Bactérias dormentes obtidas de biofinas de staphuloccus epidermidis têm menor potencial inflamatório e mantêm-se tolerantes a vancomicina e penicilina após iniciarem um crescimento planctónico

DISSERTAÇÃO – ARTIGO DE INVESTIGAÇÃO MÉDICA Mestrado Integrado em Medicina BACTÉRIAS DORMENTES OBTIDAS DE BIOFILMES DE STAPHYLOCOCCUS EPIDERMIDIS TÊM MENOR POTENCIAL INFLAMATÓRIO E MANTÊM-SE TOLERANTES A VANCOMICINA E PENICILINA APÓS INICIAREM UM CRESCIMENTO PLANCTÓNICO DORMANT BACTERIA WITHIN STAPHYLOCOCCUS EPIDERMIDIS BIOFILMS HAVE LOW INFLAMMATORY PROPERTIES AND MAINTAIN TOLERANCE TO VANCOMYCIN AND PENICILLIN AFTER ENTERING PLANKTONIC GROWTH José Filipe Dias Cerca 2014 DISSERTAÇÃO BACTÉRIAS DORMENTES OBTIDAS DE BIOFILMES DE STAPHYLOCOCCUS EPIDERMIDIS TÊM MENOR POTENCIAL INFLAMATÓRIO E MANTÊM-SE TOLERANTES A VANCOMICINA E PENICILINA APÓS INICIAREM UM CRESCIMENTO PLANCTÓNICO DORMANT BACTERIA WITHIN STAPHYLOCOCCUS EPIDERMIDIS BIOFILMS HAVE LOW INFLAMMATORY PROPERTIES AND MAINTAIN TOLERANCE TO VANCOMYCIN AND PENICILLIN AFTER ENTERING PLANKTONIC GROWTH José Filipe Dias Cerca, Aluno do 6º ano profissionalizante do Mestrado Integrado em Medicina, Instituto de Ciências Biomédicas Abel Salazar – Universidade do Porto. Orientador: Doutor Manuel Vilanova, Professor Associado, Instituto de Ciências Biomédicas Abel Salazar – Universidade do Porto. O trabalho apresentado nesta dissertação foi realizado no Laboratório de Imunologia Mário Arala Chaves – ICBAS/UP. Actualmente encontra-se submetido para publicação na revista científica Journal of Medical Microbiology (Society for General Microbiology). Dedicado a Ana e Sofia AGRADECIMENTOS Ao Professor Doutor Manuel Vilanova, pelo empenho na orientação deste trabalho e pela sua preserverança no sentido de implementar o estudo de biofilmes de Staphylococcus epidermidis no laboratório de Imunologia Mário Arala Chaves. Aos co-autores deste trabalho, pela apoio técnico e pelo sentido crítico na revisão deste manuscrito. Aos elementos do Laboratório de Imunologia Mário Arala Chaves, pelo bom ambiente e disponibilidade para ajudar. RESUMO O Staphylococcus epidermidis (S. epidermidis) é uma bactéria Grampositiva e coagulase-negativa sendo amplamente reconhecido como um comensal que coloniza o epitélio e mucosas humanas e que raramente é responsável por infecções em hospedeiros imunocompetentes. No entanto, nos últimos anos, a percepção médica de que esta bactéria seria uma espécie inócua foi-se alterando devido à sua capacidade para colonizar dispositivos médicos através da formação de biofilmes na superfície dos mesmos. Os biofilmes são definidos como uma comunidade de bactérias aderiras a uma superfície, que partilham uma arquitectura própria permanecendo embebidas por uma matriz extracelular, e que têm uma fisiologia/metabolismo distinto quando comparado com o seu estado livre/planctónico. Actualmente, dados epidemiológicos reconhecem o S. epidermidis como um dos principais agentes etiológicos de infecções nosocomiais. São exemplos de infecções por biofilmes de S. epidermidis: endocardites devido à colonização de válvula prostética; bacteriúria ou bacteriemia devido a colonização de cateteres urinários ou intravasculares respectivamente; endoftalmite devido a colonização de lente intraocular; ou infecção de prótese articular após artroplastia total do joelho ou anca. A elevada tolerância à antibioterapia que caracteriza os biofilmes determina que o tratamento destas infecções seja extremamente difícil, sendo muitas vezes necessária a remoção cirúrgica do dispositivo médico como método para eliminar o foco infeccioso. Assim, estas infecções associam-se frequentemente a elevada morbilidade para o doente assim como a elevados custos para o sistema de saúde. O ciclo de formação de biofilmes de S. epidermidis envolve 3 etapas distintas: 1) a adesão inicial à superfície de contacto, um processo molecular mediado por diversos factores de aderência da bactéria assim como por interacções físicas/hidrofóbicas entre a bactéria e a superfície de contacto; 2) crescimento e maturação do biofilme, um processo onde ocorre agregação intercelular e é mediado pela síntese bacteriana de adesinas extracelulares sendo a mais conhecida a molécula poli-N-acetilglucosamina (PNAG); 3) o 1 Dormant bacteria within Staphylococcus epidermidis biofilms have low inflammatory properties and maintain tolerance to vancomycin and penicillin after entering planktonic growth Filipe Cercaa,b, Ângela Françac, Begoña Pérez-Cabezasa,b, Adília Ribeiroa,b, Joana Azeredoc, Gerald Pierf, Nuno Cercac, Manuel Vilanovaa,b,* a ICBAS-UP - Instituto de Ciências Biomédicas de Abel Salazar - Universidade do Porto, Rua de Jorge Viterbo Ferreira n.º 228, 4050-313, 4099-003, Porto, Portugal; b IBMC - Instituto de Biologia Molecular e Celular, Rua do Campo Alegre 83, Porto, Portugal; c CEB-IBB, Centro de Engenharia Biológica - Instituto de Biotecnologia e Bioengenharia, Campus de Gualtar, Universidade do Minho, Braga, Portugal; d UMIB – Unidade Multidisciplinar de Investigação Biomédica, Universidade do Porto, Largo Prof. Abel Salazar 2, 4099-003, Porto, Portugal; e Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital/Harvard Medical School, Boston, MA 02115, USA Running title: S. epidermidis dormant cells * Corresponding author: Rua de Jorge Viterbo Ferreira n.º 228 4050-313 Porto, Portugal Tel: 351-220428214 E-mail: [email protected] 2 Abstract Staphylococcus epidermidis is the most commonly isolated etiological agent of nosocomial infections mainly due to its ability to establish biofilms on indwelling medical devices. Detachment of bacteria from S. epidermidis biofilms and subsequent growth in the planktonic form is a hallmark in the pathogenesis of these infections leading to dissemination. Here we showed that S. epidermidis cells collected from biofilms cultured in conditions that promote cell viability present marked changes in their physiological status upon initiating a planktonic mode of growth. When compared to cells growing in biofilms, they displayed an increased SYBR green I staining intensity, increased transcription of the rpiA gene, decreased transcription of icaA gene as well as higher susceptibility to vancomycin and penicillin antibiotics. When bacteria collected from biofilms with high proportions of dormant cells were subsequently cultured in the planktonic mode, a large proportion of cells maintained a low SYBR staining intensity and increased resistance to vancomycin and penicillin, a profile typical of dormant cells. This phenotype further associated with a decreased ability of these biofilm-derived cells to activate bone marrow-derived dendritic cells in vitro, as determined by pro-inflammatory cytokine quantification. These results demonstrated that cells detached from the biofilm maintain a dormant cell-like phenotype, having a low pro-inflammatory effect and decreased susceptibility to antibiotics suggesting these cells may contribute for the recalcitrant nature of biofilm infections. Keywords: biofilms; S. epidermidis; dormant bacteria; pathophysiology; antibiotic tolerance, penicillin, vancomycin, dendritic cells 3 Introduction Staphylococcus epidermidis is a commensal bacterium that colonizes the skin and mucous membranes, often being the most prevalent staphylococcal species found on human skin (Otto, 2009). The ability to colonize and establish biofilms on indwelling medical devices makes this bacterium the most commonly isolated etiological agent of nosocomial infections (Uçkay et al., 2009). Moreover, due to the intrinsic resistance of staphylococcal biofilms to antibiotics (Raad et al., 1998), staphylococcal biofilm-originated infections are associated with an increased duration of hospital admissions and use of medical resources and, consequently, of healthcare costs (Dimick et al. 2001; Rogers et al., 2009). Critically-ill immune-compromised patients (Bearman & Wenzel, 2005) and premature neonates (Fallat et al., 1998) are the individuals most vulnerable to this opportunistic pathogen. S. epidermidis biofilm formation involves initial cellular adherence to a surface followed by intercellular aggregation and accumulation in multilayered cell clusters (Otto, 2009). This process is dependent on the synthesis of adhesive extracellular molecules (Götz, 2002), such as the polysaccharide intercellular adhesin (PIA) also known as poly-N-acetyl glucosamine (PNAG), a major constituent mediating cell-to-cell adhesion in staphylococci (Mack et al., 1996; Mack et al., 1994). The final stage of the biofilm life cycle comprises cell detachment and subsequent growth in the planktonic form, a process that is crucial for S. epidermidis biofilm pathogenesis by forming the basis for dissemination of infection (Wang et al., 2011). Therefore, in this study we evaluated physiological changes occurring in S. epidermidis bacteria during the shift from the biofilm to the planktonic mode of growth. We show that dormant bacteria within S. epidermidis biofilms display a low inflammatory profile and increased tolerance to vancomycin and penicillin upon initiating a planktonic growth. 4 Material and Methods Bacterial strains and growth conditions S. epidermidis strain 9142 (Nedelmann et al., 1998) was used in this study. To establish a 48 h biofilm, a starter culture was grown overnight in tryptic soy broth (TSB) (Merck, Darmstadt, Germany) at 37ºC with agitation (80 rpm). The optical density at 640 nm of the starter culture was adjusted to 0.250 with phosphate buffered saline (PBS) and a 10 µL aliquot transferred to a 24-well polystyrene plate (Nunc, Roskilde, Denmark) containing 1 mL/well of TSB with 0.4% filtered glucose (w/v) (Merck), further supplemented with 10 mM MgCl2 (Merck). The plates were then incubated for 48 h at 37ºC with agitation (80 rpm). At 24 h of growth, the culture medium was replaced by fresh TSB supplemented with 1% of glucose and 10 mM MgCl2 (TSB 1%G + Mg2+). Similar culture conditions were used to establish 48 h biofilms with high proportions of dormant bacteria by using growth medium without MgCl2 supplementation, as previously described (Cerca et al., 2011a). Preparation of biofilm and planktonic S. epidermidis cell suspensions To assess the bacterial physiological status during the transition from the biofilm to the planktonic mode of growth, 48 h biofilms grown in TSB 1%G + Mg2+ were disaggregated as previously described (Cerca et al., 2011b) and the resulting cell suspensions were diluted in fresh TSB to a concentration of 1×108 cells/mL. These cell suspensions were allowed to grow in the planktonic form for a 6 h period (37ºC, 80 rpm). Simultaneously, the culture medium of 48 h biofilm cultures was washed out and biofilms were allowed to grow for an additional 6 h period in 1 mL fresh TSB. At the time points 0 h, 1 h 30 min, 3 h and 6 h of growth, an aliquot of bacteria was recovered from each culture (biofilm and planktonic) and used for flow cytometry analysis and gene expression quantification, as described below. 5 Flow cytometry analysis At the indicated time points, biofilms were washed twice with 1 mL of PBS, and bacteria were then recovered in 1 mL of PBS, as previously described (Cerca et al., 2011b). After a 1:10 dilution in PBS, an aliquot of 30 μL was transferred to 270 µL of PBS containing 3 µL of quantification microspheres (Invitrogen, Carlsbad, CA, USA), SYBR green I (SYBR, Invitrogen) (1:5000 commercial stock) and propidium iodide 5 µg/mL (PI, Sigma, St. Louis, MO, USA). For the planktonic cultures, an aliquot of 30 µL of cells was transferred from the culture to 270 µL of PBS containing 3 µL of quantification microspheres (Invitrogen), SYBR (1:5000 commercial stock) and PI 5 µg/mL. Bacterial fluorescence analysis was carried out by using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) containing a low-power air-cooled 15 mW blue (488 nm) argon laser. Data were acquired using the CellQuest software (Becton Dickinson) and analyzed using the Flowjo 7.2.5 software (Tree Star, Ashland, OR, USA). SYBR fluorescence was detected on the FL1 channel (BP530/30) while PI fluorescence was detected on the FL3 channel (LP650). For all detected parameters, amplification was carried out using logarithmic scales. The concentration of bacteria in the planktonic or biofilm cultures was further determined by acquiring the counts for a specific number of microspheres during flow cytometry analysis of the cell samples. Quantitative PCR Quantitative PCR (qPCR) was used to assess the expression of intercellular adhesin A (icaA) and ribose-5-phosphate isomerase A (rpiA) genes in S. epidermidis bacteria grown in biofilm or planktonic cultures. The primers used were designed with the Primer3 software (Rozen and Skaletsky, 2000) using the S. epidermidis RP62A genome as a template (PubMed NC_002976.3). The sequences of the primers used are listed in Table 1. Primer efficiency was determined by the dilution method and by performing a temperature gradient reaction from 54 to 64ºCThe set of primers having the optimal and most similar efficiency values at 60ºC were used. At each time point, total RNA from either biofilm or planktonic cultures was extracted using the FastRNA® Pro Blue Kit 6 (MP Biomedicals, Solon, OH, USA), as described previously (França et al., 2011). Contaminating genomic DNA was removed by treatment with DNase I (Fermentas, Burlington, ON, Canada) for 30 min at 37ºC. The enzyme was then heat-inactivated at 65ºC for 10 min in the presence of EDTA. Total RNA was quantified using a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and stored at –80ºC. Total RNA samples were reverse transcribed in the presence of each reverse primer and RevertAidTM M-MuLV Reverse Transcriptase (Fermentas). Control reactions lacking the reverse transcriptase enzyme (no-RT) were included. qPCR reactions contained 2 μL 1:200 diluted cDNA or no-RT control, 2 μL containing 10 pmol of each primer, 6 μL nuclease free deionized H2O, and 10 μL Maxima® SYBR Green qPCR Master Mix (Fermentas) with the following thermocycler parameters: 94ºC for 10 min, 40 cycles of 94ºC for 15 s, 60oC for 20 s and finally 72ºC for 25 s. To monitor the reaction specificity and primer dimer formation, end-products were analyzed by melting curves. Relative fold increase was calculated using 2ΔCt method, a variation of the Livak method, where ΔCt = Ct (housekeeping gene)-Ct (target gene). The data analysis was based on at least 2 independent experiments. Evaluation of the susceptibility to vancomycin and penicillin of S. epidermidis biofilm bacteria after initiating a planktonic growth S. epidermidis bacteria obtained from 48 h biofilms grown in TSB 1%G + Mg2+ were diluted in fresh TSB (1×108 cells/mL) and allowed to grow in the planktonic form for a 3 h period. Simultaneously, parallel cultures of 48 h S. epidermidis biofilms were allowed to grow for an additional 3 h period in fresh TSB. At this time point, vancomycin (40 μg/mL) (Sigma), penicillin (40 μg/mL) (Sigma) or TSB (negative control) were added to the biofilm and planktonic cultures during a 30 min period. Bacterial death was determined through flow cytometry by assessing the bacterial incorporation of PI, as described above. A similar procedure was done using 48 h biofilms grown in TSB 1%G without magnesium supplementation as a starting point. 7 Mice Male BALB/c mice 6–8 weeks of age were purchased from Charles River and kept at the animal facilities of the Institute Abel Salazar (ICBAS, Portugal) during the experiments. Hiding and nesting materials were provided for enrichment. Procedures involving mice were performed according to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS 123) and 86/609/EEC Directive and Portuguese rules (DL 129/92). Authorization to perform the experiments was issued by the competent national board (Direção Geral de Veterinária), document number 0420/000/000/2008. Bone marrow-derived dendritic cells (BMDCs) differentiation Bone marrow cells were collected from femurs and tibias of BALB/c mice by flushing with cold RPMI 1640 (Sigma). Cells (1×106/mL) were cultured in 6-well plates in RPMI supplemented with 15% (v/v) J558-cell supernatant, 10% FBS (PAA), penicillin (100 U.I./mL)-streptomycin (100 µg/mL) (Sigma), and LGlutamine (2mM) (Sigma) and incubated at 37ºC, 5% CO2. Half of the media was renewed every two days. At day 6, BMDCs were detached, adjusted at a concentration of 1×106/mL, distributed in 96-well round bottom plates (100 µL per well) and incubated overnight in supplemented RPMI medium. Stimulation of BMDCs with bacterial suspensions of S. epidermidis obtained from different cultures Bacterial suspensions were obtained from 48 h biofilms grown in TSB 1%G or TSB 1%G + Mg2+ as described above. For planktonic cultures, a single colony of S. epidermidis was inoculated into 35 mL TSB and grown overnight at 37ºC in a shaker rotator at 80 rpm. Then, cells were recovered, centrifuged during 10 min at 13000 rpm at 4ºC (Biofuge fresco HERAEUS, ThermoSCIENTIFIC, Waltham, MA, USA), and resuspended in 1mL of PBS. Before stimulation of BMDCs, all the bacterial inocula were resuspended in RPMI supplemented with 10% FBS and 2mM L-glutamine and adjusted to the concentration of 1×106 cells / mL. BMDCs were then stimulated with 100 µL of bacterial suspensions 8 (1×106/mL) obtained from biofilms grown in TSB 1%G, TSB 1%G + Mg2+ or original planktonic cultures. RPMI supplemented media and lipopolysaccharide (LPS, 1μg/mL) (Sigma) were used as negative and positive controls, respectively. After 6h of incubation (37ºC, 5% CO2), media containing bacteria was collected and replaced by fresh medium containing penicillin (200 U.I./mL) and streptomycin (200 µg/mL). At the 6 h or 18 h time points, the culture supernatants were removed and stored at -20 ºC until use. Cytokine quantification Interleukin (IL) 6, IL-12p70 and tumor necrosis factor (TNF-α) were quantified using commercially available quantification kits (eBioscience Inc, San Diego, CA, USA) according to the manufacturer’s instructions. Results were read in a Multiskan Ex spectrophotometer (Thermo Electron Corporation, Corston, UK) using the Ascent software (Thermo Electron Corporation). Statistical analysis All graphs were generated using GraphPad Prism software (GraphPad Software). Means and standard errors of the means (SEM) were calculated. Statistical analysis was carried out by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni post tests or with one-way analysis of variance with Tukey's Multiple Comparison Test. Both tests used GraphPad software. A P value of <0.05 was considered statistically significant. 9 Results and Discussion S. epidermidis cells that undergo a shift from biofilm to planktonic growth present a high SYBR staining intensity and rpiA gene expression Detachment of bacteria from a biofilm and subsequent growth in the planktonic form is considered a major event in the pathophysiology of biofilm related infections (Otto, 2013). In S. epidermidis this was previously shown to be followed by a transition from a non-aggressive, non-growing and fermentative state (biofilm stage of growth) into a growing, aggressive/inflammatory and respiratory state (planktonic stage of growth) (Yao et al., 2005). SYBR staining intensity was previously found to correlate with S. epidermidis respiratory activity, as demonstrated by co-staining studies using SYBR and the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) (Cerca et al., 2011c). Therefore, we used SYBR as a fluorescent probe to evaluate physiological changes of S. epidermidis bacteria during the shift from the biofilm to the planktonic mode of growth. For this purpose, 48 h biofilms grown in TSB media supplemented with glucose and magnesium (TSB 1%G + Mg2+) were prepared and used as the starting point of our study, as these conditions maintain the majority of cells in a culturable state (Cerca et al., 2011a). As shown in Figure 1, bacterial suspensions were prepared from the 48 h biofilms and allowed to grow in the planktonic form for a further 6 h. Simultaneously, parallel cultures of S. epidermidis biofilms continued growing for an additional 6 h in fresh TSB medium. Bacterial cells were then obtained at different time points from either the planktonic or biofilm cultures, stained with SYBR and PI, and analysed by flow cytometry. The bacteria that entered into the planktonic growth phase progressive gained increases in the mean fluorescence intensity (MFI) due to SYBR staining, reaching a detected maximum at the 3 h time point (Figure 1). In contrast, bacteria that remained growing in the biofilm mode presented a lower and constant SYBR MFI over time (Figure 1). Our results are in agreement with a previous report showing that the shift from the biofilm to the planktonic mode of growth is accompanied by an increase in the S. epidermidis respiratory activity (Yao et al., 2005). Interestingly, an increase in PI staining 16 As shown in Figure 5, bacteria obtained from biofilms with higher proportions of dormant cells induced the lower production of TNF-α, IL-12 and IL-6. Noteworthy, bacteria obtained from low dormancy biofilms activated Figure 5. S. epidermidis bacteria obtained from high dormancy biofilms induce a lower activation of murine BMDC. Cell suspensions obtained from 48 h biofilms grown in TSB 1%G (Dormant biofilms), 48 h biofilms grown in TSB 1%G + Mg2+ (Non-dormant biofilms) or original planktonic cultures (Planktonic) were used to stimulate bone marrow-derived dendritic cells (BMDCs) in vitro. Activation of BMDCs was determined by the quantification of the proinflammatory cytokines A) tumor necrosis factor α (TNF-α), B) interleukin (IL) -12 and C) IL6 in the cultures supernatants. Results shown are representative of two independent experiments that generated concordant results. Statistical analysis was carried out by one-way analysis of variance (ANOVA) with Tukey's Multiple Comparison Test. 17 BMDC to the same extent as bacteria obtained from planktonic cultures. These results are in agreement with our previous report showing that S. epidermidis bacteria obtained from biofilms enriched in dormant cells induced a low activation of murine macrophages in vitro and in vivo (Cerca et al., 2011a). Also in agreement, a previous study have shown that staphylococcal biofilms could attenuate the inflammatory response of murine macrophages, as compared to planktonic cell counterparts, by promoting differentiation of these host cells into a M2 phenotype (Thurlow et al., 2011). It would thus be interesting to ascertain in future studies whether or to what extent dormant cells may contribute to this differential role of biofilm cells on host mononuclear phagocytes. Concluding remarks The physiological shift that occurs in S. epidermidis bacteria during the transition from the biofilm to the planktonic mode of growth is considered a major event in the pathophysiology of this bacterium (Yao et al., 2005). Here, we showed that bacteria from biofilms grown in conditions that promoted cell viability displayed increased SYBR staining intensity and rpiA gene expression as well as increased susceptibility to vancomycin and penicillin. In contrast, we also showed that a high proportion of bacteria obtained from biofilms grown in high glucose, a condition promoting dormancy, maintained a low SYBR staining intensity and tolerance to vancomycin and penicillin upon initiating a planktonic growth. These data highlight that biofilms, already intrinsically tolerant to antibiotics (Høiby et al., 2010), may release cells to the surrounding environment that maintain an antibiotic-tolerant profile. This, in turn, may confer on these bacteria an increased likelihood to persist within a host and cause disease, which would be in agreement with a recent study reporting an association between the clinical symptoms of biofilm-related infections and the presence of unculturable bacteria within the biofilms (Zandri et al., 2012). As infectious biofilms may have a high frequency and prevalence of unculturable bacteria (Oliver, 2010), further studies addressing the role of dormant bacteria in the pathophysiology of S. epidermidis biofilms would help understand the clinical outcomes of biofilm-related infections. 18 Acknowledgments This work was funded by Fundação para a Ciência e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009 and FCOMP-01-0124-FEDER014309. Parts of the work were also supported by National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID) grants AI46706 and AI057159, a component of Award Number U54 AI057159. 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