayed MIC values of 0.5 and 1 µg/mL[20]. As the nonmutational resistance to linezolid, whichis due to the acquisition of the cfr gene, is extremely rareand also confers resistance to chloramphenicol [42, 43],testing for this antimicrobial agent was no longer consideredas relevant by EFSA [11]. Checking for chloramphenicolresistance should efficiently cover the hazard of an acquiredresistance to linezolid [11].

The in vitro susceptibility of bifidobacteria to quinu-pristindalfopristin has been rarely studied. Only two pre-vious reports showed that bifidobacteria are susceptibleto this semisynthetic mixture by testing 100 strains of 11


bifidobacterial species by agar overlay disc diffusion [18]and one B. thermophilum strain by broth microdilution [19]using the same test medium (LSM-C). The obtained MICvalue of 0.5 µg/mL by Klose et al. [19] was one dilutionstep higher than the MIC range (≤0.12–0.25 µg/mL) receivedwithin this study (Figure 1(e)). The EFSA breakpoint of1 µg/mL to quinupristin-dalfopristin appears to be applicablefor bifidobacteria [11].

Reduced susceptibility of bifidobacteria towards trime-thoprim was described by Ouoba et al. [37]. Susceptibilitywas also found to be variable and strain-specific by Mascoet al. investigating 100 strains of 11 bifidobacterial species[18] or to be ≥32 µg/mL by Kushiro et al. testing 10 B.longum strains [20], also using the same medium as withinthis study. Hence, a variable but species-specific susceptibilitywas detected, nearly covering the whole concentration rangetested (Figure 1(f)). Also a wide range of trimethoprimMICs with no clear breakpoint values was identified forcertain Lactobacillus species [44]. This was led back tothe presence of antagonistic components in the medium,complicating susceptibility testing concerning trimethoprim[11, 15]. Thus, MIC testing of trimethoprim was no longerconsidered as relevant by EFSA [11].

4. Conclusion

In this study, the recently published ISO 10932/IDF 233standard [22] was used to provide susceptibility data on thebasis of a representative number of animal bifidobacteria.These data could be used for reviewing or verifying presentmicrobiological breakpoints suggested by regulatory agenciesto assess the safety of microorganisms intended for theuse in probiotics. Nevertheless, more data including MICvalues of human bifidobacteria for these antimicrobial agentsapplying the same testing conditions are needed to obtainadequate breakpoints for differentiating susceptible bacteriafrom those with acquired resistance. Additionally, a broadscreening of resistance genes using molecular tools wouldalso be of importance for the definition of applicablebreakpoints.

Acknowledgements

This study was performed as a part of the EU Project“ACE-ART” (CT-2003-506214) within the 6th framework.Francoise Gavini and Matthias Upmann are gratefullyacknowledged for providing the strains isolated in the EUProject “BIFID” (CT-2000-00805). Pfizer is thanked forproviding linezolid.

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Research Article

Cardiac Conduction Safety during Coadministration ofArtemether-Lumefantrine and Lopinavir/Ritonavir inHIV-Infected Ugandan Adults

Pauline Byakika-Kibwika,1, 2, 3 Mohammed Lamorde,1, 2 Peter Lwabi,4 Wilson B. Nyakoojo,4

Violet Okaba-Kayom,1 Harriet Mayanja-Kizza,1, 3 Marta Boffito,5 Elly Katabira,1, 3

David Back,6 Saye Khoo,6 and Concepta Merry1, 2, 3

1 Infectious Diseases Institute, Makerere University, P.O. Box 22418, Kampala, Uganda2 Trinity College, Dublin 2, Ireland3 Infectious Diseases Network for Treatment and Research in Africa (INTERACT), P.O. Box 7062, Kampala, Uganda4 Uganda Heart Institute, Mulago Hospital, P.O Box 7051, Kampala, Uganda5 St. Stephen’s AIDS Trust, London SW10 9TR, UK6 University of Liverpool, Liverpool L69 3BX, UK

Correspondence should be addressed to Pauline Byakika-Kibwika, [email protected]

Received 31 August 2010; Revised 26 November 2010; Accepted 14 February 2011

Academic Editor: Kazuo Tamura

Copyright © 2011 Pauline Byakika-Kibwika et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Background. We aimed to assess cardiac conduction safety of coadministration of the CYP3A4 inhibitor lopinavir/ritonavir (LPV/r)and the CYP3A4 substrate artemether-lumefantrine (AL) in HIV-positive Ugandans. Methods. Open-label safety study of HIV-positive adults administered single-dose AL (80/400 mg) alone or with LPV/r (400/100 mg). Cardiac function was monitoredusing continuous electrocardiograph (ECG). Results. Thirty-two patients were enrolled; 16 taking LPV/r -based ART and 16 ARTnaıve. All took single dose AL. No serious adverse events were observed. ECG parameters in milliseconds remained within normallimits. QTc measurements did not change significantly over 72 hours although were higher in LPV/r arm at 24 (424 versus 406;P = .02) and 72 hours (424 versus 408; P = .004) after AL intake. Conclusion. Coadministration of single dose of AL with LPV/rwas safe; however, safety of six-dose AL regimen with LPV/r should be investigated.

1. Introduction

Malaria and HIV infection are leading causes of morbidityand mortality and remain major health problems in endemicregions. Malaria causes about 300–500 million clinical casesannually, 90% of which occur in sub-Saharan Africa [1].The Joint United Nations Program on HIV/AIDS (UNAIDS)estimated that 29.4 Million Africans are infected with HIV(UNAIDS, December 2002). Together malaria and HIVaccount for over four million deaths per year.

Studies have demonstrated increased risk for malaria inHV infected patients especially those with lower CD4 cellcounts [2–4]. More evidence suggests transient increase inHIV viral load in patients with acute malaria episodes [5]. A

major challenge to the treatment of malaria in HIV-infectedindividuals is the potential for pharmacokinetic (PK) druginteractions with concerns regarding safety and efficacy [6].

Due to the widespread resistance to older antimalarialdrugs, the World Health Organization now recommendsartemisinin combination therapy (ACT) for malaria treat-ment [7]. Artemether-lumefantrine (AL) is an oral fixed-dose combination tablet of artemether (a derivative ofartemisinin) and lumefantrine (a racemic mixture of asynthetic fluorine derivative). The drug combination ishighly efficacious against sensitive and multidrug resistantPlasmodium falciparum; with the advantage of rapid clear-ance of parasites by artemether and the slower elimination ofresidual parasites by lumefantrine [7–9].


Recommendations for antiretroviral therapy (ART)include two nucleoside reverse transcriptase inhibitors(NRTIs) plus a nonnucleoside reverse transcriptase inhibitor(NNRTI) or a protease inhibitor (PI). Lopinavir/ritonavir(LPV/r) is an oral fixed-dose combination tablet of LPV(a PI) with low dose ritonavir, a pharmacoenhancerthat significantly increases LPV plasma concentrations bycytochrome P450 3A4 (CYP3A4) inhibition.

Concerns over safety monitoring of LPV/r have becomemore crucial following the recent FDA alert on cardiotoxicityof LPV/r. Safety information on LPV/r includes warningsand precautions regarding QT/QTC interval and PR intervalprolongation. According to the revised safety label, LPV/rprolongs the PR interval, and cases of second- or third-degree atrioventricular block have been reported in somepatients. Indeed LPV/r should be used with caution inpatients who may be at increased risk of developing cardiacconduction abnormalities, such as those with underlyingstructural heart disease, preexisting conduction systemabnormalities, ischemic heart disease, or cardiomyopathies.The effect on the PR interval of coadministration of LPV/rwith other drugs that prolong the PR interval has not yetbeen determined and should be undertaken with caution.Clinical monitoring is recommended especially during coad-ministration with drugs metabolized by CYP3A [10].

Data from previous studies indicate that artemether andlumefantrine are predominantly metabolized by CYP3A4 [6,11, 12]. Knowledge of their metabolism suggests potential forPK drug-drug interactions [6]. LPV/r is a potent inhibitor ofCYP3A4, therefore, inhibition of CYP3A4 may raise plasmaconcentrations of artemether and lumefantrine but decreaseplasma concentrations of dihydroartemisinin (DHA) themetabolite of artemether. A study that investigated thepharmacokinetics of AL when administered with LPV/rin HIV-uninfected healthy volunteers demonstrated 2 to3-fold increases in lumefantrine AUC and trends towardsdecreases in artemether Cmax and AUC. Formal safetyanalysis of coadministration was not performed in this study[13]. Increased plasma concentrations of artemether andlumefantrine may enhance toxicity. Lumefantrine has somestructural similarity to halofantrine which is cardiotoxicmainly in form of QTc prolongation. Therefore, vigilantevaluation of the cardiac safety of lumefantrine, especiallywhen coadministered with a potent CYP3A4 inhibitor, iswarranted [14–17]. We aimed to assess the cardiac safety ofcoadministration of a single dose of AL (80/480 mg) withLPV/r based ART in HIV-positive Ugandan patients.


2.1. Ethical Considerations. The study was approved bythe Scientific Review Committee of the Infectious DiseasesInstitute (IDI) of Makerere University, the Uganda NationalHIV/AIDS Research Committee (ARC 056) and was regis-tered with Uganda National Council of Science and Tech-nology (HS 197) and ClinicalTrials.gov (NCT 00619944). Allparticipants gave written informed consent to participate,and all study procedures were conducted according to GoodClinical Practice (GCP).

2.2. Study Site. The study was conducted between January2008 and June 2009 at the IDI and the Uganda HeartInstitute (UHI) of Mulago National Referral Hospital inKampala, Uganda. The IDI is a regional centre of excellencefor HIV/AIDS treatment, prevention, training and research.To date, over 20,000 HIV-infected patients are registered atthe IDI with over 8,000 taking ART. About 10% these are onLPV/r-based second line ART.

2.3. Study Design and Population. This was a two-arm studyto assess the safety of coadministration of AL in HIV-positivepatients taking LPV/r-based ART and ART naıve patients.Patients were eligible to participate if they were older than18 years of age, provided written informed consent, had noevidence of systemic illness and required no medications thathad known potential for drug interactions with study drugs.Patients with abnormal ECG tracing, abnormal clinical testresults, positive blood smear for malaria, pregnant mothersand those who reported use of herbal medication wereexcluded from the study.

2.4. Study Procedures. Patients were screened and enrolledfrom the cohort of patients attending the IDI. The LPV/rarm consisted of patients stable on LPV/r 400/100 mg-basedART for at least one month and the ART naıve arm consistedof patients who had not started ART and were not yeteligible for ART according to national guidelines. Patients inboth arms took cotrimoxazole daily for prophylaxis againstopportunistic infections. Participants had detailed studyexplanation at enrolment. Adherence to study drugs wasassessed using self-report and pill count by the study phar-macist. We collected information on adverse drug events andserious adverse drug events, and a questionnaire on qualityof life was administered on each study day. On the eveningprior to the study day, participants were reminded of theirstudy day appointment, were given detailed instructions totake their medication and food at 8.00 pm, and told toarrive at the hospital by 7.00 am in a fasting state. Onthe study day, patients were admitted at the UHI and a12-lead ECG monitor was attached for continuous cardiacfunction monitoring. The intake of a standardized breakfastand morning doses of drugs was directly observed by studystaff. All patients took a single dose of AL of 80/480 mgwith 150 mL of water. Patients in the LPV/r arm took LPV/r(400/100 mg) with their AL dose. ECG monitoring wasperformed continuously for the first 12 hours after AL intake.Patients were then discharged and returned for the followingthree mornings (T = 24, 48, and 72 hours) for a single ECGtracing.

2.5. Safety Assessment. Medical history, physical examina-tion, vital signs, routine clinical laboratory tests, ECGs andurine screens for pregnancy were performed at screening.On the study day, medical history, physical examination,vital signs, and a blood smear for malaria parasites wereperformed. Adverse events were recorded continuouslythroughout the trial, and the onset, duration, severity, andrelationship to the trial drugs if any were noted. Standard


Table 1: shows a comparison of the baseline characteristics of studypatients.

VariableLPV/r arm ART naıve arm

P valuemedian (IQR) median (IQR)

Age (yrs) 38 (33–41) 34 (28–39) .2

Weight (kgs) 65 (54–73) 64 (56–71) .9

Height (cms) 163(158–172) 163 (153–169) .1

BMI 21 (19–24) 25 (22–31) .06

Viral load (c/mL) <400 26756 (5548–181186) <.01

Hb (g/dL) 14 (14–15.4) 12.2 (12–14) .003

Table 2: shows the mean electrocardiograph (ECG) parameters inmilliseconds after AL dosing.

VariableLPV/r arm ART naıve arm

P valuemean (SD) mean (SD)

Heart rate 69 (8.1) 71 (5.9) .5

PR 154 (18.4) 169 (15.9) .02

QRS 87.4 (6.6) 82.8 (6.6) .06

QTc 421 (20.0) 404 (20.7) .03

12-lead ECGs were recorded at screening, immediately priorto dosing (T = 0 hour), and continuously for 12 hoursafter dose of AL, then daily for three days. QTc-intervalswere calculated using the Bazett formula (QTc = QT/÷√RR)[37, 38] to correct for the influence of heart rate. A seniorcardiologist evaluated the PR, QRS, and QT intervals visuallyon the ECG.

2.6. Statistical Analysis. Demographic and ECG results wereentered into EpiData and exported to SPSS version 12.0and STATA version 8.0 for statistical analysis. Continuousvariables were summarized into means, and medians andcompared using the Independent T-test. A P-value < .05 wasconsidered statistically significant.

3. Results

A total of 72 HIV-positive patients (41, 65% females) werescreened between January and June 2009; 12 (17%) wereexcluded because they had other concurrent illnesses thatrequired treatment, 28 (39%) were excluded because theyhad abnormal ECG tracings and 32 (44%) were enrolled,16 in each arm. Patients in the two study arms werecomparable on majority of baseline characteristics (Table 1);however, patients in the LPV/r arm had significantly higherhemoglobin levels with lower viral load.

There were no serious adverse events during the studyperiod. ECG parameters (heart rate, PR-interval, QRS-complex and QTc) remained well within normal limits inboth study arms (Table 2). The mean QRS-complex and QTcinterval after AL administration were higher in the LPV/rarm compared to the ART naıve arm (87.4 versus 82.8,P = .06 and 421 versus 404, P = .03, resp.) but the meanPR-interval was significantly higher in the ART naıve arm(154 versus 169, P = .02) (Table 2). Mean (SD) change in

Table 3: shows the median QTc interval measurements in millisec-onds over 72 hours period after AL dosing.

TimeQTc (ms) median (IQR)

P valueLPV/r arm ART naıve arm

Screening 415 (403–439) 395 (388–425) .14

12 hours 415 (404–439) 419 (403–427) .7

24 hours 424 (401–434) 406 (393–411) .02

48 hours 411 (396–432) 409 (401–419) .7

72 hours 424 (416–441) 408 (392–417) .004

QTc interval values from the pre-AL QTc interval values wasgreater for the ART naıve arm compared to the LPV/r arm(6.7 (15.4) versus −0.8 (13), P = .17). The QTc intervalmeasurements for participants in both study arms remainedwithin normal ranges over the 72 hours period (Table 3);with none above the upper limit of normal (450 ms for malesand 470 ms for females).

4. Discussion

LPV/r is a potent inhibitor of CYP3A4, therefore, coadmin-istration with AL which is predominantly metabolized byCYP3A4 may potentially result in enhanced pharmacologicaland toxicological effects. We aimed to assess the cardiac safetyof coadministration of a single dose (80/480 mg) of AL inHIV-infected patients taking LPV/r based ART and HIVpositive ART naıve patients. Since LPV/r is a potent CYP3A4inhibitor, only a single dose of AL was given in order to avoidany unknown potential adverse effects of the latter.

We found that HIV-positive patients taking LPV/r had ahigher QTc interval prior to administration of AL comparedto HIV-positive ART naıve patients, nevertheless, the differ-ence was not statistically significant. It is possible that thiscould have been a result of the effects of LPV/r on the heart;however, we cannot establish a causal relationship since wedid not have QTc measurements for these patients prior toinitiation of LPV/r. This however, raises concern especiallyin view of the recent FDA alert over the effects of LPV/ron the heart. Indeed the label for LPV/r includes warningsand precautions regarding QT/QTc interval and PR intervalprolongation [10].

Although the QTc interval for the LPV/r arm wassignificantly higher than that for the ART naıve arm at72 hours, the difference could not be attributed to LPV/rcoadministration with AL because baseline QTc intervalwas higher in the LPV/r arm and both study arms hadan increment in QTc interval values from baseline whichremained well within normal limits (Table 3). It is possiblethat the increment in the QTc intervals could have been moreif patients had received the full six-dose regimen of AL. TheLPV/r label clearly states that LPV/r should be avoided inpatients using drugs that prolong the QT interval. Since wedo not know what levels and effects of lumefantrine wouldresult if the full six-dose AL regimen is coadministered withLPV/r, we suggest close clinical monitoring of HIV-positivepatients taking LPV/r with AL concomitantly until more databecomes available.


This is one of the very few studies that have assessed thecardiac safety of coadministration of AL and LPV/r in HIVpositive patients. Previous studies have evaluated safety of ALin healthy volunteers and patients with malaria. Bindschedlerand others found that the QTc interval remained unchangedafter a single dose of AL in healthy males [14]. Thedifference in results may be explained by the difference in thestudy populations. Bindschedler and others demonstratedsignificant exposure dependent increase in the QTc intervalin healthy males after halofantrine. It is possible that LPV/rcoadministered with a full six-dose regimen of AL may causeincreased concentrations of lumefantrine causing an expo-sure dependent QTc interval prolongation. Previous datashowed no evidence of cardiotoxicity during AL treatmentin healthy volunteers [18]. However, these were conductedin patients with malaria without coadministration of LPV/r.It is possible that results may be different with concomitanttreatment with the full six-dose AL regimen and LPV/r.

5. Conclusion and Recommendation

Our data suggests no evidence of cardiac conduction abnor-malities after concomitant treatment with LPV/r and a singledose AL. There is need to assess the safety of the full six-doseregimen of AL in HIV positive patients receiving LPV/r basedART.

Acknowledgments

The authors thank the clinical pharmacology research team:Deborah Ekusai, Jamillah Nakku, Rhoda Namakula, andJohn Magoola. They are very indebted to the InfectiousDiseases Network for Treatment and Research in Africa(INTERACT) for financial and scientific support. They aregrateful to the staff of the Infectious Diseases Clinic, UgandaHeart Institute and Makerere University-Johns Hopkins Uni-versity Laboratory for the clinical and laboratory support.They are very thankful to the study participants. This studywas supported by a grant from the University of Liverpool,UK.

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Review Article

Antibiotic Combinations with Daptomycin for Treatment ofStaphylococcus aureus Infections

Kristina Nadrah and Franc Strle

Department of Infectious Diseases, University Medical Centre Ljubljana, Japljeva 2, 1525 Ljubljana, Slovenia

Correspondence should be addressed to Kristina Nadrah, [email protected]

Received 19 November 2010; Accepted 23 January 2011

Academic Editor: Athanassios Tsakris

Copyright © 2011 K. Nadrah and F. Strle. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Daptomycin is a lipopeptide antibiotic with a unique mechanism of action on Gram-positive bacteria. It is approved for treatmentof skin and soft-tissue infections with Gram-positive bacteria, bacteraemia and right-sided infective endocarditis caused byStaphylococcus aureus. Diminishing susceptibility of S. aureus to daptomycin during treatment of complicated infections andclinical failure have been described. Combinations of daptomycin with other antibiotics including gentamicin, rifampin, beta-lactams, trimethoprim/sulfamethoxazole (TMP-SMX), or clarithromycin present a new approach for therapy. In vitro and animalstudies have shown that such combinations may, in some cases, be superior to daptomycin monotherapy. In this paper we focuson the antibiotic combinations for complicated S. aureus infections.

1. Introduction

Daptomycin, an antibiotic with a new mechanism of actionand low occurrence of spontaneous resistance, came tomarket in 2003 in the USA and in 2006 in Europe. Inspite of its many promises for treatment of infectionscaused by Gram-positive bacteria, reports on clinical failureand diminished in vitro susceptibility soon came forward.Alongside, it became clear that daptomycin monotherapyof biofilm-related and deep-seated infections is often noteffective. Due to limited clinical settings in which dapto-mycin is effective, diminishing susceptibility to daptomycin,emerging resistance to linezolid, and slow development ofnew antibiotics, clinicians have difficulties treating seriousStaphylococcus aureus infections. Therefore, combinations ofdaptomycin with other antibiotics are extensively studiedas a potential new therapeutic strategy. In this review, wewill focus on the antibiotic combinations for complicated S.aureus infections.

2. Daptomycin Pharmacology

Daptomycin is a novel lipopeptide antibiotic approved fortreatment of complicated skin and soft-tissue infections

caused by Gram-positive bacteria, and for S. aureus bacter-aemia and right-sided endocarditis [1]. It is not active in lowrespiratory tract infections, because lung surfactant formscomplexes with daptomycin thereby inactivating it [2].

Daptomycin mechanism of action is complex and notentirely understood. Research so far indicates that it actson cell membrane [3, 4] and also inhibits the synthesis oflipoteichoic acid, necessary for cell wall synthesis [4].

Daptomycin structure consists of an anionic core anda lipophilic tail. By binding divalent calcium and formingCa2+-complexes, the net charge of the molecule becomespositive and thus enables electrostatic interactions withnegatively charged cell membrane. It then binds to cellmembrane, where it forms oligomers and subsequentlychannels through which intracellular ions, like potassium,leak out of the cell, diminishing cell membrane negativepotential and causing cell death [3]. This initial model waslater modified according to the observation that daptomycinforms micelle-like structures in the medium and binds to themembrane in the already oligomerized form [5]. However,Hobbs et al. found that changes in dissipation of membranepotential and leakage of intracellular material occur ratherlate, after cell death, and with no significant alterations inmembrane integrity [6]. Similarly, no significant cell lysis was


found by electron microscopy and the membrane integrityprobes used on S. aureus treated in vitro with daptomycin[7]. In addition, analogous to cell wall active antibiotics,daptomycin was found to upregulate cell wall stress stimulongenes [8]. The gene expression profile of S. aureus after beingexposed to daptomycin is similar to profiles developed afterexposure to cell wall active antibiotics, such as beta-lactams,as well as to compounds which disrupt cell membrane,like carbonyl cyanide m-chlorophenylhydrazone [8]. Thus,beside acting on the cell membrane, daptomycin likely actson several other targets.

Daptomycin activity is sensitive to the inoculum of theinfectious micro-organism [9]. This is probably due to thedecrease of the local effective antibiotic concentration at highinoculum [10]. Therefore, sufficient serum concentrationsare essential for clearance of bacteria from the infection site.

The current susceptibility cutoff MIC of S. aureusisolates to daptomycin is set at 1 mg/L [11]. In some cases,susceptibility profile is heterogeneous [12]. No MIC creeplike in the case of vancomycin has been noticed [13–15],even in the isolates collected over several decades [11]. Sinceunequivocal criteria for resistance have not been defined yet,the term non-susceptibility is used instead of resistance.

3. Mechanisms of Non-susceptibilityto Daptomycin

Strains of S. aureus non-susceptible to daptomycin havebeen obtained from clinical cases, by in vitro selection,and by chemical mutagenesis. Frequency of spontaneousdaptomycin resistance in S. aureus is low (10−10) [12]. Serialpassages in subinhibitory concentrations of daptomycin giverises of MICs by a factor of 8–32; the same is valid forchemical mutagenesis [12, 16, 17].

Several genes have been implicated in S. aureus non-susceptibility to daptomycin. Overexpression and mutationsin mprF [18–20] which encodes lysyl-phosphatidylglycerol(LPG) synthetase and flippase, and yycG which encodeshistidine kinase in a two-component sensor regulatorysystem of YycF/YycG [16, 20] were identified in clinicalisolates and laboratory-derived strains, while rpoB [20, 21]and rpoC [16] which encode β and β

′subunits of RNA

polymerase were detected only in in vitro selected laboratorynon-susceptible strains. In vitro insertional mutation ofcspB [22], a cold shock gene, led to increased susceptibilityto daptomycin in a daptomycin non-susceptible strain ofS. aureus. Several mutants obtained in vitro and some ofthe clinical isolates contain at least one of these geneticchanges; a combination of the genetic alterations seems tohave an additive effect on the MIC value [20]. However,some daptomycin non-susceptible strains do not have any ofthese alterations [23, 24], indicating that the mechanism of“resistance” is probably multifactorial.

The phenotypic alterations in non-susceptible S. aureuscan be grouped into (1) changes in cell wall structure andturnover; (2) changes in membrane composition, membranestructure, and membrane potential; (3) modifications insensitivity to depolarization, autolysis, and permeabilization.

In some cases, thicker cell wall had been correlated to non-susceptibility [18, 20, 21, 25], but this was not a uniformfinding; for example, Yang et al. found no changes in cell wallthickness in a clinical meticillin-resistant S. aureus (MRSA)isolate with diminished susceptibility to daptomycin whichdeveloped during treatment [19]. S. aureus strains non-susceptible to daptomycin had an increased synthesis of LPGand increased translocation of LPG to the outer membrane,and hence modifications in membrane fluidity and elec-trostatic potential [19, 20, 26]. A loss of an unidentified81 kD membrane protein has also been described in a non-susceptible clinical isolate [17].

So far, most cases of clinically acquired non-susceptibilityof S. aureus to daptomycin occurred in a setting of inade-quate dosing [17] and/or deep-seated, high-inoculum, andbiofilm-related infective, such as infectious endocarditis (IE)[17, 19, 26, 27] or bone infections [24]. In these cases,the effective concentration of daptomycin at the site of thehighest bacterial density is low [10], and activity is furtherdiminished by the stationary phase of bacteria in biofilmwhich has been associated with such infections [28].

4. Relationship between DiminishedSusceptibility toVancomycin and Daptomycin

Diminished susceptibility of S. aureus to vancomycin hasbeen associated with a development of diminished sus-ceptibility to daptomycin. This has been established formultidrug resistant (MDR) S. aureus, for MRSA, as well asfor meticillin-susceptible S. aureus (MSSA). Daptomycin isoften prescribed for treatment of MDR S. aureus infectionsif vancomycin therapy fails. Clinical experience indicatesthat strains of S. aureus with diminished susceptibility tovancomycin develop a diminished susceptibility to dapto-mycin during treatment [27, 29–31] or even in absenceof daptomycin exposure [32, 33]. Several studies foundthat diminished susceptibility to vancomycin in MRSA isassociated with diminished susceptibility to daptomycin[32], and, according to some reports, that it correlates wellwith increased cell wall thickness [25, 27]. The highestMICs of vancomycin intermediate S. aureus (VISA)/MRSA,observed in deep-seated infections such as septic arthritisand osteomyelitis, are often associated with higher MICs fordaptomycin or even with non-susceptibility to daptomycin[33]. In the case of MRSA IE and septic thrombophlebitis,the patient’s strain developed non-susceptibility to gly-copeptides during treatment with glycopeptides, and, afterswitching therapy to daptomycin, a non-susceptibility todaptomycin. MRSA was successfully eradicated from thebloodstream only after therapy with linezolid and fusidicacid [27]. A similar relationship was found for MSSA:diminished susceptibility to vancomycin which developedduring therapy of patients with osteomyelitis [34] or IE[26] was associated with a higher MIC to daptomycineven though the patients had never received it. Subsequentanalysis found that vancomycin and daptomycin exhibiteda reduced bactericidal activity towards the non-susceptible


Table 1: Data on previous vancomycin/daptomycin therapy in MRSA and MSSA clinical isolates non-susceptible to daptomycin.

Strain InfectionPreviousVAN therapy(days)

Reason forchange

DAP therapyMIC of DNSstrain (days∗)

Outcome Ref.

MRSA IE Yes (No data) Failure YesMIC 2b (Nodata)

No data[26] (Casereport)

MRSA IE Yes (47∗∗) Failure Yes MIC 4a (14)Died (C. albicans septicshock)

[27] (Casereport)

MRSA IE Yes (46∗∗∗) Failure6 mg/kgq24 h

MIC 4a (24)Survived on alternativetreatment

[30] (Casereport)

MRSA BSI Yes (15) Failure

6 mg/kgq24 h, then8 mg/kgq24 h

MIC 4a (6) Died[31] (Casereport)

MRSA BSI Yes (No data) No change NoMIC 2a (Nodata)

No data[32] (Caseseries)

MRSA UTI Yes (12) Failure6 mg/kgq24 h

MIC 4a (7) Died[29] (Casereport)

MSSA OM Yes (60) Failure NoMIC 4a (Nodata)

Survived on alternativetreatment(nafcillin+rifampin)

[34] (Casereport)

DAP: daptomycin; VAN: vancomycin; DNS: daptomycin non-susceptible; UTI: urinary tract infection; BSI: bloodstream infection; IE: infective endocarditis;OM: osteomyelitis. MRSA: meticillin-resistant S. aureus; MSSA: meticillin-susceptible S. aureus; ∗duration (days) of treatment with daptomycin priorto isolate was obtained; ∗∗vancomycin for 12 days, then teicoplanin 35 days; ∗∗∗2 courses: 6 weeks initially, additional 4 days after relapse; (a) brothmicrodilution (b) Etest. All initial isolates were susceptible to daptomycin; MIC: minimal inhibitory concentration (mg/L).

examined strain and a lesser degree of its chemical autolysiswas observed [34]. Nevertheless, in vitro time-kill studiesshow that daptomycin retains bactericidal activity in spite ofincreased MIC provided that the concentrations exceed MICby several times [31] (Table 1).

However, not all studies confirmed that diminishedsusceptibility of S. aureus to vancomycin is associated withdiminished susceptibility to daptomycin. In a retrospectivestudy of the influence of previous vancomycin therapyon MIC and bactericidal activity of daptomycin and van-comycin in S. aureus (isolates from patients who receivedvancomycin within 30 days prior to development of MRSAbacteraemia were compared with isolates from those whodid not receive the drug), higher MICs and decreased killingwas established only for vancomycin but not for daptomycin[35].

5. Antibiotic Combinations with Daptomycin

In some groups of patients, such as those infected withS. aureus strains which have diminished susceptibility toboth vancomycin and daptomycin, especially in the case ofMDR S. aureus, or in patients with deep-seated daptomycinsusceptible S. aureus infections, monotherapy with dapto-mycin at approved doses (4–6 mg/kg/day) is frequently noteffective and may even yield non-susceptible isolates. In suchpatients, treatment options are highly limited, and additionof another antibiotic to daptomycin may be beneficial.The combinations can be divided into three groups: (1)classical combinations of daptomycin and gentamicin orrifampicin; (2) combinations of daptomycin and beta-lactams; (3) combinations of daptomycin with bacteriostatic

antibiotics such as trimethoprim/sulfamethoxazole (TMP-SMX) or clarithromycin. Findings on in vivo and in vitrosynergy of antibiotic combinations are depicted on Tables 2and 3.

5.1. Combinations of Daptomycin with

Gentamicin or Rifampin

5.1.1. Infective Endocarditis. Standard treatment of left-sidedIE caused by S. aureus usually includes two antibioticswith a synergistic bactericidal action, typically a cell wallsynthesis inhibitor and an aminoglycoside, with the additionof rifampicin in case of infected prosthetic material [36].Study on a small number of severely ill patients withleft-sided S. aureus endocarditis revealed that daptomycinmonotherapy was inferior to standard therapy, and that bothtreatment groups had a very low success rate (11% and 22%,resp.) [1]. A retrospective analysis of data from daptomycinoutcomes registry [37, 38] suggested that the overall successof treatment of the left-sided IE caused by S. aureus may beas high as 60% with the majority of the patients receivingdaptomycin in combination with other antibiotics. Thesefindings were a stimulus for the assessment of the efficacy ofdaptomycin in combination with other antibiotics, initiallywith gentamicin or rifampicin, that is, antibiotics used forstandard treatment.

Combination of daptomycin with gentamicin orrifampicin was tested in vitro as well as in in vivoanimal models of IE [39]. Etest with MSSA/heterogene-ous glycopeptide-intermediate S. aureus (hGISA) andMRSA/GISA showed synergy of daptomycin-gentamicin


Table 2: Information on in vivo synergy of antibiotic combinations.

Model Strain Combination Observation Ref.

Experimental modelof IE

MRSADaptomycin 6 mg/kg q24 h + rifampin300 mg q8 h

Rifampin and gentamicinantagonized/delayed the bactericidalactivity of daptomycin

[39]

Daptomycin 6 mg/kg q24 h + gentamicin1.3 mg/kg q12 h

Rabbit model of IE MRSADaptomycin 6 mg/kg q24 h + gentamicin1 mg/kg q8 h

60% vegetations sterilized [41]

Daptomycin 6 mg/kg q24 h + rifampin300 mg q8 h

20% vegetations sterilized

Rabbit model of IE DNS MRSADaptomycin 12 mg/kg q24 h + oxacillin200 mg/kg q8 h

Enhanced bacterial clearance from tissues [56]

Case report on IE

MRSA withprogressive lossof susceptibilityduringtreatment

Treated with vancomycin, thendaptomycin 6 mg/kg q24 h, thendaptomycin 12 mg/kg q24 h + rifampin300 mg q8 h

Clinical success [42]

Rabbit acuteosteomyelitis model

MRSADaptomycin 6 mg/kg q24 h Failure to eradicate bacteria [50]

Daptomycin 6 mg/kg q24 h + rifampin20a mg/kg q12 h

Eradication of bacteria: bone 100%, bonemarrow 89%, and joint fluid 44%

Guinea pigforeign-bodyinfection model

MRSA

Daptomycin 20 mg/kg q24 h + rifampin12.5 mg/kg q12 h

25% cure [51]

Daptomycin 30b mg/kg q24 h + rifampin12.5 mg/kg q12 h

67% cure

Vancomycin 15 mg/kg q12 h + rifampin12.5 mg/kg q12 h

8% cure

Rat foreign-bodyinfection model

MRSA

Daptomycin 100c mg/kg q24 h +rifampin 25 mg/kg q12 h

94% cure [53]

Daptomycin 45 mg/kg q24 h + rifampin25 mg/kg q12 h

64% cure

Vancomycin 50 mg/kg q12 h +rifampin25 mg/kg q12 h

25% cure

In vitro PK/PD modelof SEV

DNS MRSADaptomycin 6 mg/kg q24 h + TMP/SMX160/800 mg q12 h

Bactericidal activity reached at 8 hours [59]

Daptomycin 6 mg/kg q24 h + cefepime2 g q8 h


MSSA, MRSADaptomycin 6–8 mg/kg q24 h +gentamicin 5 mg/kg as a single dose; or +gentamicin 1 mg/kg q8 h, three doses only

Daptomycin 6–8 mg/kg/day combinedwith one 5mg/kg dose of gentamicin wasbactericidal in 4 h

[66]


GISA, MRSADaptomycin 3–6 mg/kg q24 h +arbekacin 100 mg q12 h

Synergy, but regrowth in 48 h in theregimen with daptomycin 4 mg/kg/day

[67]

Equivalent dosing to (a) rifampin 10 mg/kg q12 h oral administration (b) daptomycin 6 mg/kg q24 h (c) daptomycin 10 mg/kg q24 h in humans; PK/PD:pharmacokinetic/pharmacodynamic; SEV: simulated endocardial vegetations; TMP/SMX: trimethoprim-sulfamethoxazole; IE: infective endocarditis, DNS:daptomycin non-susceptible; MRSA: meticillin-resistant S. aureus; GISA: glycopeptide-intermediate S. aureus.

combination for both types of strains used while thetime-kill studies confirmed the synergy only in the caseof MSSA/hGISA [40]. Combinations of daptomycin andrifampicin were indifferent in both assays [40]. In an in vitropharmacodynamic model of IE with simulated endocardialvegetations and daptomycin susceptible MRSA strain, theaddition of rifampicin and gentamicin substantially delayedor even antagonized the bactericidal effect of daptomycin[39]. Similar results were found in an in vivo rabbit model ofaortic IE caused by daptomycin-susceptible MRSA: not only

was the addition of gentamicin or rifampicin to daptomycinat 6 mg/kg/day of no beneficial value, the combinationtreatment was less successful in lowering the bacterial cellcounts than daptomycin monotherapy [41].

However, in a clinical case of a mitral valve endocarditiswith heterogeneous population of S. aureus, where one pop-ulation was fully susceptible to vancomycin and daptomycinand the other non-susceptible, the addition of rifampicinto a high dose of daptomycin (12 mg/kg/day) resulted intreatment success with sterile blood cultures in 72 hours [42].


5.1.2. Bone Infections, Foreign-Body Infections, and Biofilms.In general, antibiotic combinations with rifampicin moresuccessfully treat prosthetic devices-related staphylococcalinfections [43, 44] and more efficiently eradicate S. aureusbiofilm than monotherapy without rifampicin [45]. Becausepenetration of antistaphylococcal antibiotics into the biofilmis variable [46] and the bacteria are in a senescent state [28],serum concentrations of the anti-staphylococcal drug mayplay a role in successful eradication of staphylococci froma biofilm [46]. In addition, activity of several antibiotics,including daptomycin, is concentration dependent, and thusenhanced with higher serum concentrations [4]. Penetrationof rifampicin [47] and daptomycin [48] in an S. epidermidisbiofilm is good, but, in the absence of data, we may onlyspeculate that the same is valid for S. aureus biofilms.

Information on the efficacy of daptomycin alone andin combination with rifampicin for treatment of S. aureusbone and foreign-body infections is rather incomplete,and limited to in vitro testing and in vivo animal modelinvestigations; the findings of the studies are not completelyunequivocal. An in vivo study using a rat model of foreign-body infection with a fully susceptible MSSA has found thatdaptomycin monotherapy at low doses (corresponding tohuman dose of 4 mg/kg/day) is as effective as vancomycin butcan lead to diminished susceptibility to daptomycin whichdevelops during treatment [49]. In an animal model of acuteosteomyelitis caused by MRSA susceptible to vancomycin,daptomycin, and rifampicin, monotherapy with daptomycin(6 mg/kg/day) was not successful and in some cases dimin-ished susceptibility occurred during therapy. Addition ofrifampicin resulted in a more frequent eradication of bacteriafrom the bone and prevented the emergence of resistantstrains [50]. Similarly, in a guinea pig foreign-body infectionmodel with MRSA susceptible to daptomycin, the addi-tion of rifampicin to an intermediate dose of daptomycin(6 mg/kg/day) resulted in treatment success in two thirdsof the animals whereas monotherapy was ineffective [51].Higher doses of daptomycin seem to be better in eradicatingthe bacteria, but combinations with rifampicin are even moreefficient. A comparison of daptomycin (corresponding tohuman dose of 10 mg/kg/day) and rifampicin monotherapyin a rat model of foreign-body infection with MRSAsusceptible to daptomycin and rifampicin has shown thatboth therapies are equally effective, but rifampicin resis-tance developed during treatment in 60% [52]. In a ratmodel of foreign-body infection with MRSA susceptibleto vancomycin, daptomycin, and rifampicin, monotherapywith a high dose of daptomycin (10 mg/kg/day) effectivelydecreased bacterial cell counts (over 3 log CFU/ml by day 11);the combination of high-dose daptomycin and rifampicinwas even more successful (a decrease in bacterial cell countsover 4.5 log CFU/mL by day 11) and cured 94% of the ani-mals whereas the standard dose of daptomycin (6 mg/kg/day)combined with rifampicin cured only two-thirds althoughbeing similarly bactericidal when compared to the high-dose daptomycin combined with rifampicin in vitro [53].However, in an in vitro model of biofilm with MRSAsusceptible to daptomycin, monotherapy with daptomycinat a high dose (10 mg/kg/day) was only minimally effective

in diminishing the biofilm cell counts within 72 h whereasthe addition of rifampicin enhanced the bactericidal activityof daptomycin against bacteria in biofilm [54].

The reason for the discrepancy in rifampicin actionamong the results obtained in animal models of IE andforeign-body infection is not clear. Recently, Olson et al.proposed that rifampicin exerts the main bactericidal actionon a biofilm while anti-staphylococcal antibiotics in thecombination with rifampicin only prevent the outgrowthof rifampicin-resistant mutants which are known to emergefrequently and in high numbers during monotherapy withrifampicin in a laboratory setting [55].

5.2. Combinations of Daptomycin and Beta-Lactams. Com-binations of daptomycin with beta-lactams have a basis inan observation that in some cases MRSA, which developsa diminished susceptibility to vancomycin or daptomycin,becomes more susceptible to oxacillin, a so-called “see-saw”effect [56]. An example of such pathogen is MDR S. aureuswhich becomes non-susceptible to daptomycin.

Various combinations have been tested in in vitro andanimal models, most commonly daptomycin combined witha beta-lactam at a fraction of its MIC.

In in vitro time-kill experiments with MRSA susceptibleto daptomycin, synergy was found between daptomycinat 0.5 MIC and 32 mg/L oxacillin (oxacillin MIC for themajority of tested strains ≥256 mg/l). Aminopenicillins,such as ampicillin, showed synergy at even lower con-centrations (2–8 mg/l ampicillin in a combination withampicillin/sulbactam) [57].

Similarly, addition of subtherapeutic levels of beta-lactams to daptomycin delayed or prevented the emergenceof diminished susceptibility to daptomycin in in vitro selec-tion studies performed on four homogenously daptomycinsusceptible MRSA which were also vancomycin-susceptible(VSSA) and two heterogeneously daptomycin susceptibleand methicillin-resistant GISA strains. The MICs increased2–4 times over the baseline in case of MRSA and only two-fold in case of GISA [58], which is substantially less thanthat seen after several passages of S. aureus in subinhibitoryconcentrations of daptomycin alone [12, 16, 17]. The effectwas the most pronounced in case of aminopenicillins, suchas ampicillin or amoxicillin/clavulanic acid [58].

Interestingly, in an in vitro pharmacodynamic model ofIE with simulated endocardial vegetations, the combinationof daptomycin 6 mg/kg/day with cefepime 2 g bid wassuperior to daptomycin monotherapy in eradication ofMRSA/VSSA but not of MRSA/hVISA [59]. Both strainswere heterogeneously non-susceptible to daptomycin withthe former having two-fold higher MIC than the latter.

In in vitro time-kill assays using clinical strains of S.aureus which developed non-susceptibility during treatment(daptomycin MIC 2–4 mg/L) and one in vitro selected non-susceptible strain (MIC 8 mg/L), treatment with a combi-nation of daptomycin and oxacillin at 0.25 MIC showedmodest increase of in vitro oxacillin bactericidal activitywithin 4–6 hours (1-2 log CFU/mL decrease in cell counts).An in vivo study of a rabbit model of aortic IE was then


Table 3: In vitro synergy of antibiotic combinations.

Method Strain Combination Observation Ref.

Etest and time-kill study hGISA/MSSA

Daptomycin withvancomycin,gentamicin, rifampin,linezolid,quinupristin/dalfopristin,ampicilin-sulbactam

Etest: additive effect with daptomycin +vancomycin and daptomycin + gentamycin,other combinations with daptomycinindifferentTime-kill study: additive effect withdaptomycin + gentamycin, othercombinations with daptomycin indifferent

[40]

Etest and time-kill study GISA/MRSA

Daptomycin withvancomycin,gentamicin, rifampin,linezolid,quinopristine/dalfopristine,ampicilin-sulbactam

Etest: additive effect only with daptomycin+ gentamycin, other combinations withdaptomycin indifferentTime-kill study: indifference

[40]

Time-kill study MRSADaptomycin +gentamicin

Synergy in all three strains [41]

Time-kill study MRSADaptomycin +rifampin

Antagonism in one strain and indifferencein two other strains

[41]

Time-kill study MRSA

Daptomycin at 0.0625MIC to 2 MIC +oxacillin orampicillin- sulbactam

Synergy of daptomycin at 0.5 MIC +oxacillin 32 mg/L or ampicillin-sulbactam2–8 mg/L (ampicillin)

[57]

In vitro model of biofilm MSSA

Daptomycin10 mg/kg/d +clarithromycin250 mg q12 h

Sustained bactericidal activity againstplanctonic and biofilm bacteria

[54]

In vitro model of biofilm MRSA

Daptomycin10 mg/kg/d +rifampin at 600 mgq24 h

Sustained bactericidal activity againstplanctonic and biofilm bacteria

[54]

In vitro selection forresistance

MRSA andGISA

Daptomycin + 0.25MIC ampicillin,amoxicillin-clavulanicacid, gentamicin,rifampin

Combination with ampicillin,amoxicillin-clavulanic aciddelayed/prevented occurrence ofnon-susceptibility; rifampin delayednon-susceptibility

[58]

Checkerboard method MRSA, MSSA

Daptomycin +rifampicin,moxifloxacin orfusidic acid

Daptomycin + fusidic acid: antagonism inone MSSA strain reported

[68]

MRSA: meticillin-resistant S. aureus; MSSA: meticillin-susceptible S. aureus; GISA: glycopeptide-intermediate S. aureus; hGISA: heteroresistant glycopeptide-intermediate S. aureus; MIC: minimal inhibitory concentration (mg/L).

performed using initial daptomycin susceptible and non-susceptible clinical MRSA isolates. An increased bactericidaleffect of the combination on the daptomycin non-susceptibleS. aureus strains was established; it was more pronounced invivo than in vitro. For the daptomycin-susceptible parentalstrains, the combination treatment with beta-lactams wasnot studied because daptomycin alone was found to besufficiently bactericidal to cure the infection [56]. In spiteof the observed “see-saw” effect (increased susceptibility tooxacillin) in daptomycin non-susceptible strains oxacillinmonotherapy was not successful. All examined strainsretained mecA gene.

5.3. Combinations of Daptomycin with Bacteriostatic Antibi-otics. A somewhat unusual combination of daptomycin and

TMP-SMX was studied in an in vitro pharmacodynamicmodel of IE with simulated endocardial vegetations. TheTMP-SMX monotherapy was found inferior to the stan-dard treatment of S. aureus IE [60]. The combination ofdaptomycin at 6 mg/kg/day and TMP-SMX at 160/800 mgbid was more rapidly bactericidal against daptomycin non-susceptible strains of S. aureus than daptomycin alone [59].

The efficacy of combination of clarithromycin and abeta-lactam or vancomycin for eradication of S. aureusbiofilms has already been demonstrated [61]. A similarincrease in bactericidal activity was observed in an in vitromodel of biofilm using an MSSA strain. The combinationof daptomycin 10 mg/kg/day and clarithromycin 250 mg bidwas more effective in eradication of bacteria in the biofilmthan daptomycin alone [53].


The mechanism behind the efficacy of some of theunusual combinations of antibiotics presented in this reviewis not clear. A concept of a mutant prevention concentrationand mutant selection window hypothesis was proposed adecade ago [62]. Mutant prevention concentration is definedas the minimal concentration of antibiotic at which nosingle-step resistant mutants are recovered on agar plateswhen plated at high inoculum (>1010 cells) [62] whilemutant selection window denotes a window between MIC99

and mutant prevention concentration in which resistantmutants are enriched (MIC99/mutant prevention concentra-tion) [63]. It is thought to represent the MICs of susceptibleand single-step resistant mutants. Both parameters are agentand bacterial-strain dependent. Mutant selection window isin some cases small with mutant prevention concentrationwell below the clinically achievable antibiotic concentrations,such as those of penicillin in case of MSSA. On the otherhand, mutant selection window of rifampicin for MSSA isvery broad (160,000) with mutant prevention concentrationof 480 mg/L [63] which is far too high for clinical use. Thismay explain the high resistance observed in the laboratory.Combined treatment with two or more antibiotics mayhave a lower mutant prevention concentration, since twoor more concomitant resistance mutations are necessary forgrowth; thus, reaching concentrations of antibiotics thatexceed mutant prevention concentration is then feasible in aclinical setting [63]. Mutant selection window hypothesis hasbeen confirmed for daptomycin in vitro, where the mutantselection window is about 2–5 with clinically achievable con-centrations well above the mutant prevention concentrationwith a currently highest approved daily dose of 6 mg/kg[64, 65]. Nevertheless, with the high protein binding of dap-tomycin [4], the observed development of non-susceptibilityin vitro and in clinical cases at a dose of 6 mg/kg/day suggeststhat the effective free drug concentrations are lower (asmuch as 90%) and that mutant prevention concentrationis not reached. This speculation is further strengthened bythe success of high doses of daptomycin (10 mg/kg/day),which are, even so, not approved by regulatory agenciesfor clinical use. A combination of daptomycin with otherantibiotics may lower the mutant prevention concentrationas has already been described for tobramycin-rifampicincombination [63].

6. Conclusions and Future Outlook

We have discussed combinations of daptomycin with otherantibiotics, a new therapeutic strategy for treatment ofcomplicated infections with S. aureus. Classical combinationof daptomycin with rifampicin seems to be effective only forforeign-body infections while data for IE is somewhat con-tradictory. Combinations of daptomycin with beta-lactams,TMP-SMX, or clarithromycin provide a fresh strategy incombating infections with MDR S. aureus; the value of thenew approaches is supported by the findings of in vitro andin vivo studies but is yet to be assessed and proven in clinicalsettings.

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Research Article

Activity of Antimicrobial Peptides andConventional Antibiotics against Superantigen PositiveStaphylococcus aureus Isolated from the Patients withNeoplastic and Inflammatory Erythrodermia

Wioletta Baranska-Rybak,1 Oscar Cirioni,2 Malgorzata Dawgul,3

Malgorzata Sokolowska-Wojdylo,1 Lukasz Naumiuk,3 Aneta Szczerkowska-Dobosz,1

Roman Nowicki,1 Jadwiga Roszkiewicz,1 and Wojciech Kamysz3

1 Department of Dermatology, Venereology and Allergology, Medical University of Gdansk, 80-210 Gdansk, Poland2 Institute of Infectious Diseases and Public Health, Marche Polytechnic University, 60121 Ancona, Italy3 Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Gdansk, Al. Gen. J. Hallera 107,80-416, Gdansk, Poland

Correspondence should be addressed to Wojciech Kamysz, [email protected]

Received 30 October 2010; Revised 14 March 2011; Accepted 15 March 2011

Academic Editor: G. J. Peters

Copyright © 2011 Wioletta Baranska-Rybak et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Superantigens are proteins comprising a group of molecules produced by various microorganisms. They are involved inpathogenesis of several human diseases. The aim of the study was the comparison of susceptibility to antibiotics and antimicrobialpeptides (AMPs) of Staphylococcus aureus (SA) strains producing staphylococcal enterotoxins SEA, SEB, SEC, SED, and TSST-1and nonproducing ones. In the group of the total 28 of the patients with erythrodermia the presence of SA was confirmed in 24cases. The total of 14 strains of SA excreted enterotoxins SEA, SEC, SED, and TSST-1. We did not observe that strains producingmentioned superantigens were less susceptible to AMPs (aurein 1.2, citropin 1.1, lipopeptide, protegrin 1, tachyplesin 3, temporinA, and uperin 3.6). The opposite situation was observed in conventional antibiotics. SA strains excreting tested superantigenshad higher MICs and MBCs than nonproducing ones. The interesting finding considering the high efficacy of AMPs, against allexamined strains of SA, makes them attractive candidates for therapeutic implication.

1. Introduction

Superantigens are proteins comprising a group of molecu-les produced by various microorganisms, such as bacte-ria (staphylococci, streptococci, and mycoplasma), fungi(yeasts), and viruses. They are involved in the pathogenesisof several human diseases (atopic eczema, toxic shock syn-drome, psoriasis, and Kawasaki disease) [1]. Superantigensare characterized by their capacity to stimulate a large num-ber of T-cells. In contrast to conventional antigens, superan-tigens bypass avoid intracellular processing and bind directlyto the major histocompatibility complex (MHC) class II mol-ecule, on the surface of the antigen processing cell, outside

the antigen-binding groove [2]. T-cells belonging to boththe CD4 and CD8 subtype are activated. T-cell activation inthe presence of superantigens may lead to the activation ofseveral percent of the total T-cell population, and therebyactivate by a factor of more than 10–100 the number of T-cells activated in the presence of conventional antigens [3].

Some 80 to 100 percent of atopic dermatitis (AD) pa-tients have skin colonization with Staphylococcus aureus (SA)[4]. It has been found on both the healthy and lesional skin ofthose patients. SA superantigens are a well-known AD-ex-acerbating factor. The pathogens concentration (cfu/cm2) onthe skin of atopic dermatitis patients is significantly higherthan on that of healthy population [5]. Suppressed levels


of ceramides, free lipoid acids, superficial polar lipids, skinnatural antimicrobial peptides (LL-37, β-defensin), as well asthe pH shifted to alkaline region (7-8), fibronectin receptorsexposure of adhesin-binding cell wall of SA, and destructionof the skin barrier by substances excreted by these germs areresponsible for SA skin colonization in AD [6–8].

The aggravating role of SA superantigens is well known inAD but has not been well documented in psoriasis. There aresingle reports concerning correlation between the severity ofAD and psoriasis and enterotoxin production of isolated SAstrains [9].

There are single reports confirming the relationship be-tween erythrodermic cutaneous T-cell lymphoma (CTCL)and superantigens producing SA colonization [10]. It hasbeen demonstrated that antibiotic therapy in CTCL cansuppress inflammation and the size of neoplastic tumoursin those cases. The relationship between staphylococcal skininfections and erythrodermic CTCL needs exploration. It canbe hypothesized that like other T-cell-mediated skin diseases,CTCL occurs in the setting of a genetically determined host(HLA determinants), a trigger (antigens or superantigens),and an immune response with cytokine and chemokineproduction. In CTCL, T-cells are attracted into the epider-mis, and they may be unable to limit their proliferation(absent apoptosis). SA superantigen presented either byLangerhans cells or by class-II-bearing keratinocytes resultsin cytokines that stimulate T-cells. It is reasonable that per-sistent colonization with toxigenic bacteria would expand thepopulation of epidermotropic T-cells and elicit productionof T-cell-activating cytokine/chemokines [11, 12].

Bering in mind that the enhanced resistance of bacteriato conventional antibiotics is a serious problem in present-day healthcare, the development of novel antimicrobial ther-apies, such as those based on various antimicrobial peptides,seems to be reasonable.

Humans express a blend of antimicrobial peptides(AMPs), which are found at biological boundaries prone toinfection. One of the most important innate defense mech-anisms of the human skin is the production of AMPs.They are produced mainly by keratinocytes in the stratumcorneum, neutrophils, or by sweat glands and are eitherexpressed constitutively like RNase 7, psoriasin, or dermcidinor after an inflammatory stimulus like β-defensin-2 (HBD-2) and -3 (HBD-3) or the cathelicidin LL-37 [13]. AMPskill bacteria by permeating their membranes, and thus,the lack of a specific molecular microbial target minimizesresistance development [14]. Actually, several peptides andpeptide-based compounds are passing clinical trials [15].Expression levels of these natural antibiotics correlate wellwith susceptibility to skin infections [16].

Herein, we investigated SA colonization in patients witherythrodermia (a skin inflammation of more than 90% ofbody surface) that developed in the course of psoriasis, atopicdermatitis, cutaneous T-cell lymphoma, or Sezary Syndrome(SS) [17, 18]. The isolated bacterial strains were analyzedfor the superantigen excretion and susceptibility to conven-tional antibiotics and selected AMPs (aurein 1.2, citropin1.1, lipopeptide Pal-KK-NH2, protegrin 1, tachyplesin 3,temporin A, and uperin 3.6).


2.1. Antimicrobial Peptides. Peptides (aurein 1.2, citropin1.1, lipopeptide Pal-KK-NH2, protegrin 1, tachyplesin 3,temporin A, and uperin 3.6) included in the study weresynthesized manually in a microwave reactor by the solid-phase method using the 9-fluorenylmethoxycarbonyl chem-istry (Fmoc) [19, 20]. The completeness of each couplingreaction was monitored by the chloranil test. The peptideswere cleaved from the solid support by trifluoroacetic acid(TFA) in the presence of water (2.5%) and triisopropylsilane(2.5%) as scavengers. The cleaved peptides were precipitatedwith diethyl ether, and cysteine-containing ones peptideswere oxidized by 0.1 M iodine in methanol. The peptideswere purified by high-performance liquid chromatography(HPLC). The resulting fractions of purity greater than 95%–98% were tested by HPLC and thin layer chromatography(TLC) for lipopeptide. The peptides were analyzed also bymatrix-assisted laser desorption ionization time of flightmass spectrometry (MALDI-TOF MS).

2.2. Bacterial Isolates and Antibiotics Disk Susceptibility Test.Twenty-eight patients with erythrodermia, hospitalized atthe Department of Dermatology, Venereology, and Aller-gology from January 2007 to October 2008, were enrolledin the study. From each patient, skin swabs were taken.All samples were cultured on the Columbia agar plates(Becton Dickinson, Germany) using standard microbiologicalprocedures. SA was identified on the basis of colony mor-phology, production of catalase, and positive slide coagu-lation test (Staphaurex and Biomerieux). The susceptibilityto antibiotics was determined by disk diffusion method asrecommended by CLSI (Clinical Laboratory Standards Insti-tute) guidelines. The following antimicrobials were tested:penicillin, oxacillin, erythromycin, doxycycline, clindamycin,rifampicin, chloramphenicol, linezolid, daptomycin, tigecy-cline, and ciprofloxacin (Oxoid, UK). The susceptibility testswere performed on the Mueller-Hinton II (Becton Dick-inson). The results obtained by disk diffusion were comparedto those of the broth microdilution in the case of linezolid,daptomycin, tigecycline, rifampicin, chloramphenicol, ery-thromycin, and clindamycin.

2.3. Enterotoxin Detection. A staphylococcal enterotoxin testkit (SET-RPLA KIT TOXIN DETECTION KIT, Oxoid) wasused for the detection of staphylococcal enterotoxins A, B,C, and D in culture by reversed passive latex agglutination.Clinical strains of SA were incubated in Tryptone SoyBroth (Becton Dickinson) and incubated at 37◦C for 18–24hours, with shaking on a water bath. After growth, theywere centrifuged at 900 g for 20 minutes at 4◦C, and thesupernatants were used as the test sample. Latex sensitisedwith antienterotoxin A, B, C, or D was added to filter-sterilized supernatants on V-well microtiter plates (Sigma-Aldrich, Germany). A visible agglutination on the bottom ofthe well was considered as a positive result.

2.4. TSST Detection. A staphylococcal toxic shock syndrometoxin kit (TST-RPLA TOXIN DETECTION KIT, Oxoid) was


used for the detection of a staphylococcal toxic shock syn-drome toxin in culture by reversed passive latex agglutina-tion. Clinical strains of S. aureus were incubated in a brain-heart infusion broth (Becton Dickinson) and incubated at37◦C for 18–24 hours, with shaking on a water bath. Aftergrowth, they were centrifuged at 900 g for 20 minutes at 4◦C,and the supernatants were used as the test sample. Latexsensitized with antitoxin was added to filter-sterilized super-natants on V-well microtiter plates (Sigma-Aldrich). A visibleagglutination on the bottom of the well was considered as apositive result.

2.5. Microorganisms and Antimicrobial Assay. A total numberof 24 SA strains were obtained from patients with erythro-dermia and 3 reference SA ones from the American Type Cul-ture Collection: 6538P ATCC, 9144 ATCC, and 25923 ATCC(Institute of Experimental Therapy, Wroclaw, Poland). MICwas determined using a microbroth dilution method withthe Mueller Hinton Broth II (MHB II) (Becton Dickinson)and initial inoculums of 5×105 CFU/mL. Polypropylene 96-well plates (Sigma-Aldrich) were incubated for 18 h at 37◦C.MIC was taken as the lowest drug concentration at whicha noticeable growth was inhibited. MBC was taken as thelowest concentration of each drug that resulted in more than99.9% reduction of the initial inoculums. The experimentswere performed in triplicate.

3. Results

3.1. Staphylococcus Aureus Isolation. In a group of 28 patientswith erythrodermia (11 in the course of psoriasis, 9 withatopic dermatitis, 6 with CTCL, and 2—Sezary syndrome)the presence of SA was confirmed in 24 cases. Negativecultures for SA were noticed in three patients with psoriasis.

3.2. Superantigens Detection. A total number of 14 SA strainsexcreted enterotoxins SEA (8 strains), SEC (3 strains), and/orSED (5 strains) and only one TSST-1 in the group of 24.Intermediate results (+/−) were considered as negative. Inthe group of 9 patients with AD, the superantigen-producingstrains were detected in 6 patients, in the group of 6 patientswith CTCL—in 3 cases and in 8 patients with psoriasis—in3 cases. SA strains isolated from two patients with SS did notproduce the above-mentioned superantigens.

3.3. Antibiotics and Antimicrobial Peptides Susceptibility. Thesusceptibility to antibiotics determined by the disk diffusionmethod and broth microdilution gave identical results (datanot shown). The antibiotics used in the study, rifampicin,tigecyline, vancomycin, daptomycin, ciprofloxacin, chloram-phenicol, clindamycin, and erythromycin, exhibited diverseactivities against clinical isolates of SA. The rifampicin,tigecycline, vancomycin, and daptomycin MICs values,which were the lowest among the tested antibiotics, variedbetween 1 and 4 mg/L. The other ones were higher than thetested antimicrobial peptides: tachyplesin 3, lipopeptide, andprotegrin 1 were extremely effective against all the testedbacterial strains (MIC values between 1 and 8 mg/L); see

1 0.51 1 20.5 320.5321 0.52 22 3264256

512

MICs90%

of antibiotics for clinicalisolates from erythrodermia

Superantigen negative S. aureus (10)Superantigen positive S. aureus (14)

Rif

ampi

cin

Tig

ecyc

line

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ezol

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Van

com

ycin

Dap

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Cip

rofl

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lora

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Ery

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Clin

dam

ycin

Figure 1: The relationship between superantigen production andsusceptibility to conventional antibiotics.

Table 1. The reference strains were more susceptible to bothconventional antibiotics and AMPs than the clinical ones; seeTable 2.

3.4. Correlation Study. We did not notice that strains pro-ducing tested superantigens (SEA, SEC, SED, and TSST-1)were less susceptible to AMPs than nonproducing ones. Theopposite situation was observed in conventional antibiotics.SA strains excreting those superantigens had higher MICsand MBCs Figures 1 and 2.

4. Discussion

Bacterial superantigens, which stimulate clonal expansionof T-cells by mechanisms involving specific HLA molecules,have also been hypothesized to cause inflammatory skin dis-eases [10]. The mechanisms by which these toxins act remainstill unknown. This is the first report of the occurrence ofstaphylococcus superantigens in erythrodermic skin diseases(AD, psoriasis, CTCL, and SS).

There are many studies that explain the effect of SA onAD [21]. Most SA strains isolated from AD patients canproduce superantigenic toxins such as staphylococcal entero-toxin SEA, SEB, SEC, SED, and the toxic shock syndrometoxin-1 (TSST-1) that correspond well with our findings(66.7% of strains excreted tested superantigens). Coloniza-tion and infection with Staphylococcus and Streptococcus havebeen reported to exacerbate psoriasis [22, 23]. The presenceof SA in psoriatic erythrodermia was confirmed in 8 out of 11patients, while the ability to produce examined superantigenswas detected in 3 strains. CTCL patients resemble those withacquired immunodeficiency syndrome who cannot clearthe skin off staphylococcus and have protracted pruritusand erythrodermic psoriasis [10]. The association betweenstaphylococcal colonization and the erythrodermic form ofCTCL deserves further attention and study. The strainsexcreting specified superantigens colonized 50% of patientswith CTCL in our study.


Table 1: The activity of antimicrobial peptides and conventional antibiotics against S. aureus clinical isolates.

Strain (no. of isolates) and agentMIC (mg/liter) MBC (mg/liter)

Range 50% 90% Range 50% 90%

Superantigen negative S. aureus (10)

Tachyplesin 3 1–4 2 4 2–4 2 4

Lipopeptide 1–8 2 4 2–8 4 8

Protegrin 1 1–4 2 4 2–8 4 8

Temporin A 8–32 8 16 16–64 16 32

Citropin 1.1 8–32 16 32 16–64 16 64

Aurein 1.2 32–128 64 128 64–128 64 128

Uperin 3.6 128–256 128 128 128-128 128 128

Rifampicin 0.25–1 0.25 1 1-2 0.25 1

Tigecycline 0.5–1 0.5 0.5 1-2 1 1

Linezolid 0.5–2 1 1 NT NT NT

Vancomycin 0.5–1 1 1 1–4 1 2

Daptomycin 1–4 1 2 1–4 2 2

Ciprofloxacin 0.25–0.5 0.25 0.5 1–8 1 4

Chloramphenicol 2–32 4 32 8–32 8 32

Erythromycin 0.25–64 0.5 0.5 1–4 1 2

Clindamycin 16–64 32 32 64–128 32 64

Superantigen positive S. aureus (14)

Tachyplesin 3 1–4 2 4 2–4 2 4

Lipopeptide 1–8 2 4 2–8 4 8

Protegrin 1 1–4 2 4 2–8 4 8

Temporin A 8–32 8 16 16–64 32 64

Citropin 1.1 8–32 16 32 16–64 16 64

Aurein 1.2 32–128 64 128 64–128 64 128

Uperin 3.6 128–256 128 128 128-128 128 128

Rifampicin 0.25–1 0.25 1 0.5–2 1 2

Tigecycline 0.5–1 0.5 0.5 1-2 1 1

Linezolid 0.5–2 1 2 NT NT NT

Vancomycin 0.5–2 1 2 1–4 2 4

Daptomycin 1–4 1 2 1–4 2 4

Ciprofloxacin 0.25–128 2 32 2–128 4 64

Chloramphenicol 4–128 8 64 8–128 16 128

Erythromycin 0.25–512 1 256 4–512 8 128

Clindamycin 8–512 64 512 16–512 64 128

We found that 24 out of 28 erythrodermic patients hada staphylococcal culture positive from the skin, and testedsuperantigens were detected in SA strains isolated from 14patients. The purpose of our study was to investigate whetheror not the strains producing SEA, SEC, SED, and TSST-1 aremore resistant to conventional antibiotics and AMPs. Con-sidering susceptibility to antimicrobial peptides, we did notnotice any significant differences between strains producingtested superantigens and nonproducing strains. The oppositesituation was noticed in susceptibility to conventional antibi-otics. The SA strains producing specified superantigens hadhigher MICs and MBCs as compared to the nonproducingones. Especially alarming is the higher resistance of thosestrains to macrolides and lincosamides which could not onlykill bacteria and diminish the rate of colonization but also

inhibit their superantigen and toxin production [24, 25].One study showed that β-lactams which target cell walldevelopment in bacteria and are the basis for the treatmentof skin and soft-tissue infections could even increase the pro-duction of toxins [25]. SA strains which can produce super-antigens and toxins and additionally acquire the mechanism(i.e., resistance) protecting their production are the mostdifficult to control. Adachi et al. speculated that inhibitorsof protein synthesis may have an antimicrobial effect andalso inhibitory effect on superantigen production from SA[24]. In fact, the inhibition of superantigen production byantibiotics may not be sufficient to justify clinical efficacy.

Over 50% incidence of production of tested superanti-gens in strains from AD patients is in accordance with pre-vious studies [26].


Table 2: The activity of antimicrobial peptides and conventionalantibiotics against S. aureus referential strains.

MIC (mg/liter)

ATCC 6538P ATCC 9144 ATCC 25923

Tachyplesin 3 2 2 2

Lipopeptide 2 2 4

Protegrin 1 4 2 4

Temporin A 8 16 16

Citropin 1.1 8 32 32

Aurein 1.2 64 64 64

Uperin 3.6 64 64 128

Rifampicin 0.25 0.25 0.25

Tigecycline 0.25 0.25 0.25

Linezolid 0.5 1 0.5

Vancomycin 1 2 1

Daptomycin 2 2 2

Ciprofloxacin 1 2 1

Chloramphenicol 4 4 4

Erythromycin 1 1 1

Clindamycin 4 2 2

Several studies on the effect of antimicrobial treatmenton the colonization with SA and the severity of inflammationgave conflicting results. In several open or double-blindplacebo-controlled trials, topical or systemic antibiotics wereable to reduce colonization density and led to a partialimprovement of skin lesions [27–29]. On the other hand,treatment with oral antibiotics did not lead to a significantimprovement of AD in two double-blind placebo-controlledstudies [30, 31]. No matter what kind of the treatmenthas been adopted, recolonization occurred after 4–8 weeks[32].

5. Conclusions

From a clinical point of view, our study has several implica-tions. Considering that erythrodermic patients are frequentlytreated with various antibiotics, the question may be raisedwhether excessive use of antibiotics and induction of resis-tance are associated with cross-resistance to AMPs. We foundno evidence for the development of the AMPs resistancein relation to antibiotic susceptibility, likely reflecting thefact that the mode of action of the antibiotics investigatedherein is not shared with AMPs. An interesting finding ofthe high efficacy of AMPs, especially lipopeptides, againstall tested strains of SA makes them attractive candidates fortherapeutic application.

Conflict of Interests

The authors declare that they have no conflict of interests.

1 0.51 1 20.5 320.5 321 0.52 2 2 3264

256

512

MICs90% of antibiotics for clinicalisolates from erythrodermia


Rif

ampi

cin

Tig

ecyc

line

Lin

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id

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com

ycin

Dap

tom

ycin

Cip

rofl

oxac

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thro

myc

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Clin

dam

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(a)

4 4 4 16 32

128128

4 44 16 32

128128

MICs90% of peptides for clinicalisolates from erythrodermia


Tach

yph

elas

in3

Lip

opet

ide

Pro

tegr

in1

Tem

pori

nA

Cit

ropi

n1.

1

Au

rein

1.2

Up

erin

3.6

(b)

Figure 2: The relationship between superantigen production andsusceptibility to antimicrobial peptides.

Abbreviations

SA: Staphylococcus aureusSEA: Staphylococcal enterotoxin ASEB: Staphylococcal enterotoxin BSEC: Staphylococcal enterotoxin CSED: Staphylococcal enterotoxin DTSST: Staphylococcal toxic shock syndrome toxinAMPs: Antimicrobial peptidesAD: Atopic dermatitisCTCL: Cutaneous T-cell lymphomaSS: Sezary syndromeMIC: Minimal inhibitory concentrationMBC: Minimal bactericidal concentration.

Acknowledgments

This work was supported by grant from the Polish ScientificCommittee (Project Nr NN402195235). The authors aregrateful to Phizer company for supplying us with linezolid.They also thank Lukas Szulka for technical assistance.


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