- Email: [email protected]
Lactobacilli: Important in biofilm formation on voice prostheses
Otolaryngology–Head and Neck Surgery (2007) 137, 505-507
SHORT SCIENTIFIC COMMUNICATION
Lactobacilli: Important in biofilm formation on voice prostheses Kevin J.D.A. Buijssen, MD, Hermie J.M. Harmsen, PhD, Henny C. van der Mei, PhD, Henk J. Busscher, PhD, and Bernard F.A.M. van der Laan, MD, PhD, Groningen, the Netherlands OBJECTIVE: We sought to identify bacterial strains responsible for biofilm formation on silicone rubber voice prostheses. STUDY DESIGN: We conducted an analysis of the bacterial population in biofilms on used silicone rubber voice prostheses by using new microbiological methods. METHODS: Two microbiological methods were used: polymerase chain reaction⫺denaturing gradient gel electrophoresis and fluorescence in situ hybridization. Twenty-six Provox2 and eight Groningen Ultra Low Resistance voice prostheses that were removed because of leakage through the prosthesis or because of increased airflow resistance were used in this study. RESULTS: The results showed that 33 of the 34 explanted voice prosthetic biofilms contained lactobacilli in close association with the Candida sp. present. CONCLUSION: Lactobacilli are general colonizers of tracheoesophageal voice prostheses in vivo, growing intertwined with Candida. This knowledge may be important in the development of new pathways directed to prevent or to influence biofilm formation on tracheoesophageal voice prostheses and elongate their lifespan. © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
T
racheoesophageal shunt speech is the most frequently used method of voice rehabilitation in laryngectomized patients. There are several types of silicone rubber voice prostheses in use worldwide, such as the Provox2, BlomSinger, and Groningen Ultra Low Resistance prosthesis. The esophageal side of all voice prostheses becomes rapidly covered by a microbial biofilm, which limits the lifespan of the prostheses. Microbial biofilms are held responsible for deterioration of the silicone rubber and malfunctioning of the valve mechanism, causing either increased airflow resistance or leakage through the prosthesis. Candida sp. are ubiquitously accepted as the predominant causative organisms for prosthesis failure,1 although Candida as a solitary species is incapable of forming a biofilm and many other microbial strains and species can be found in voice prosthetic biofilms. However, there is no general consensus on
the strains and species occurring in voice prosthetic biofilms. Rothia dentocariosa and Staphylococcus aureus have been suggested as causative bacteria in early failure of prostheses, but usually traditional, culture-based techniques have not been able to identify all bacterial strains isolated from voice prosthetic biofilms. Rod-shaped organisms, for instance, have often been observed but have never been identified.2 Moreover, in vitro culture conditions may not allow the growth of all bacteria present in a sample. Nowadays, new techniques have become available for studying mixed bacterial communities, without the bias of culture.
METHODS We used two microbiological methods, polymerase chain reaction⫺denaturing gradient gel electrophoresis (PCRDGGE) and fluorescence in situ hybridization (FISH), to identify the microbial diversity in biofilms from explanted tracheoesophageal voice prostheses. DGGE is a molecular fingerprinting method that separates PCR-generated DNA products based on their melting behavior in a denaturing gradient. FISH is a visualization method, based on the use of fluorescently labeled ribosomal RNA⫺targeted probes to identify and visualize specific microorganisms in a biofilm. In this study, the Lab158 probe (specifically designed to visualize lactobacilli) in combination with an EUK516 probe (indicating yeasts) was used. All oligonucleotide probes were commercially obtained (Eurogentec, Seraing, Belgium), and the specificity of the probes has been confirmed by testing against a panel of reference strains,3,4 using the EUB338 probe as a positive and the antisense probe non-EUB338 as a negative control. Twenty-six Provox2 and eight Groningen Ultra Low Resistance voice prostheses removed in the outpatient clinic of the University Medical Center Groningen, the Netherlands, were used for this study. All replacements were necessary because of
Received March 21, 2007; revised May 1, 2007; accepted May 14, 2007.
0194-5998/$32.00 © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2007.05.051
506
Otolaryngology–Head and Neck Surgery, Vol 137, No 3, September 2007
increased airflow resistance or leakage through the prosthesis. Prostheses had a mean in situ lifetime of 86 days, with the minimum equal to 5 days and the maximum lifetime of 576 days.
RESULTS A DGGE gel of three voice prosthetic biofilms, including a marker lane, is shown in Figure 1. The marker lane contains bands from strains cultured from explanted prostheses and identified by sequence analysis of the 16S rRNA gene as Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus fermentum, Staphylococcus aureus, Staphylococcus epi-
Figure 2 Overlay-images of a biofilm from an explanted voice prosthesis (life time 318 days) hybridized with the FITC-labeled EUK516 probe indicating all yeasts (with hyphae) and Cy3-labeled Lab158 probe illustrating presence of lactobacilli. The frame in the left lower corner is a magnification that clearly shows the association of lactobacilli (red) and yeasts (green).
Figure 1 Example of a part of a DGGE-gel showing lanes from three tracheoesophageal voice prosthetic biofilms and a marker lane, indicated by the arrow. The marker contains bands from a mixture of single species isolated from voice prostheses that have been identified by 16S rRNA-gene sequence analysis The corresponding species are indicated at the right. Lactobacillus bands are positioned in the box and are present in each of the three voice prosthetic biofilms shown.
dermidis, and Streptococcus salivarius. Because 33 of the 34 explanted voice prosthetic biofilms yielded bands that migrated to the same height as did the DNA from the lactobacilli marker strains, it can be concluded for the first time that lactobacilli are ubiquitously present in tracheoesophageal voice prosthetic biofilms. Visualization of the biofilms by FISH and using confocal laser scanning microscopy (Fig 2) confirmed that most bacteria present in voice prosthetic biofilms are lactobacilli, colonizing prostheses in close association with Candida. The Candida⫺lactobacilli interaction likely plays an important role in the mixed biofilm on tracheoesophageal voice prostheses, although it is unclear whether the presence of lactobacilli in voice prosthetic biofilms should be associated with failure of the prostheses or not. It is well known that lactobacilli inhibit the growth of Candida albicans at varying concentrations and lactobacilli are among the most widely used probiotic organisms. Each probiotic strain is different and may have different clinical effects. Lactobacilli as probiotics exert their beneficial effects through competitive displacement and exclusion of pathogens, affinity for the tissues and materials to be protected, coaggregation with pathogens to be eliminated, and H2O2, lactic acid, bacteriocin, and biosurfactant production. Lactobacillus therapy has been applied especially to improve the intestinal microbial balance and is used for the prevention of symptoms of lactose intolerance, treatment of acute diarrhea, attenuation of antibiotic-associated gastrointestinal side effects, and prevention and treatment of allergy manifestations. Also, lactobacillus therapy is known to restore a healthy urogenital microflora, preventing urinary
Buijssen et al
Lactobacilli: Important in biofilm formation . . .
tract infections. Recently, the consumption of a fermented dairy product containing a particular strain of probiotic lactobacilli, Lactobacillus casei Shirota, has been associated with a prolonged life span of voice prostheses in patients.5
507
Corresponding author: Kevin J. D. A. Buijssen, MD, Department of Otorhinolaryngology, University Medical Center Groningen, and University of Groningen, Hanzeplein 1, 9713 GZ Groningen, the Netherlands. E-mail address: [email protected].
FINANCIAL DISCLOSURE CONCLUSION
None.
This is the first time that lactobacilli have been identified to be general colonizers of tracheoesophageal voice prostheses in vivo. Lactobacilli form biofilms in close association with the Candida sp. present. This knowledge may be important in the development of new pathways directed to prevent or to influence biofilm formation on tracheoesophageal voice prostheses and elongate their life time.
REFERENCES
AUTHOR INFORMATION From the Departments of Biomedical Engineering (Buijssen, van der Mei, and Busscher), Otorhinolaryngology and Head and Neck Surgery (Buijssen and van der Laan), and Medical Microbiology (Harmsen), University Medical Center Groningen and University of Groningen, Groningen, the Netherlands.
1. Mahieu HF, Van Saene HK, Rosingh HJ, et al. Candida vegetations on silicone voice prostheses. Arch Otolaryngol Head Neck Surg 1986;112: 321–5. 2. Elving GJ, Van der Mei HC, Busscher HJ, et al. Comparison of the microbial composition of voice prosthesis biofilms from patients requiring frequent versus infrequent replacement. Ann Otol Rhinol Laryngol 2002;111:200 –3. 3. Harmsen HJ, Elfferich P, Schut F, et al. A 16S rRNA-targeted probe for detection of lactobacilli and enterococci in faecal samples by fluorescent in situ hybridization. Microbiol Ecol Health Dis 1999;11:3–12. 4. Amann RI, Binder BJ, Olson RJ, et al. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 1990;56:1919–25. 5. Schwandt LQ, Van Weissenbruch R, Van der Mei HC, et al. Effect of dairy products on the lifetime of Provox2 voice prostheses in vitro and in vivo. Head Neck 2005;27:471–7.
SHORT SCIENTIFIC COMMUNICATION
Lactobacilli: Important in biofilm formation on voice prostheses Kevin J.D.A. Buijssen, MD, Hermie J.M. Harmsen, PhD, Henny C. van der Mei, PhD, Henk J. Busscher, PhD, and Bernard F.A.M. van der Laan, MD, PhD, Groningen, the Netherlands OBJECTIVE: We sought to identify bacterial strains responsible for biofilm formation on silicone rubber voice prostheses. STUDY DESIGN: We conducted an analysis of the bacterial population in biofilms on used silicone rubber voice prostheses by using new microbiological methods. METHODS: Two microbiological methods were used: polymerase chain reaction⫺denaturing gradient gel electrophoresis and fluorescence in situ hybridization. Twenty-six Provox2 and eight Groningen Ultra Low Resistance voice prostheses that were removed because of leakage through the prosthesis or because of increased airflow resistance were used in this study. RESULTS: The results showed that 33 of the 34 explanted voice prosthetic biofilms contained lactobacilli in close association with the Candida sp. present. CONCLUSION: Lactobacilli are general colonizers of tracheoesophageal voice prostheses in vivo, growing intertwined with Candida. This knowledge may be important in the development of new pathways directed to prevent or to influence biofilm formation on tracheoesophageal voice prostheses and elongate their lifespan. © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
T
racheoesophageal shunt speech is the most frequently used method of voice rehabilitation in laryngectomized patients. There are several types of silicone rubber voice prostheses in use worldwide, such as the Provox2, BlomSinger, and Groningen Ultra Low Resistance prosthesis. The esophageal side of all voice prostheses becomes rapidly covered by a microbial biofilm, which limits the lifespan of the prostheses. Microbial biofilms are held responsible for deterioration of the silicone rubber and malfunctioning of the valve mechanism, causing either increased airflow resistance or leakage through the prosthesis. Candida sp. are ubiquitously accepted as the predominant causative organisms for prosthesis failure,1 although Candida as a solitary species is incapable of forming a biofilm and many other microbial strains and species can be found in voice prosthetic biofilms. However, there is no general consensus on
the strains and species occurring in voice prosthetic biofilms. Rothia dentocariosa and Staphylococcus aureus have been suggested as causative bacteria in early failure of prostheses, but usually traditional, culture-based techniques have not been able to identify all bacterial strains isolated from voice prosthetic biofilms. Rod-shaped organisms, for instance, have often been observed but have never been identified.2 Moreover, in vitro culture conditions may not allow the growth of all bacteria present in a sample. Nowadays, new techniques have become available for studying mixed bacterial communities, without the bias of culture.
METHODS We used two microbiological methods, polymerase chain reaction⫺denaturing gradient gel electrophoresis (PCRDGGE) and fluorescence in situ hybridization (FISH), to identify the microbial diversity in biofilms from explanted tracheoesophageal voice prostheses. DGGE is a molecular fingerprinting method that separates PCR-generated DNA products based on their melting behavior in a denaturing gradient. FISH is a visualization method, based on the use of fluorescently labeled ribosomal RNA⫺targeted probes to identify and visualize specific microorganisms in a biofilm. In this study, the Lab158 probe (specifically designed to visualize lactobacilli) in combination with an EUK516 probe (indicating yeasts) was used. All oligonucleotide probes were commercially obtained (Eurogentec, Seraing, Belgium), and the specificity of the probes has been confirmed by testing against a panel of reference strains,3,4 using the EUB338 probe as a positive and the antisense probe non-EUB338 as a negative control. Twenty-six Provox2 and eight Groningen Ultra Low Resistance voice prostheses removed in the outpatient clinic of the University Medical Center Groningen, the Netherlands, were used for this study. All replacements were necessary because of
Received March 21, 2007; revised May 1, 2007; accepted May 14, 2007.
0194-5998/$32.00 © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2007.05.051
506
Otolaryngology–Head and Neck Surgery, Vol 137, No 3, September 2007
increased airflow resistance or leakage through the prosthesis. Prostheses had a mean in situ lifetime of 86 days, with the minimum equal to 5 days and the maximum lifetime of 576 days.
RESULTS A DGGE gel of three voice prosthetic biofilms, including a marker lane, is shown in Figure 1. The marker lane contains bands from strains cultured from explanted prostheses and identified by sequence analysis of the 16S rRNA gene as Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus fermentum, Staphylococcus aureus, Staphylococcus epi-
Figure 2 Overlay-images of a biofilm from an explanted voice prosthesis (life time 318 days) hybridized with the FITC-labeled EUK516 probe indicating all yeasts (with hyphae) and Cy3-labeled Lab158 probe illustrating presence of lactobacilli. The frame in the left lower corner is a magnification that clearly shows the association of lactobacilli (red) and yeasts (green).
Figure 1 Example of a part of a DGGE-gel showing lanes from three tracheoesophageal voice prosthetic biofilms and a marker lane, indicated by the arrow. The marker contains bands from a mixture of single species isolated from voice prostheses that have been identified by 16S rRNA-gene sequence analysis The corresponding species are indicated at the right. Lactobacillus bands are positioned in the box and are present in each of the three voice prosthetic biofilms shown.
dermidis, and Streptococcus salivarius. Because 33 of the 34 explanted voice prosthetic biofilms yielded bands that migrated to the same height as did the DNA from the lactobacilli marker strains, it can be concluded for the first time that lactobacilli are ubiquitously present in tracheoesophageal voice prosthetic biofilms. Visualization of the biofilms by FISH and using confocal laser scanning microscopy (Fig 2) confirmed that most bacteria present in voice prosthetic biofilms are lactobacilli, colonizing prostheses in close association with Candida. The Candida⫺lactobacilli interaction likely plays an important role in the mixed biofilm on tracheoesophageal voice prostheses, although it is unclear whether the presence of lactobacilli in voice prosthetic biofilms should be associated with failure of the prostheses or not. It is well known that lactobacilli inhibit the growth of Candida albicans at varying concentrations and lactobacilli are among the most widely used probiotic organisms. Each probiotic strain is different and may have different clinical effects. Lactobacilli as probiotics exert their beneficial effects through competitive displacement and exclusion of pathogens, affinity for the tissues and materials to be protected, coaggregation with pathogens to be eliminated, and H2O2, lactic acid, bacteriocin, and biosurfactant production. Lactobacillus therapy has been applied especially to improve the intestinal microbial balance and is used for the prevention of symptoms of lactose intolerance, treatment of acute diarrhea, attenuation of antibiotic-associated gastrointestinal side effects, and prevention and treatment of allergy manifestations. Also, lactobacillus therapy is known to restore a healthy urogenital microflora, preventing urinary
Buijssen et al
Lactobacilli: Important in biofilm formation . . .
tract infections. Recently, the consumption of a fermented dairy product containing a particular strain of probiotic lactobacilli, Lactobacillus casei Shirota, has been associated with a prolonged life span of voice prostheses in patients.5
507
Corresponding author: Kevin J. D. A. Buijssen, MD, Department of Otorhinolaryngology, University Medical Center Groningen, and University of Groningen, Hanzeplein 1, 9713 GZ Groningen, the Netherlands. E-mail address: [email protected].
FINANCIAL DISCLOSURE CONCLUSION
None.
This is the first time that lactobacilli have been identified to be general colonizers of tracheoesophageal voice prostheses in vivo. Lactobacilli form biofilms in close association with the Candida sp. present. This knowledge may be important in the development of new pathways directed to prevent or to influence biofilm formation on tracheoesophageal voice prostheses and elongate their life time.
REFERENCES
AUTHOR INFORMATION From the Departments of Biomedical Engineering (Buijssen, van der Mei, and Busscher), Otorhinolaryngology and Head and Neck Surgery (Buijssen and van der Laan), and Medical Microbiology (Harmsen), University Medical Center Groningen and University of Groningen, Groningen, the Netherlands.
1. Mahieu HF, Van Saene HK, Rosingh HJ, et al. Candida vegetations on silicone voice prostheses. Arch Otolaryngol Head Neck Surg 1986;112: 321–5. 2. Elving GJ, Van der Mei HC, Busscher HJ, et al. Comparison of the microbial composition of voice prosthesis biofilms from patients requiring frequent versus infrequent replacement. Ann Otol Rhinol Laryngol 2002;111:200 –3. 3. Harmsen HJ, Elfferich P, Schut F, et al. A 16S rRNA-targeted probe for detection of lactobacilli and enterococci in faecal samples by fluorescent in situ hybridization. Microbiol Ecol Health Dis 1999;11:3–12. 4. Amann RI, Binder BJ, Olson RJ, et al. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 1990;56:1919–25. 5. Schwandt LQ, Van Weissenbruch R, Van der Mei HC, et al. Effect of dairy products on the lifetime of Provox2 voice prostheses in vitro and in vivo. Head Neck 2005;27:471–7.