JAC Advance Access originally published online on December 1, 2006
Journal of Antimicrobial Chemotherapy 2007 59(2):301-304; doi:10.1093/jac/dkl482
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Differences in biofilm development and antibiotic susceptibility among Streptococcus pneumoniae isolates from cystic fibrosis samples and blood cultures
Servicio de Microbiología, Hospital Universitario Ramón y Cajal Ctra. Colmenar Km 9.1, Madrid 28034, Spain
*Corresponding author. Tel: +34-91-3368542; Fax: +34-91-3368809; E-mail: rosacampo{at}yahoo.com
Received 9 August 2006; returned 12 September 2006; revised 24 October 2006; accepted 3 November 2006
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Abstract |
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Objectives: To compare the capability of biofilm development between Streptococcus pneumoniae isolates from cystic fibrosis (CF) respiratory samples and those from non-CF blood cultures. Antibiotic susceptibility of biofilm-forming isolates, as well as differences between antibiotic susceptibility of sessile cells [minimum biofilm inhibitory concentration (MBIC)] and their planktonic counterparts (conventional MIC), were also assessed.
Methods: Biofilm formation was performed using a microtitre method in 20 CF and 22 non-CF blood culture S. pneumoniae isolates.
Results and conclusions: Biofilm formation occurs more frequently among S. pneumoniae isolates from CF (80%) than among non-CF blood culture isolates (50%) (P = 0.04). Moreover MBICs were significantly higher than conventional planktonic MICs among CF but not among non-CF blood isolates, suggesting a high adaptability of CF strains to form biofilms in adverse conditions.
Keywords: S. pneumoniae , minimum biofilm inhibitory concentrations , CF
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Introduction |
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Biofilms are complex and organized bacterial communities able to grow in association with different biological or inert surfaces. The major clinical consequence of biofilms developed by pathogenic bacteria relates to the difficulty of therapeutic eradication of sessile cells forming such supra-cellular structures.1,2 Bacterial biofilms play a relevant role in persistent infections such as chronic sinusitis and chronic otitis, as well as chronic bronchitis and bronchial colonization-infection of cystic fibrosis (CF) patients.3 Biofilm formation by certain CF pathogens has been related with both long-term persistence and significant increase in antibiotic resistance.4,5
S. pneumoniae might play a role in the early stages of bronchial colonization of CF patients. Persistence of identical clones with increasing rates of antibiotic resistance during prolonged periods affirms the possibility of a biofilm type of growth among CF S. pneumoniae isolates.6 Furthermore, biofilm development is facilitated by the anaerobic or microaerophilic environment that is characteristic in the CF airways. The capability of S. pneumoniae isolates to form biofilms has been evaluated in laboratory strains that have not included collections of clinical isolates.7,8
The aim of this work was to compare the capability for biofilm development between S. pneumoniae isolates from CF respiratory samples and from blood cultures of non-CF patients. Susceptibility to several antibiotics by standard procedures (MIC) was determined against both groups of isolates. For those able to form biofilms, differences in susceptibility between sessile cells (minimum biofilm inhibitory concentration, MBIC) and their planktonic counterparts (conventional MIC) were assessed.
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Materials and methods |
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A total of 22 and 20 S. pneumoniae isolates from blood cultures of non-CF patients and bronchial secretions from CF patients, respectively, were included. Blood isolates, recovered during 2005 from 22 different patients, were genetically unrelated by PFGE patterns. Chromosomal DNA was prepared for PFGE following the classical Gram-positive protocol with some modifications (R. del Campo, unpublished data). DNA restriction was made with the endonuclease SmaI (Amersham Biosciences Europe GmbH, Freiburg, Germany). Electrophoresis was carried out in a CHEF DR-III apparatus (Bio-Rad, Birmingham, UK) for 23 h at 14°C, and the following settings were applied at 6 V/cm: 1–30 s.
CF isolates corresponded to sixteen different paediatric patients (1–2 isolates per patient) who attended the Cystic Fibrosis Unit at Ramón y Cajal University Hospital between 1995 and 2003 and were resolved into 16 different clones (Table 1). Sputum samples from CF patients, collected either during scheduled clinical assessments or pulmonary exacerbations, were homogenized with N-acetyl-cysteine, seeded in bacitracin–chocolate agar and incubated in a 5% CO2 atmosphere for 48 h at 37°C. S. pneumoniae isolates recovered were identified on the basis of standard laboratory protocols using optochin and sodium deoxycholate solubility tests. Pneumococcal serotyping was performed with the Quellung reaction using antisera from the Statens Serum Institut (Copenhagen, Denmark) at the Spanish National Reference Center for Microbiology (Instituto de Salud Carlos III, Majadahonda, Madrid).
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MICs of penicillin, erythromycin, telithromycin, tetracycline, levofloxacin, linezolid and rifampicin were determined by broth microdilution following the CLSI guidelines.9 All compounds were supplied by their corresponding manufacturers or purchased from Sigma (Sigma Chemical Co., St Louis, MO, USA). Susceptibility panels were incubated overnight at 35°C in ambient air after inoculation. S. pneumoniae ATCC 49619 and 6330 strains were used as controls in each run.
A biofilm susceptibility assay was performed as described previously, with some modifications.10 Briefly, 96-well microtitre plates (Alpha Laboratories Ltd, Hampshire, UK) were inoculated with 100 µL of a 1/100 dilution of an overnight brain heart infusion (BHI) broth (Difco, Detroit, MI, USA) culture. Bacterial biofilms were formed by immersing the pegs of a modified polystyrene microtitre lid (catalogue no. 445497; Nunc TSP system, Nunc, Roskilde, Denmark) into this biofilm growth plate incubated for 20 h at 37°C. Lids were then washed three times in sterile water to eliminate the planktonic organisms and placed into another microtitre plate containing serial dilutions of antibiotic, and incubated for 18–20 h at 37°C. Biofilms formed in the lids were transferred to the microtitre wells by centrifugation at 3000 rpm for 10 min. The lid was rejected and replaced with another clean lid and incubated for another 6 h. BHI broth was the medium used in all experiments. An adequate biofilm growth for the positive growth-control well was defined as a mean OD600 difference (OD600 at 6 h minus OD600 at 0 h) higher than 0.05. Optical density was measured in a microtitre optical colorimeter (Flow Titertek Multiskan Plus, Flow Laboratories, Finland). MBIC was defined as the lowest concentration of drug that resulted in an OD600 difference at or below 10% of the mean of two positive growth-control well readings.10 All isolates were tested for antibiotic susceptibility by standard procedures (MIC) as described above while the comparison between MBIC50–MIC50 and MBIC90–MIC90 values was only carried out for those strains that were able to form biofilms. Antibiotic mode and range values were also compared. Statistical significance was calculated by the 
2 test.
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Results |
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Different capabilities of biofilm formation for S. pneumoniae isolates were observed depending on their clinical origin. In fact 80% of CF isolates (16 out of 20) were able to form biofilm whereas only 50% of the non-CF blood isolates (11 out of 22) (P = 0.04) did. No relation between specific serotypes or clones and biofilm formation was observed. Moreover, biofilm former and non-former clones sharing the same serotype were found among both groups of isolates (Table 1).
No significant differences in the MIC values among CF or non-CF blood isolates were observed for penicillin (55% and 47.6%), erythromycin (40% and 42.8%) and tetracycline (50% and 47.6%), respectively (Table 2). Almost all isolates were susceptible to the other antibiotics tested. Differences in susceptibility MIC values were not observed when we compared biofilm- and non-biofilm-forming S. pneumoniae isolates (data not shown). However, antibiotic resistance rates in biofilm-forming clones (MBIC) exhibited different trends depending on their origin. In CF isolates the MBICs were higher than the MICs of penicillin (P = 0.03), tetracycline (P = 0.01) and rifampicin (P = 0.09), whereas in non-CF blood isolates MBIC values were not statistically higher than MICs of penicillin, erythromycin and tetracycline (Table 2).
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Discussion |
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The pathogenic role of S. pneumoniae in early stages of lung colonization or infection in CF patients has been discussed previously.6 According to results obtained in this study, the capability of biofilm formation with penicillin and tetracycline resistance appears to be a trait among CF pneumococcal isolates. Moreover, in a previous study conducted in the same hospital, CF S. pneumoniae isolates presented higher frequencies of rifampicin mutation than non-CF isolates, which may denote a certain capability for adaptation under selective pressure.6 Characterization of pneumococcal biofilms has recently been performed suggesting that diverse architectures observed among these structures may be related to different serotypes.7
S. pneumoniae MBICs tended to be higher than MICs in the CF isolates, reaching statistical significance for penicillin and tetracycline. This result suggests that CF isolates forming biofilms may have reached a high level of tolerance to certain antibiotics resulting in reduced cell lysis. Mutations in a number of S. pneumoniae genes produce pleiotropic effects involving lysis and it has been demonstrated previously that S. pneumoniae isolated from CF patients tend to have increased mutation rates.6 High tolerance to antibiotic action may then explain the high MBICs for CF isolates forming biofilms. Conversely, in the case of non-CF blood isolates able to form biofilms, retention of their normal autolytic activity should be deleterious for an important part of the population as such lysis capacity could be favoured by the slow mode of growth inside the biofilm structure.
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Transparency declarations |
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F. B. and R. C. received grants from Sanofi-Aventis. The other participants have nothing to declare.
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References |
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1 Costerton JW, Stewart PS, Greenberg EP. (1999) Bacterial biofilms: a common cause of persistent infections. Science 284:1318–22.
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Donlan R, Piede MJA, Heyes CD, et al. (2004) Model system for growing and quantifying Streptococcus pneumoniae biofilms in situ and in real time. Appl Environ Microbiol 70:4980–8.
3 Singh PK, Schaefer AL, Parsek MR, et al. (2000) Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407:762–4.[CrossRef][Medline]
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Hill D, Rose B, Pajkos A, et al. (2005) Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions. J Clin Microbiol 43:5085–90.
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Lee B, Haagensen JA, Ciofu O, et al. (2005) Heterogeneity of biofilms formed by nonmucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis. J Clin Microbiol 43:5247–55.
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del Campo R, Morosini MI, de la Pedrosa EG, et al. (2005) Population structure, antimicrobial resistance, and mutation frequencies of Streptococcus pneumoniae isolates from cystic fibrosis patients. J Clin Microbiol 43:2207–14.
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Allegrucci M, Hu FZ, Shen K, et al. (2006) Phenotypic characterization of Streptococcus pneumoniae biofilm development. J Bacteriol 188:2325–35.
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Budhani RK and Struthers JK. (1997) The use of Sorbarod biofilms to determine the antimicrobial susceptibilities of a strain of Streptococcus pneumoniae. J Antimicrob Chemother 40:601–2.
9 Clinical and Laboratory Standards Institute. (2006) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Seventh Edition: Approved Standard M7-A7(CLSI, Wayne, PA, USA).
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Moskowitz SM, Foster JM, Emerson J, et al. (2004) Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis. J Clin Microbiol 42:1915–22.
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