Viridans streptococci in endocarditis and neutropenic sepsis: biofilm formation and effects of antibiotics

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1 Journal of Antimicrobial Chemotherapy (2005) 55, doi: /jac/dkh479 Advance Access publication 24 November 2004 Viridans streptococci in endocarditis and neutropenic sepsis: biofilm formation and effects of antibiotics E. Presterl 1 *, A. J. Grisold 2, S. Reichmann 1, A. M. Hirschl 3, A. Georgopoulos 1 and W. Graninger 1 JAC 1 Department of Medicine I, Division of Infectious Diseases, Medical University of Vienna, Waehringer Guertel 18 20, 1090 Vienna; 2 Institute of Hygiene, Medical University of Graz, Universitätsplatz 4, 8010 Graz; 3 Division of Clinical Microbiology, Institute of Hygiene and Medical Microbiology, University of Vienna, Allgemeines Krankenhaus, Waehringer Guertel 18 20, 1090 Vienna, Austria Received 16 July 2004; accepted 23 September 2004 Objectives: Viridans group streptococci (VGS) are a frequent cause of bacterial endocarditis or sepsis in patients with neutropenia. Endocarditis in particular, is associated with plaque formation on the endocardium and valve leaflets whereas VGS septicaemia in neutropenic patients is caused by the influx of oral flora bacteria through mucositic lesions. This study examined the in vitro potency for biofilm formation of clinical VGS bloodstream isolates, and the effects of antibiotics on these biofilms. Methods: During the years , 40 VGS bloodstream isolates from 18 patients with endocarditis and 22 patients with severe sepsis and neutropenia were collected. The MICs of penicillin, teicoplanin and moxifloxacin were determined using the microdilution broth method according to NCCLS criteria. Biofilms were grown in microtitre plates, dyed with Crystal Violet, and the mean optical density (OD) was used for quantification. Biofilms were incubated with penicillin, teicoplanin and moxifloxacin at various concentrations starting with the MICs for the respective isolates tested. Results: Isolates from eight out of 18 patients with endocarditis and six out of 22 patients with neutropenia formed biofilms (not significant). For the 14 isolates, the MIC 90 s (range) of penicillin, teicoplanin and moxifloxacin were 0.5 mg/l ( ), mg/l ( ) and 0.5 mg/l ( ), respectively. Generally, biofilms persisted although incubated with the antibiotics up to concentrations of 1283 MIC. However, the ODs of biofilms after incubation with an antibiotic were significantly lower than the ODs of biofilms without antibiotic (P < 0.05). A significant decrease in the biofilms with increasing antibiotic concentrations was observed for teicoplanin and moxifloxacin, but not for penicillin G. Conclusions: VGS isolated from patients with endocarditis and patients with sepsis and neutropenia form biofilms. Biofilms persist even when exposed to antibiotics at concentrations up to 1283 MIC. Nevertheless, teicoplanin and moxifloxacin reduced the density of the biofilms at concentrations >_ 163MIC. Thus, testing the effects of antibiotics on biofilms may supply useful information in addition to standard in vitro testing, particularly in diseases where biofilm formation is involved in the pathogenesis. Keywords: neutropenia, penicillin, teicoplanin, moxifloxacin Introduction Viridans group streptococci (VGS) are a frequent cause of bacterial endocarditis, and bacteraemia with associated multiorgan failure in patients with neutropenia. 1 Endocarditis is a serious disease associated with a mortality of up to 50% despite antibiotic treatment. 1 A main feature of endocarditis is the endocarditic plaque on the leaflets of one or more heart valves. The formation of the endocarditic plaque is unique, involving bacteria, platelets, coagulation factors and leucocytes, and is... *Corresponding author. Tel: ; Fax: ; Elisabeth.Presterl@meduniwien.ac.at JAC vol.55 no.1 q The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.

2 E. Presterl et al. considered a rather special kind of biofilm. 2 Another clinical entity with serious outcome is bacteraemia and sepsis caused by VGS in patients with profound neutropenia due to leukaemia. Cytotoxic chemotherapy causes severe mucositis enabling colonizing bacteria of the oral flora to enter the bloodstream. Serious but rare complications are septic shock and the adult respiratory distress syndrome. 3 Bacteria growing in biofilms are more resistant to killing by antibiotics than bacteria growing under planktonic conditions. 4 Normally, VGS are part of the surface flora of the respiratory tract and gastrointestinal tract. There, they form biofilms on mucosal surfaces in dynamic equilibria with host defences and contribute to the maintenance of the integrity. The ability of VGS to form biofilms has been discussed as a virulence factor in periodontal disease, however little is known about VGS isolates from endocarditis or patients with septicaemia. 5 The ability to form biofilms may be a prerequisite for the pathogenesis of endocarditis. Because of the biofilm itself and the altered metabolic state of the VGS in the biofilm, 6 endocarditis is thus difficult to treat and is associated with considerable mortality. However, in patients with neutropenia and sepsis, VGS seem to be swept into the bloodstream through the damaged mucosa causing sepsis and bacteraemia with a usually good response to treatment. 7 The treatment of choice for infections caused by VGS is still penicillin, and vancomycin or teicoplanin has been proposed as a second-line alternative in the case of hypersensitivity. 1 Meanwhile, new antibiotics with particular activity against Gram-positive cocci have become available. Moxifloxacin, a new quinolone, has particular activity against Gram-positive cocci, and has been used in clinical and experimental encodarditis. 8 This study examined 18 VGS bloodstream isolates from patients with endocarditis and compares their potency to form biofilms with that of 22 VGS bloodstream isolates from patients with neutropenia. Further, the MICs and the effects on biofilms of penicillin, teicoplanin and moxifloxacin were tested. Materials and methods During the years , 40 VGS bloodstream isolates (in >_2 blood cultures) were collected at the University Hospital of Vienna, from patients with endocarditis and patients with a neutrophil count <500/mm 3 for more than 5 days due to leukaemia. Endocarditis was defined according to the criteria of Durack et al. 9 with at least two blood cultures positive for a typical organism and echocardiographic evidence for endocardial involvement (vegetations). Aerobic and anaerobic blood cultures (VITAL; biomérieux, Paris, France) were incubated at 378C for 28 days. The VGS isolates were identified by API Strep and Ani/VITEK (both biomérieux, Marcy L Étoile, France). MICs were tested using the standard method of the National Committee for Clinical Laboratory Standards. 10 For microbroth dilution, cation-adjusted Mueller Hinton broth (MHB) with 2 5% (v/w) lysed horse blood was used (supplemented Mueller Hinton broth; SMHB). Breakpoints for the clinical susceptibility testing are available for penicillin only (<_ 0.12 mg/l). There are no approved breakpoints for teicoplanin and moxifloxacin for viridans streptococci. Thus, for teicoplanin, an MIC <_ 8 mg/l, and for moxifloxacin an MIC of <_1 mg/l were considered as susceptible. 11 Biofilms were studied using the static microtitre plate model established by Christensen and colleagues with a few modifications with regard to fixation. 12,13 Briefly, bacteria were prepared in MHB at a concentration equivalent to a 0.5 McFarland standard and diluted 1:10 with MHB. Each well of a 96-well microtitre plate was filled with 50 ml of diluted bacteria suspension and 50 ml of cation-adjusted MHB. Lysed horse blood [2 5% (v/w)] was added as most of the isolates would not grow or form biofilms in any other medium described for biofilm formation before. 14 After incubation for 24 h in a microaerobic atmosphere (room air with 5% CO 2 )at 358C, media and planktonic cells were removed. The wells with the presumably formed biofilms were fixed with formalin (37%, diluted 1:10) plus 2% natriumacetate, and each well was stained with ml of 1% Crystal Violet for 3 min. Then the dyed biofilms were washed two times with approximately 300 ml of distilled water. Wells were then visually checked for the presence or absence of a biofilm based on the presence of staining at the bottom of the well. The mean optical density (OD) was used for quantification using a routine microtitre plate reader at a wavelength of 550 nm. All biofilm experiments were carried out eight times for each isolate to minimize variability in OD measurements. Biofilms were fixed with 2% glutaraldehyde and biofilm formation was verified by electron microscope scanning (Philips XL30 ESEM). To test the antimicrobial activity of penicillin, teicoplanin and moxifloxacin, biofilms were prepared as described and grown for 24 h. After removal of the medium, 100 ml of SMHB with penicillin, teicoplanin and moxifloxacin at concentrations of 1, 2, 4, 8, 16, 32, 64, 128 MIC for the respective isolate tested was added to the biofilms (eight wells with biofilm per isolate per each concentration for every antimicrobial substance tested). These biofilms were incubated with the antibiotics at the defined concentrations for 24 h at 358C under microaerobic conditions, and then quantified as described above. To correct for the individual biofilm formation of each isolate, a ratio of the biofilm OD of the isolate incubated with antibiotic to the biofilm OD of the same isolate without antibiotic (control) was calculated. This OD ratio was used to measure changes in the thickness of the biofilms with increasing concentrations of the antibiotics tested. To test for bacterial growth in the biofilms, the biofilms were incubated with the increasing antibiotic concentrations, but not fixed and dyed, but scraped off and seeded on to Columbia agar and examined for growth. Statistics The data were analysed with SSPS 11.0 software. Groups (endocarditis and neutropenia isolates) were compared using the Wilcoxon s test, and the level of significance (P = probability) was set to The general linear model for repeated measurements (for not normally distributed data) was used to calculate differences in the ODs of biofilms incubated with increasing concentrations of the antimicrobial agents; the significance level was set at Results Eighteen patients with endocarditis caused by VGS and 22 patients with sepsis during neutropenia and leukaemia were identified. The patients data on the underlying conditions, treatment and outcome are given in Table 1. The duration of therapy was 38 days for the patients with endocarditis and 9 days for the patients with sepsis and neutropenia. While all but one of the patients with neutropenia and sepsis were cured, three patients with endocarditis died despite adequate antibiotic therapy and cardiac surgery. Two isolates (Streptococcus mitis, Streptococcus anginosus) showed MICs of penicillin of 1.0 mg/l, thus being considered intermediately susceptible to penicillin. The patient with endocarditis due to S. mitis died despite treatment with 46

3 Viridans streptococci in endocarditis and neutropenic sepsis Table 1. Demographic data of the 40 patients with VGS bloodstream infections Patients with endocarditis (n = 18) Patients with neutropenia (n = 22) Male/female 13/5 9/13 Age, years [median (range)] 60 (24 83) 42 (16 69) Death due to endocarditis/sepsis 3 1 Acute respiratory distress syndrome 1 Concurrent chemotherapy 13 Body temperature (8C) 37.9 ( ) 38.6 ( ) Leucocytes/mm 3 [median (range)] 9700 ( ) 690 ( ) Vegetations in sonography 10 Native valve disease 13 Prosthetic valve disease 2 Acute valve surgery 4 Leukaemia 22 Mucositis 22 Antibiotic therapy penicillin 3 penicillin+gentamicin 1 teicoplanin 14 piperacillin/tazobactam 11 third-generation cephalosporins 7 vancomycin 8 teicoplanin to which the pathogen was highly susceptible in vitro (MIC mg/l). Teicoplanin treatment for this patient was initiated after the empirical combination therapy with penicillin plus gentamicin. The identification of the 18 VGS species causing endocarditis was as follows: Streptococcus acidominimus (n = 1), S. anginosus (n = 2), Streptococcus bovis (n = 3), Streptococcus intermedius (n = 1), S. mitis (n = 3), Streptococcus oralis (n = 3), Streptococcus salivarius (n = 1), Streptococcus sanguis (n = 4). The identification of the species isolated in patients with neutropenia and sepsis was as follows: S. acidominimus (n = 1), S. intermedius (n = 1), S. mitis (n = 9), S. oralis (n = 5), S. salivarius (n = 1), S. sanguis (n = 4), other Streptococcus sp. not specified (n = 1). The susceptibility of these 40 VGS bloodstream isolates to penicillin, teicoplanin and moxifloxacin was tested. The MIC 90 s (MIC range) of penicillin, teicoplanin and moxifloxacin were 0.25 mg/l ( ), 0.25 mg/l ( ) and 0.5 mg/l ( ), respectively. Biofilm formation was tested for all VGS isolates. Eight out of the 18 endocarditis isolates and six out of the 22 neutropenia isolates formed biofilms (not significant, P = 0.2). For these 14 isolates, MIC 90 s (range) of penicillin, teicoplanin and moxifloxacin were 0.5 mg/l ( ), mg/l ( ) and 0.5 mg/l ( ), respectively. Detailed MICs with regard to the species are given in Table 2. Then, the effects of penicillin, teicoplanin and moxifloxacin were tested in the biofilm model. Generally, biofilms persisted despite an antibiotic concentration up to 128 the planktonic MIC. There was a significant reduction in the biofilms after incubation with any antibiotic compared with the control biofilm without antibiotic (P < 0.05). With increasing concentrations of the antibiotics, the densities of biofilms declined significantly when incubated with Table 2. MICs for the 14 VGS isolates that formed biofilms MIC 90 mg/l (range) penicillin teicoplanin moxifloxacin EC S. bovis (n = 3) 0.25 ( ) ( ) 0.25 ( ) S. oralis (n = 3) 0.25 ( ) ( ) 0.25 ( ) S. sanguis (n = 2) 0.25 ( ) ( ) 0.25 ( ) SN S. intermedius (n = 1) S. mitis (n = 3) 0.25 ( ) ( ) 0.5 ( ) S. oralis (n = 2) ( ) ( ) ( ) Patients with endocarditis (EC), n = 8; patients with sepsis and neutropenia (SN), n = 6. MICs were determined using the microdilution method, according to the NCCLS. 47

4 E. Presterl et al. with sepsis and neutropenia (S. mitis). With regard to their MICs, all isolates were highly susceptible. Discussion Figure 1. Decrease in the biofilm density given as the change in the optical density (DOD) measured after 24 h of incubation with penicillin, teicoplanin or moxifloxacin at concentrations of 2, 4, 8, 16, 32, 64 and 128 MIC in reference to the OD of the biofilms measured after an incubation at a concentration of 1 MIC of the respective antimicrobial substance. Penicillin, crossed diamonds; teicoplanin, filled squares; moxifloxacin, open circles. The symbols represent the means, the whiskers the S.E.M. teicoplanin and moxifloxacin, but not when incubated with penicillin (P < 0.05) (Figure 1). A significant decrease in the OD was observed at 16 the MIC for moxifloxacin, and at 32 the MIC for teicoplanin (P < 0.05; Figure 1). There was no significant difference between endocarditis or neutropenia isolates, neither in the OD of biofilms nor in the behaviour of the biofilms when incubated with increasing concentrations of all three antibiotics (Table 3). Bacterial growth was demonstrated up to 128 the MIC of penicillin. However, an inhibition of bacterial growth was detected for teicoplanin and moxifloxacin with increasing antibiotic concentration. As for the clinical course, three isolates of four patients who died from their underlying disease (three from endocarditis and one from acute respiratory distress syndrome and neutropenic sepsis) did form biofilms: two in the endocarditis group (S. sanguis, S. oralis) and one in the group Because of the high mortality in the pre-antibiotic era, the treatment of endocarditis has always been considered as unique among bacterial infections. Although the bacteria exhibit low MICs and susceptibility in vitro to the antibiotics normally used, complete eradication of bacteria in the plaques takes weeks, and clinical relapse is common. Possible explanations include the impaired host defences and the high density of bacteria exhibiting an altered metabolism within the plaque. 15 In this study, VGS isolated from patients with endocarditis as well as VGS isolated from patients with sepsis and neutropenia formed biofilms. However, biofilm formation was more common within the endocarditis isolates though not significant. Biofilm formation of VGS is possibly not a prerequisite for the pathogenesis of endocarditis. Nevertheless, the ability to form biofilm may be a virulence factor of the causative pathogens and contribute to the serious course of the disease and a lack of response to antimicrobial therapy. Biofilm-forming VGS may need longer periods of therapy and higher doses of antibiotics as recommended in the guidelines for treatment of endocarditis. 1 In our study, all patients with endocarditis irrespective of the potency to form biofilms received treatment for 4 6 weeks but endocarditis due to non-biofilm-forming VGS may need therapy for a shorter period of 14 days as described previously. 16 Treatment of endocarditis is considered complex with regard to the choice of antibiotics, dosage and duration of therapy. 1 Our effort was not aimed to carry out biofilm MICs but to investigate the behaviour of biofilms of specific clinical isolates challenged with antibiotics. As described in other studies, 4,17 there was a persistence of the biofilms in our study, even when incubated with an antibiotic concentration of 128 the MIC determined at planktonic conditions. Yet, a decrease in the biofilm OD was demonstrated when incubated with antibiotics. This decrease was marked for increasing concentrations of teicoplanin and moxifloxacin, but not for penicillin. The reason for this may be that penicillin has a short half-life and becomes inactivated quite rapidly at body temperature. 18 However, for all antibiotics Table 3. Optical density (OD) of the biofilms of the VGS isolates from patients with endocarditis (n = 8; EC) and from patients with sepsis and neutropenia (n = 6; SN) starting with the MIC determined at planktonic conditions OD [median (range)] penicillin teicoplanin moxifloxacin EC SN EC SN EC SN MIC 0.66 ( ) 0.86 ( ) 0.69 ( ) 0.73 ( ) 0.75 ( ) 0.97 ( ) 2 MIC 0.67 ( ) 0.92 ( ) 0.67 ( ) 0.71 ( ) 0.60 ( ) 0.93 ( ) 4 MIC 0.63 ( ) 0.95 ( ) 0.81 ( ) 0.78 ( ) 0.81 ( ) 0.85 ( ) 8 MIC 0.77 ( ) 0.88 ( ) 0.56 ( ) 0.71 ( ) 0.91 ( ) 0.88 ( ) 16 MIC 0.65 ( ) 0.73 ( ) 0.49 ( ) 0.75 ( ) 0.58 ( ) 0.70 ( ) 32 MIC 0.69 ( ) 0.75 ( ) 0.62 ( ) 0.77 ( ) 0.64 (0.32 1) 0.67 ( ) 64 MIC 0.64 ( ) 0.78 ( ) 0.52 ( ) 0.72 ( ) 0.62 ( ) 0.69 ( ) 128 MIC 0.66 ( ) 0.73 ( ) 0.56 ( ) 0.78 ( ) 0.67 ( ) 0.70 ( ) 48

5 Viridans streptococci in endocarditis and neutropenic sepsis tested, reduction in the biofilms incubated with any concentration was observed compared with biofilms. Compared with the other antibiotics tested, the reduction at low concentrations of penicillin is not different to that of the other antibiotics at low concentrations, but a further decrease does not occur with penicillin with increasing concentrations. Likewise, a further decrease in the biofilms is not observed at concentrations above 32 the MIC of teicoplanin and moxifloxacin. Overall, there was no difference in the decrease in biofilm densities between isolates from patients with endocarditis and from patients with neutropenia when incubated with the increasing antibiotic concentrations. In the current recommendations for antibiotic treatment of endocarditis, parenteral bactericidal antibiotics are considered superior to the use of oral drugs because of the importance of sustained antibacterial activity. 19 Short-term therapy has been associated with relapse, thus most current recommendations emphasize extended drug administration. Newer antibiotics with good activity against Gram-positive cocci and orally available were reported to be useful in the treatment of experimental and clinical endocarditis. 8,20 Linezolid was shown to have a decreasing effect on biofilm, however ciprofloxacin produced the greatest effects in the decreasing concentration experiment, but only in the susceptible strains. 21 Moxifloxacin has been shown to be the most active quinolone in experimental endocarditis caused by both methicillin-susceptible and methicillin-resistant Staphylococcus aureus. 8 In this study, moxifloxacin decreased the density of the formed biofilms, but controlled clinical studies would be needed to evaluate moxifloxacin s value in the treatment of endocarditis, particularly in the light of controversial clinical reports. 22 Generally, biofilm formation was detected in VGS isolated from both patients with endocarditis and with sepsis and neutropenia. The bacterial ability to form biofilms may be important for the antimicrobial treatment: VGS biofilms persist even when incubated with antibiotics to which VGS are susceptible at planktonic conditions. Penicillin had no significant effect on the biofilm OD. Teicoplanin and moxifloxacin at higher concentrations (>_ 16 MIC) caused a significant decrease in the biofilm OD although a further decrease in the OD was not observed at concentrations above 32 MIC. Whether standard in vitro testing at planktonic conditions is valid in biofilm-associated diseases like endocarditis is unclear and requires further investigation. Testing the effects of antibiotics on biofilms may supply useful information in addition to standard in vitro testing, particularly in diseases where biofilm formation may be involved in the pathogenesis. Acknowledgements We thank Michaela Eder, Institute of Material Sciences and Process Engineering, University of Natural Resources and Applied Life Sciences Vienna, for electron microscope scanning of the biofilms, Waltraud Schmidt at our department for determining MICs, and Christian Joukhadar, Department of Clinical Pharmacology, Medical University Vienna, for manuscript/editorial/analysis support. References 1. Bayer, A. S. & Scheld, W. M. (2000). Endocarditis and intravascular infections. In Principles and Practice of Infectious Diseases (Mandell, G. L., Bennett, J. E. & Dolin, R., Eds), pp Churchill-Livingstone, Philadelphia, PA, USA. 2. Donlan, R. M. & Costerton, J. W. (2002). Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical Microbiology Reviews 15, Marron, A., Carratala, J., Gonzalez-Barca, E. et al. (2000). Serious complications of bacteremia caused by viridans streptococci in neutropenic patients with cancer. 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