Folia Microbiol DOI 10.1007/s12223-013-0296-9
Antimicrobial activity of tigecycline alone or in combination with rifampin against Staphylococcus epidermidis in biofilm Ewa Szczuka & Adam Kaznowski
Received: 14 November 2012 / Accepted: 11 December 2013 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014
Abstract Staphylococcus epidermidis is a commensal inhabitant of the healthy human skin, but in the recent years, it has been recognized as a nosocomial pathogen especially in immunocompromised patients. The pathogenesis of S. epidermidis is thought to be based on its capacity to form biofilms on the surface of medical devices, where bacterial cells may persist, protected from host defence and antimicrobial agents. Rifampin has been shown to be one of the most active antimicrobial agents in the eradication of the staphylococcal biofilm. However, this antibiotic should not be used in monotherapy. Therefore, one of the objectives of our research was to study the efficacy of the tigecycline/rifampin combination against methicillin-resistant S. epidermidis embedded in biofilms. Of the 80 clinically significant S. epidermidis isolates, 75 strains possess the ability to form a biofilm. These bacteria formed the biofilm via ica-dependent mechanisms. However, other biofilmassociated genes, including aap (encoding accumulation-associated protein) and bhp (coding cell wall-associated protein), were present in 85 and 29 % of isolates, respectively. The biofilm structures of S. epidermidis strains were also analyzed in confocal laser scanning microscopy (CLSM) and the obtained image demonstrated differences in their architecture. In vitro studies showed that the MIC value for tigecycline against S. epidermidis growing in the biofilm ranged from 0.125 to 2 μg/mL. Tigecycline in combination with rifampin demonstrated higher activity against bacteria embedded in biofilms than tigecycline alone. E. Szczuka (*) : A. Kaznowski Department of Microbiology, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University, ul. Umultowska 89, 61-614 Poznań, Poland e-mail: [email protected]
Abbreviations CLSM CNS CRA icaADBC MPU S
PBS PCR PI PIA
Confocal laser scanning microscopy Coagulase negative staphylococci Congo red agar icaADBC gene Bacteriology collection of the Department of Microbiology, A. Mickiewicz University, Poznań Phosphate-buffered saline Polymerase chain reaction Propidium iodide Polysaccharide intercellular adhesion
Introduction Staphylococcus epidermidis is a coagulase-negative staphylococcus (CNS) that has emerged as an important opportunistic pathogen. It is frequently associated with bacteremia and hospital-acquired infections, particularly in patients with catheters and indwelling medical devices. This is related to its ability to form biofilm, which is composed of surfaceadherent microorganisms embedded in a self-synthesized extracellular polymeric matrix, usually comprising polysaccharide, proteins, enzymes, extracellular DNA and teichoic acid (Mack et al. 2007; Rodhe et al. 2006; Voung and Otto 2002). The formation of biofilms is a multistep process that involves initial adherence of cells to the surface to be colonized, accumulation in multilayered cell clusters, biofilm maturation and detachment of planktonic cells from the biofilm, which may then initiate a new cycle of biofilm formation elsewhere (Mack et al. 2006; Rodhe et al. 2010). The adherence of cells to biomaterials can occur early after implantation or later, when the surface of an artificial device has undergone
Folia Microbiol
significant changes and when proteins such as fibrinogen, fibronectin or vitronectin are absorbed on the artificial surface. Specific binding to proteins involves cell wall-associated adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). In the accumulative phase, a polysaccharide intercellular adhesin (PIA) composed of β-1,6-linked 2-amino-2-deoxy-D-glucopyranosyl residues is the major component mediating intercellular adhesion in S. epidermidis strains. PIA is synthesized by products of icaADBC operon, particularly by the enzyme Nacetylglucosaminyltransferase encoded by the icaA gene (Arciola et al. 2001; Costa et al. 2009). Also, the accumulation-associated protein (Aap) and the cell wallassociated protein (Bhp) related to the Bap of Staphylococcus aureus can mediate cell aggregation during the process of biofilm development (Mack et al. 2007; Rodhe et al. 2004). Moreover, recent reports have demonstrated that extracellular DNA fragments and extracellular teichoic acid are structural components in the S. epidermidis biofilm matrix (Rodhe et al. 2010; Izano et al. 2008). The extracellular matrix protects microbial cells from phagocytes and antibiotics leading to chronic infections. The biofilm harbours slow or nongrowing bacteria which are less susceptible to antibiotics. Also, the concentration of enzymes which inactivate antibiotics is significantly higher within biofilms. Consequently, biofilm-associated bacteria are 100 to 10,000 times more resistant to antibiotics than planktonic bacteria. Previous studies showed that rifampin has the ability to penetrate through the biofilm, but the use of this antibiotic alone was associated with the emergence of rifampin-resistant strains, and for this reason, this antibiotic compound should be combined with other antibiotic agents. Tigecycline, a novel glycylcycline antibiotic, has demonstrated excellent in vitro and in vivo activity against a large number of Gram-positive bacteria, including methicillin-resistant S. aureus (MRSA) strains (Aybar et al. 2012). The tigecycline/rifampin combination has been investigated in vitro and in animal models. In in vitro studies, the addition of rifampin to tigecycline resulted in an antagonism for two of four tested S. aureus strains (Mercier et al. 2002). In other studies, antagonism was not observed for the tigecycline/rifampin combination against MRSA strains (Petersen et al. 2006). Data from the rabbit model experiment, although limited to one strain, demonstrated that the combination of tigecycline with rifampin exhibited complete infection clearance from the tibia. However, the authors underline that the eradication of the staphylococcal biofilm was also very high, when the tigecycline alone was tested (Yin et al. 2005). In this study, we examined the biofilm formation by S. epidermidis clinical isolates and the effect of tigecycline alone or in combination with rifampin against adhered bacteria in the biofilm.
Materials and methods Bacterial strains Eighty methicillin-resistant S. epidermidis strains were collected from treated patients in the 700-bed Provincial Hospital in Poznań, Poland, over a period of 2 years. The majority of isolates were isolated from the blood of patients with bacteremia. The isolates were identified by conventional methodology and using Vitek 2 system (bioMérieux, France). All the isolates were resistant to oxacillin, which was confirmed by the presence of the mecA gene (Geha et al. 1994). The biofilm-positive S. epidermidis strain ATCC 35984 and biofilm-negative S. epidermidis strain ATCC 12228 were included in this study as a control. Detection of the icaA, bhp, aap, fbe, embp, atlE, aap and mecA genes and insertion sequence IS256 by PCR Isolation of DNA was performed using the Genomic DNA Prep Plus kit (A&A Biotechnology, Gdynia, Poland). The PCR was applied to determine the presence or absence of the icaA, bhp, aap, fbe, embp, atlE (Rodhe et al. 2007), aap (Vandecasteele et al. 2003) and mecA genes (Geha et al. 1994) and the insertion sequence IS256 (Rodhe et al. 2007). The amplification products were electrophoresed in 1.5 % agarose gel. The DNA was stained with ethidium bromide, visualized on a UV light transilluminator and documented with V.99 Bio-Print system (Vilber Lourmat, Torcy, France). Determination of biofilm production by microtiter plate assay Biofilm production was determined as described previously (Christensen et al. 1985; Fredheim et al. 2009; Kim et al. 2008). In brief, the overnight culture grown in tryptic soy broth (TSB; Difco, Becton Dickinson, France) supplemented with 0.25 % glucose was diluted 1:100 in TSB with 0.25 % glucose and 200 μL transformed into 96-well polystyrene microtiter plates. After 24 h of incubation at 37 °C, the cultures were gently removed and wells were washed three times with phosphate-buffered saline (PBS), air-dried and stained with the 0.4 % crystal violet solution for 10 min. The plate was washed, the adherent cells were resuspended in the ethanol-acetone mixture (70:30) and finally, the absorbance at 490 nm was determined. The strains were considered biofilm positive if they had an absorbance of >0.25. Each isolate was tested in triplicate. The biofilm-producing strain ATCC 35984 was used as the positive control, whereas the ATCC 12228 was used as the negative control. Confocal laser scanning microscopy (CLSM) Overnight cultures of various strains were inoculated into a cover glass cell culture chamber (Nunc) as described by Qin et al. (2007). After 24 h of incubation at 37 °C, the chamber cover glasses were washed gently three times with PBS to remove planktonic cells. Biofilms and bacteria were stained using SYTO and PI (Live/Dead BacLight Bacterial Viability kits,
Folia Microbiol
Invitrogen) for 15 min and observed under a microscope (Carl Zeiss LSM 510-Axiovert 200M) equipment with detector and filter sets for monitoring SYTO9 and PI. The Carl Zeiss confocal software was used for the analysis of the biofilm, which permitted the collections of image stacks and threedimensional (3D) visualization of the biofilms. Susceptibility testing with adherent cells The biofilm minimal inhibitory concentration (MICb) and minimal bactericidal concentration (MBCb) were determined according to the methods of Giacometti et al. (2005). Briefly, the biofilms were washed three times with PBS to remove non-adherent cells. Serial twofold dilutions of antimicrobial agents in MuellerHinton (MH) broth were added to wells containing adherent bacteria. The 96-well polystyrene plates were incubated for 18 h at 37 °C. The MICb was defined as the lowest concentration of antibiotics at which there was no observable bacterial growth. In order to determine the MBCb, the MH broth with antibiotics was removed from wells and antibiotic-free MH broth was added. The plates were incubated for 18 h at 37 °C. The MBC was defined as the lowest drug concentration at which there was no bacterial growth after removal of the antibiotics. To determine the changes due to the antibiotic and combination of antibiotics on isolates, a general linear model was calculated. All tests were performed using STATISTICA software (10.00 StatSoft, Tulsa, OK, USA). A P value of
Antimicrobial activity of tigecycline alone or in combination with rifampin against Staphylococcus epidermidis in biofilm Ewa Szczuka & Adam Kaznowski
Received: 14 November 2012 / Accepted: 11 December 2013 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014
Abstract Staphylococcus epidermidis is a commensal inhabitant of the healthy human skin, but in the recent years, it has been recognized as a nosocomial pathogen especially in immunocompromised patients. The pathogenesis of S. epidermidis is thought to be based on its capacity to form biofilms on the surface of medical devices, where bacterial cells may persist, protected from host defence and antimicrobial agents. Rifampin has been shown to be one of the most active antimicrobial agents in the eradication of the staphylococcal biofilm. However, this antibiotic should not be used in monotherapy. Therefore, one of the objectives of our research was to study the efficacy of the tigecycline/rifampin combination against methicillin-resistant S. epidermidis embedded in biofilms. Of the 80 clinically significant S. epidermidis isolates, 75 strains possess the ability to form a biofilm. These bacteria formed the biofilm via ica-dependent mechanisms. However, other biofilmassociated genes, including aap (encoding accumulation-associated protein) and bhp (coding cell wall-associated protein), were present in 85 and 29 % of isolates, respectively. The biofilm structures of S. epidermidis strains were also analyzed in confocal laser scanning microscopy (CLSM) and the obtained image demonstrated differences in their architecture. In vitro studies showed that the MIC value for tigecycline against S. epidermidis growing in the biofilm ranged from 0.125 to 2 μg/mL. Tigecycline in combination with rifampin demonstrated higher activity against bacteria embedded in biofilms than tigecycline alone. E. Szczuka (*) : A. Kaznowski Department of Microbiology, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University, ul. Umultowska 89, 61-614 Poznań, Poland e-mail: [email protected]
Abbreviations CLSM CNS CRA icaADBC MPU S
PBS PCR PI PIA
Confocal laser scanning microscopy Coagulase negative staphylococci Congo red agar icaADBC gene Bacteriology collection of the Department of Microbiology, A. Mickiewicz University, Poznań Phosphate-buffered saline Polymerase chain reaction Propidium iodide Polysaccharide intercellular adhesion
Introduction Staphylococcus epidermidis is a coagulase-negative staphylococcus (CNS) that has emerged as an important opportunistic pathogen. It is frequently associated with bacteremia and hospital-acquired infections, particularly in patients with catheters and indwelling medical devices. This is related to its ability to form biofilm, which is composed of surfaceadherent microorganisms embedded in a self-synthesized extracellular polymeric matrix, usually comprising polysaccharide, proteins, enzymes, extracellular DNA and teichoic acid (Mack et al. 2007; Rodhe et al. 2006; Voung and Otto 2002). The formation of biofilms is a multistep process that involves initial adherence of cells to the surface to be colonized, accumulation in multilayered cell clusters, biofilm maturation and detachment of planktonic cells from the biofilm, which may then initiate a new cycle of biofilm formation elsewhere (Mack et al. 2006; Rodhe et al. 2010). The adherence of cells to biomaterials can occur early after implantation or later, when the surface of an artificial device has undergone
Folia Microbiol
significant changes and when proteins such as fibrinogen, fibronectin or vitronectin are absorbed on the artificial surface. Specific binding to proteins involves cell wall-associated adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). In the accumulative phase, a polysaccharide intercellular adhesin (PIA) composed of β-1,6-linked 2-amino-2-deoxy-D-glucopyranosyl residues is the major component mediating intercellular adhesion in S. epidermidis strains. PIA is synthesized by products of icaADBC operon, particularly by the enzyme Nacetylglucosaminyltransferase encoded by the icaA gene (Arciola et al. 2001; Costa et al. 2009). Also, the accumulation-associated protein (Aap) and the cell wallassociated protein (Bhp) related to the Bap of Staphylococcus aureus can mediate cell aggregation during the process of biofilm development (Mack et al. 2007; Rodhe et al. 2004). Moreover, recent reports have demonstrated that extracellular DNA fragments and extracellular teichoic acid are structural components in the S. epidermidis biofilm matrix (Rodhe et al. 2010; Izano et al. 2008). The extracellular matrix protects microbial cells from phagocytes and antibiotics leading to chronic infections. The biofilm harbours slow or nongrowing bacteria which are less susceptible to antibiotics. Also, the concentration of enzymes which inactivate antibiotics is significantly higher within biofilms. Consequently, biofilm-associated bacteria are 100 to 10,000 times more resistant to antibiotics than planktonic bacteria. Previous studies showed that rifampin has the ability to penetrate through the biofilm, but the use of this antibiotic alone was associated with the emergence of rifampin-resistant strains, and for this reason, this antibiotic compound should be combined with other antibiotic agents. Tigecycline, a novel glycylcycline antibiotic, has demonstrated excellent in vitro and in vivo activity against a large number of Gram-positive bacteria, including methicillin-resistant S. aureus (MRSA) strains (Aybar et al. 2012). The tigecycline/rifampin combination has been investigated in vitro and in animal models. In in vitro studies, the addition of rifampin to tigecycline resulted in an antagonism for two of four tested S. aureus strains (Mercier et al. 2002). In other studies, antagonism was not observed for the tigecycline/rifampin combination against MRSA strains (Petersen et al. 2006). Data from the rabbit model experiment, although limited to one strain, demonstrated that the combination of tigecycline with rifampin exhibited complete infection clearance from the tibia. However, the authors underline that the eradication of the staphylococcal biofilm was also very high, when the tigecycline alone was tested (Yin et al. 2005). In this study, we examined the biofilm formation by S. epidermidis clinical isolates and the effect of tigecycline alone or in combination with rifampin against adhered bacteria in the biofilm.
Materials and methods Bacterial strains Eighty methicillin-resistant S. epidermidis strains were collected from treated patients in the 700-bed Provincial Hospital in Poznań, Poland, over a period of 2 years. The majority of isolates were isolated from the blood of patients with bacteremia. The isolates were identified by conventional methodology and using Vitek 2 system (bioMérieux, France). All the isolates were resistant to oxacillin, which was confirmed by the presence of the mecA gene (Geha et al. 1994). The biofilm-positive S. epidermidis strain ATCC 35984 and biofilm-negative S. epidermidis strain ATCC 12228 were included in this study as a control. Detection of the icaA, bhp, aap, fbe, embp, atlE, aap and mecA genes and insertion sequence IS256 by PCR Isolation of DNA was performed using the Genomic DNA Prep Plus kit (A&A Biotechnology, Gdynia, Poland). The PCR was applied to determine the presence or absence of the icaA, bhp, aap, fbe, embp, atlE (Rodhe et al. 2007), aap (Vandecasteele et al. 2003) and mecA genes (Geha et al. 1994) and the insertion sequence IS256 (Rodhe et al. 2007). The amplification products were electrophoresed in 1.5 % agarose gel. The DNA was stained with ethidium bromide, visualized on a UV light transilluminator and documented with V.99 Bio-Print system (Vilber Lourmat, Torcy, France). Determination of biofilm production by microtiter plate assay Biofilm production was determined as described previously (Christensen et al. 1985; Fredheim et al. 2009; Kim et al. 2008). In brief, the overnight culture grown in tryptic soy broth (TSB; Difco, Becton Dickinson, France) supplemented with 0.25 % glucose was diluted 1:100 in TSB with 0.25 % glucose and 200 μL transformed into 96-well polystyrene microtiter plates. After 24 h of incubation at 37 °C, the cultures were gently removed and wells were washed three times with phosphate-buffered saline (PBS), air-dried and stained with the 0.4 % crystal violet solution for 10 min. The plate was washed, the adherent cells were resuspended in the ethanol-acetone mixture (70:30) and finally, the absorbance at 490 nm was determined. The strains were considered biofilm positive if they had an absorbance of >0.25. Each isolate was tested in triplicate. The biofilm-producing strain ATCC 35984 was used as the positive control, whereas the ATCC 12228 was used as the negative control. Confocal laser scanning microscopy (CLSM) Overnight cultures of various strains were inoculated into a cover glass cell culture chamber (Nunc) as described by Qin et al. (2007). After 24 h of incubation at 37 °C, the chamber cover glasses were washed gently three times with PBS to remove planktonic cells. Biofilms and bacteria were stained using SYTO and PI (Live/Dead BacLight Bacterial Viability kits,
Folia Microbiol
Invitrogen) for 15 min and observed under a microscope (Carl Zeiss LSM 510-Axiovert 200M) equipment with detector and filter sets for monitoring SYTO9 and PI. The Carl Zeiss confocal software was used for the analysis of the biofilm, which permitted the collections of image stacks and threedimensional (3D) visualization of the biofilms. Susceptibility testing with adherent cells The biofilm minimal inhibitory concentration (MICb) and minimal bactericidal concentration (MBCb) were determined according to the methods of Giacometti et al. (2005). Briefly, the biofilms were washed three times with PBS to remove non-adherent cells. Serial twofold dilutions of antimicrobial agents in MuellerHinton (MH) broth were added to wells containing adherent bacteria. The 96-well polystyrene plates were incubated for 18 h at 37 °C. The MICb was defined as the lowest concentration of antibiotics at which there was no observable bacterial growth. In order to determine the MBCb, the MH broth with antibiotics was removed from wells and antibiotic-free MH broth was added. The plates were incubated for 18 h at 37 °C. The MBC was defined as the lowest drug concentration at which there was no bacterial growth after removal of the antibiotics. To determine the changes due to the antibiotic and combination of antibiotics on isolates, a general linear model was calculated. All tests were performed using STATISTICA software (10.00 StatSoft, Tulsa, OK, USA). A P value of
