Home » Volumes » Volume 45 July/August 2012 » Rifampicin fails to eradicate mature biofilm formed by methicillin-resistant Staphylococcus aureus

Rifampicin fails to eradicate mature biofilm formed by methicillin-resistant Staphylococcus aureus

Keli Cristine ReiterI,II; Gustavo Enck SambranoII; Bárbara VillaII; Thiago Galvão da Silva PaimI,II; Caio Fernando de OliveiraI,II; Pedro Alves d'AzevedoI,II

IPrograma de Pós-graduação em Ciências da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS IILaboratório de Cocos Gram-positivos, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS

DOI: 10.1590/S0037-86822012000400011


INTRODUCTION: Antimicrobial activity on biofilms depends on their molecular size, positive charges, permeability coefficient, and bactericidal activity. Vancomycin is the primary choice for methicillin-resistant Staphylococcus aureus (MRSA) infection treatment; rifampicin has interesting antibiofilm properties, but its effectivity remains poorly defined.
METHODS: Rifampicin activity alone and in combination with vancomycin against biofilm-forming MRSA was investigated, using a twofold serial broth microtiter method, biofilm challenge, and bacterial count recovery.
RESULTS: Minimal inhibitory concentration (MIC) and minimal bactericidal concentration for vancomycin and rifampicin ranged from 0.5 to 1mg/l and 0.008 to 4mg/l, and from 1 to 4mg/l and 0.06 to 32mg/l, respectively. Mature biofilms were submitted to rifampicin and vancomycin exposure, and minimum biofilm eradication concentration ranged from 64 to 32,000 folds and from 32 to 512 folds higher than those for planktonic cells, respectively. Vancomycin (15mg/l) in combination with rifampicin at 6 dilutions higher each isolate MIC did not reach in vitro biofilm eradication but showed biofilm inhibitory capacity (1.43 and 0.56log10 CFU/ml reduction for weak and strong biofilm producers, respectively; p<0.05).
 In our setting, rifampicin alone failed to effectively kill biofilm-forming MRSA, demonstrating stronger inability to eradicate mature biofilm compared with vancomycin.

Keywords: Staphylococcus aureus. Rifampicin. Vancomycin. Biofilm. Resistance.


INTRODUÇÃO: A atividade dos antimicrobianos em biofilmes depende do seu peso molecular, de cargas positivas, coeficiente de permeabilidade e atividade bactericida. Vancomicina é a escolha primária para o tratamento de infecções causadas por Staphylococcus aureus resistentes à meticilina (MRSA) e rifampicina possui interessante propriedade antibiofilme, apesar da sua efetividade ainda ser fracamente definida.
MÉTODOS: Foi investigada a atividade da rifampicina sozinha e em combinação com vancomicina frente à MRSA formadores de biofilme, utilizando o método das microplacas com diluição seriada e recuperação bacteriana em biofilme após exposição antimicrobiana.
RESULTADOS: Concentração inibitória minima (MIC) e concentração bactericida mínima (MBC) para vancomicina e rifampicina foi de 0,5-1mg/l e 0,008-4mg/l; 1-4mg/l e 0,06-32mg/l, respectivamente. Biofilmes maduros foram expostos à vancomicina e rifampicina, e a concentração mínima para erradicar o biofilme (MBEC) foi 64-32.000 e 32-512 vezes maior do que para células planctônicas, respectivamente. A combinação de vancomicina (15mg/l) com rifampicina (6-diluições maior do que o MIC de cada isolado) não atingiu erradicação do biofilme in vitro, porém apresentou capacidade inibitória do biofilme formado (redução de 1,43 e 0,56log10 UFC/ml para produtores fracos e fortes, respectivamente; p<0,05).
 Rifampicina sozinha falhou em efetivamente matar MRSA formadores de biofilme, demonstrando fraca habilidade para erradicação de biofilmes maduros comparado com vancomicina.

Palavras-chaves: Staphylococcus aureus. Rifampicina. Vancomicina. Biofilme. Resistência.




Biofilms provide bacterial cell attachment to an abiotic surface very rapidly, and growth-dependent accumulation form multilayered cell clusters surrounded by a slime-like glycocalix matrix1. This matrix confers increased protection against antimicrobials in addition to facilitating adherence to medical devices and cause persistent infections2. Antimicrobial activity on biofilms depends on their molecular size, positive charges, permeability coefficient, and bactericidal activity3, indicating the importance of testing new drugs antibiofilm activity or even trying alternative drug combinations.

Vancomycin is the primary choice for methicillin-resistant Staphylococcus aureus (MRSA) infections treatment, although recent studies have demonstrated treatment failures even when the bacteria still is in vitro susceptible to vancomycin4-7. This antimicrobial antibiofilm activity already was evaluated and seemed to be highly powerless regarding complete biofilm eradication requirement8,9.

Rifampicin has putative antibiofilm properties, ability to penetrate staphylococcal biofilm10, and had demonstrated promising utility as agent for eradicating S. aureus biofilm alone8 or in combination with other drugs especially for device-related infections11-14. Nevertheless, its effectivity remains poorly defined because few and limited supporting human studies have been performed11,14. Moreover, recently, in vitro studies have demonstrated antagonistic rifampicin effects in experimental foreign body infection models15.

To evaluate antimicrobial behavior in biofilm, rifampicin and vancomycin activities alone and in combination against device-related MRSA were investigated.



Bacterial isolates

Five known biofilm-producing MRSA (H142SA, H290SA, H369SA, H403SA, and H410SA) previously obtained from five different patients with device-related bloodstream infections at Complexo Hospitalar Santa Casa de Misericordia de Porto Alegre (Porto Alegre, Brazil) were evaluated. These isolates were selected from positive blood cultures and previously assessed for biofilm-producing ability, mecA and SCCmec typing, and antimicrobial susceptibility pattern (Table 1)16.



Minimum inhibitory concentration and MBC testing

Conventional minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of vancomycin and rifampicin were determinate by twofold serial broth microdilution according to CLSI (2009) guidelines17Staphylococcus aureus ATCC 29213 was tested as quality control. Vancomycin and rifampicin analytical powder was provided by Sigma-Aldrich (St. Louis, MO, USA).

Biofilm susceptibility tests

Minimal inhibitory concentration in biofilm (MICADH) and minimum biofilm eradication concentration (MBEC) experiments were performed as described elsewhere8, with a serial twofold dilution of each antimicrobial in cation-adjusted Mueller-Hinton broth. Minimum inhibitory concentrationADH was defined as the minimal antimicrobial concentration at which there was no observable bacterial growth in the wells containing adherent microcolonies, in other words, the minimal concentration that inhibits the bacterial growth. Minimum biofilm eradication concentration was defined as the minimal antimicrobial concentration at which bacteria fail to regrow after antimicrobial exposure, that is, the minimal concentration required to eradicate the biofilm. All determinations were performed in duplicate. Rifampicin MBEC values also were determined using an alternative method18, to compare and confirm the results. It was also performed in duplicate.

Biofilm challenge and recovery

Standard vancomycin concentration corresponding to clinical pharmacokinetic trough concentration goal of 15mg/l19, rifampicin at 6-dilution higher each microorganism MIC, and vancomycin 15mg/l in combination with rifampicin 6-dilution higher each microorganism MIC were used in biofilm challenge according to Raad et al.20 with some modifications. Briefly, biofilms formed on the MRSA microtiter plates’ bottom were rinsed twice with sterile saline and submitted to antimicrobial exposure. Challenged biofilms were washed twice in sterile saline and placed with fresh trypticase soy broth (TSB), and the remaining biofilm was mechanically disrupted. Bacterial count recovery was determined by 1-µl culture on trypticase soy agar (upper detection limit 6log10 colony-forming units per mililiter (CFU/ml)), in quadruplicate. Bactericidal activity was defined as a 3log10 CFU/ml) or greater reduction (99.9% kill) from the untreated biofilms21. Only rifampicin-susceptible isolates were tested and organized into weak (H290SA and H410SA) and strong/moderate (H142SA and H403SA) biofilm producers.

Statistical analysis

The difference between positive control (without antimicrobial exposure) and each isolate after antimicrobial exposure was characterized as ∆log reduction, in log10 CFU/ml. The variables investigated were the antimicrobial tested (vancomycin, rifampicin or the association of both) and intensity of biofilm production (weak or strong), which were analyzed by applying two-tailed independent samples t Student test with significant p value of 0.05 or lower. All statistical tests were performed using SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA).



All isolates were susceptible to vancomycin by MIC deter-mination. Only H142SA was the one not considered multiresistant but demonstrated strong biofilm formation ability and SCCmec type I.

Vancomycin MBC was constantly one dilution higher than MIC values for all tested isolates, and MBEC ranged from two to six dilutions higher than MICADH values. Only H410SA on biofilm remained within vancomycin susceptibility breakpoint. However, its MBEC was six dilutions higher than MICADH (Table 2).



High rifampicin MBEC/MIC ratio and MBEC measurements six to fifteen dilutions higher than MIC were observed. Strong biofilm producers presented higher MBEC values than weak biofilm producers, same with MICADH values. Both methods used for rifampicin MBEC testing showed very similar results (Table 2).

Rifampicin-susceptible isolates CFU/ml counting was performed. Rifampicin at 0.5mg/l and vancomycin at 15mg/l did not achieve bactericidal activity at 24h, same with combination of both drugs. Log10 CFU/ml reduction was significantly different between weak and strong biofilm producers (p < 0.05) and among all antimicrobials tested (p < 0.05) (Figure 1).




Device-related infections have been associated with bacteria embedded in biofilm11,22,23, and rifampicin could be used as additional therapy in foreign body-related infections due to MRSA24. Otherwise, in our setting, vancomycin is preferable as antimicrobial coverage, and rifampicin is unusually prescribed. Because studies have demonstrated that rifampicin in combination with other drugs might be more effective12,13 despite contradictory results15, we decided to investigate rifampicin activity alone and in combination with vancomycin against biofilm-forming MRSA.

Distinct research groups have investigated anti-Gram-positive drug activity, alone or in combination with other agents, against biofilm-forming bacteria. However, not all studies are comparable in terms of results concordance8,12,20,25-28. In this study, vancomycin was not able to inhibit adherent cells or eradicate mature biofilms at the same concentration necessary for killing planktonic cells. Likewise, MICADH and MBEC values were widely distant from each other; biofilm-eradicating concentrations varied from 8- to 64-fold higher than biofilm-inhibiting concentrations. Vancomycin susceptibility against biofilm-forming staphylococci was previously studied in Brazil9and showed alarming results—as also demonstrated in this study—because this drug is the primary choice for antimicrobial and empirical treatment.

Unlike other studies8,12,13, we demonstrated that rifampicin alone is worse than vancomycin for inhibiting staphylococci embedded in biofilm. On the other hand, rifampicin in combination with vancomycin at 15mg/l inhibited bacterial grown in biofilm and, therefore, improved vancomycin activity, because of rifampicin’s better biofilm penetration10,20. Rifampicin associated with other antimicrobials, for example, gentamicin and clindamycin, may be a better strategy and also more effective than rifampicin alone29, but all MRSA in our study were resistant to both drugs, and this combination would not be appropriate in this case.

Bacterial growth inhibition occurred with rifampicin in combination with vancomycin, but absence of biofilm eradication may contribute to persistence of biofilm-forming bacteria in the human body. Further and more specific studies in our setting regarding rifampicin activity in biofilm are necessary to fully understand its place in biofilm-related MRSA infection treatment, but this antimicrobial could be considered an interesting candidate for enhancer of antistaphylococcal activity combined with more bactericidal agents.



The authors declare that there is no conflict of interest.



This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS).



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 Address to:
Dra. Keli Cristine Reiter
Laboratório de Cocos Gram-positivos/UFCSPA
Rua Sarmento Leite 245
90050-170 Porto Alegre, RS, Brasil
Phone: 55 51 3303-8742; Fax: 55 51 3303-8810
e-mail: kelicrisr@gmail.comkelir@ufcspa.edu.br

Received in 31/08/2011
Accepted in 27/10/2011