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

E. Presterl1,*, A. J. Grisold2, S. Reichmann1, A. M. Hirschl3, A. Georgopoulos1 and W. Graninger1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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 1998–2000, 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 MIC90s (range) of penicillin, teicoplanin and moxifloxacin were 0.5 mg/L (0.001–0.5), 0.125 mg/L (0.025–0.125) and 0.5 mg/L (0.05–0.5), respectively. Generally, biofilms persisted although incubated with the antibiotics up to concentrations of 128x 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 128x MIC. Nevertheless, teicoplanin and moxifloxacin reduced the density of the biofilms at concentrations ≥16xMIC. 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
During the years 1998–2000, 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/mm3 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 37°C 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 µL of diluted bacteria suspension and 50 µL 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% CO2) at 35°C, 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 150–250 µL of 1% Crystal Violet for 3 min. Then the dyed biofilms were washed two times with approximately 300 µL 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 µL of SMHB with penicillin, teicoplanin and moxifloxacin at concentrations of 1x, 2x, 4x, 8x, 16x, 32x, 64x, 128x 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 35°C 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 0.05. 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 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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 teicoplanin to which the pathogen was highly susceptible in vitro (MIC 0.001 mg/L). Teicoplanin treatment for this patient was initiated after the empirical combination therapy with penicillin plus gentamicin.


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Table 1. Demographic data of the 40 patients with VGS bloodstream infections

 
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 MIC90s (MIC range) of penicillin, teicoplanin and moxifloxacin were 0.25 mg/L (0.001–1), 0.25 mg/L (0.001–0.25) and 0.5 mg/L (0.001–1), 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, MIC90s (range) of penicillin, teicoplanin and moxifloxacin were 0.5 mg/L (0.001–0.5), 0.125 mg/L (0.025–0.125) and 0.5 mg/L (0.05–0.5), 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 128x 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 teicoplanin and moxifloxacin, but not when incubated with penicillin (P < 0.05) (Figure 1). A significant decrease in the OD was observed at 16x the MIC for moxifloxacin, and at 32x 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 128x 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 with sepsis and neutropenia (S. mitis). With regard to their MICs, all isolates were highly susceptible.


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Table 2. MICs for the 14 VGS isolates that formed biofilms

 


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Figure 1. Decrease in the biofilm density given as the change in the optical density ({Delta}OD) measured after 24 h of incubation with penicillin, teicoplanin or moxifloxacin at concentrations of 2x, 4x, 8x, 16x, 32x, 64x and 128x MIC in reference to the OD of the biofilms measured after an incubation at a concentration of 1x 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.

 

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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

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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 128x 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 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 32x 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 (≥ 16x MIC) caused a significant decrease in the biofilm OD although a further decrease in the OD was not observed at concentrations above 32x 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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.


    Footnotes
 
* Corresponding author. Tel: +43-1-40400-4440; Fax: +43-1-40400-4418; Email: elisabeth.presterl{at}meduniwien.ac.at


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 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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8 . Entenza, J. M., Que, Y. A., Vouillamoz, J. et al. (2001). Efficacies of moxifloxacin, ciprofloxacin, and vancomycin against experimental endocarditis due to methicillin-resistant Staphylococcus aureus expressing various degrees of ciprofloxacin resistance. Antimicrobial Agents and Chemotherapy 45, 3076–83.[Abstract/Free Full Text]

9 . Durack, D. T., Bright, D. K. & Lukes, A. S. (1994). New criteria for diagnosis of infective endocarditis: utilization of specific echocardiographic findings. American Journal of Medicine 96, 200–9.[ISI][Medline]

10 . National Committee for Clinical Laboratory Standards. (2004). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA.

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14 . Li, Y. H., Lau, P. C., Tang, N. et al. (2002). Novel two-component regulatory system involved in biofilm formation and acid resistance in Streptococcus mutans. Journal of Bacteriology 184, 6333–42.[Abstract/Free Full Text]

15 . Wen, Z. T. & Burne, R. A. (2002). Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans. Applied and Environmental Microbiology 68, 1196–203.[Abstract/Free Full Text]

16 . Francioli, P., Ruch, W. & Stamboulian, D. (1995). Treatment of streptococcal endocarditis with a single daily dose of ceftriaxone and netilmicin for 14 days: a prospective multicenter study. Clinical Infectious Diseases 21, 1406–10.[ISI][Medline]

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19 . Shanson, D. C. (1998). New guidelines for the antibiotic treatment of streptococcal, enterococcal and staphylococcal endocarditis. Journal of Antimicrobial Chemotherapy 42, 292–6.[Free Full Text]

20 . Dailey, C. F., Dileto-Fang, C. L., Buchanan, L. V. et al. (2001). Efficacy of linezolid in treatment of experimental endocarditis caused by methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 45, 2304–8.[Abstract/Free Full Text]

21 . Gander, S., Hayward, K. & Finch, R. (2002). An investigation of the antimicrobial effects of linezolid on bacterial biofilms utilizing an in vitro pharmacokinetic model. Journal of Antimicrobial Chemotherapy 49, 301–8.[Abstract/Free Full Text]

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