Non-Culture-Based Analysis of Bacterial Populations from Patients with Chronic Rhinosinusitis
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微生物临床杂志 2005年第11期
Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
BLIS Technologies Limited, Center for Innovation, University of Otago, Dunedin, New Zealand
Otolaryngology and Head and Neck Surgery, Department of Medical and Surgical Sciences, University of Otago, Dunedin, New Zealand
ABSTRACT
Middle meatus aspirates from patients with chronic rhinosinusitis were analyzed by bacterial culture, denaturing gradient gel electrophoresis (DGGE), and antibiotic sensitivity techniques. DGGE detected a greater bacterial diversity than culture methods. Although resistance to antibiotics was low, there was evidence of changes in the composition of the bacterial microbiota over time, and the presence of noncultured bacteria was demonstrated.
CASE REPORT
Using a glass canula, aspirates were taken from the middle meatus of six patients who had previously undergone surgery for chronic rhinosinusitis. These patients had continued infection after surgery despite ongoing nasal toilet and antibiotic treatment. The antibiotics prescribed to the patients within the 2 months prior to the aspirates being taken are listed in Table 1. Four of the patients had a second aspirate taken 2 to 3 months after the first sample. The aspirates were first suspended in 2 ml phosphate-buffered saline and then thoroughly homogenized by being vortexed before being divided into two equal volumes for use in culture-based and DNA-based analyses. For the isolation of staphylococci, mannitol salt agar (Difco, Sparks, MD) was incubated at 37°C aerobically. Total bacterial populations were isolated on chocolate agar (Columbia agar base [Difco] with 5% heat-lysed defibrinated sheep blood [New Zealand Venous Supplies, Tuakau, New Zealand]) by incubation at 37°C either aerobically, in air supplemented with 5% CO2, or in an anaerobic atmosphere (85% N2, 10% H2, 5% CO2). For the isolation of Streptococcus pneumoniae, sheep blood agar (Columbia agar base containing 5% defibrinated sheep blood) with 5 μg/ml gentamicin (Sigma, St. Louis, MO) was used with anaerobic incubation at 37°C. Representative bacterial isolates were then identified by sequencing of the 16S rRNA gene according to the method of Wilson et al. (18). Where isolates were identified as belonging to the anginosus group of streptococci, multiplex PCR according to the protocol of Takao et al. (14) was used to differentiate the subgroups. The antibiotic sensitivities of the isolates were tested using Etest Strips (AB BIODISK, Solna, Sweden) according to the manufacturer's instructions.
The DNA for the non-culture-based analysis was extracted as previously described (3). The extracted DNA was then used as a template for PCR-denaturing gradient gel electrophoresis (DGGE) according to the protocol of Walter et al. (17), except that a 40 to 55% denaturing gradient was used in a CBS Scientific (Del Mar, CA) denaturing gradient gel electrophoresis system (model DGGE-2001). DNA fragments of interest were excised from the gel and sequenced. The nucleotide sequences were analyzed using SeqMan software (version 5.51; DNASTAR, Inc., Madison, WI) and compared against sequences in the GenBank DNA database using the BLAST algorithm (1).
For each of the samples tested by culture, at least one bacterial colony type was detected, and two of the samples yielded two different bacterial species. The bacteria present included S. pneumoniae (n = 4), Staphylococcus aureus (n = 4), anginosus group streptococci (n = 3), Staphylococcus epidermidis (n = 1), and Haemophilus influenzae (n = 1) (Table 1). All three anginosus group streptococci were subsequently identified as Staphylococcus constellatus subsp. constellatus using the group-specific multiplex PCR. When tested against antibiotics commonly prescribed in New Zealand for upper respiratory tract infections, most of the isolates were sensitive (Table 1). However, four showed decreased sensitivity or resistance to at least one of the tested antibiotics. None of the isolates displayed resistance to amoxicillin. At least one isolate showed either resistance or decreased sensitivity to erythromycin, cefaclor, or trimethoprim-sulfamethoxazole.
PCR-DGGE detected three or more bacterial types (Fig. 1) in every sample. For the patients who had aspirates taken on two occasions, there was a clear difference between the profiles in the two samples. When major fragments (indicated in Fig. 1) were sequenced, a variety of bacteria were identified (Table 2). The most common identification was that of streptococci belonging to the mitis-sanguinis group. The potential pathogens S. aureus and H. influenzae were both detected by PCR-DGGE and by culture. Other bacteria of interest detected included the potential oral pathogens Porphyromonas, Fusobacterium, and Prevotella. In addition, two DNA fragments were identified as corresponding to bacteria previously identified as noncultured, high-G+C bacteria from aquatic or soil environments.
Discussion. The bacterial species isolated in the culture-based part of this study were consistent with those recovered in previous studies of the microbiology of infectious rhinosinusitis (2, 5, 6, 8, 9, 19). Although most appeared sensitive to amoxicillin, erythromycin, cefaclor, and trimethoprim-sulfamethoxazole, 4 of the 13 representative isolates tested showed resistance or decreased sensitivity to at least one of these antibiotics. Only patient 5 yielded an isolate that was resistant to a recently prescribed antibiotic. In this case, an H. influenzae isolate resistant to erythromycin was recovered from a patient who had received ongoing treatment with the antibiotic. Failure of antibiotics to resolve the infection could be due to other confounding factors, such as the chronic inflammatory process that characterizes this disease or perhaps its being a biofilm infection, which has been suggested by Ramadan et al. (11) to interfere with the action of antibiotics.
PCR-DGGE has previously been used for analysis of the human microbiota of the gastrointestinal tract (10, 16, 20), vagina (4), oral cavity (12), and eye (13). However, this is the first report of the application of this technique to samples from the sinuses. The results of the PCR-DGGE analysis show a greater variety of bacterial species to be present in all of the samples than was detected by direct culture. This indicates that there may be some bacterial species involved in the infection, or that are commensal organisms of the sinuses, not regularly detected by standard culture methods. This could be due to their being present in smaller numbers, being difficult to culture, or possibly being outcompeted in culture by more dominant bacterial species. The bacterial types not detected so readily in culture included H. influenzae and the relatively slow-growing oral pathogen Prevotella sp. In two cases, putative environmental bacteria were also shown to be present. It is unclear whether these uncultured bacteria are commensals or are actively involved in the disease process or whether they exist as transient inhabitants of the sinuses. The detection of these bacteria by PCR-DGGE suggests that they must be present in substantial numbers (greater than 1% of the population), and thus, the potential for their direct involvement in the disease process appears to merit further investigation. The inability to distinguish between the various members of the mitis-sanguinis group of streptococci by PCR-DGGE is due to the high level of similarity within their 16S rRNA genes (7). This makes differentiation between common pathogens, such as S. pneumoniae, and nonpathogenic members of this group quite difficult.
Although staphylococci were readily isolated by culture-based methods, there was only one specimen in which they were identified by PCR-DGGE. A previous study involving PCR-DGGE (4) also failed to detect staphylococci in specimens in which they appeared dominant when tested by other means. Possible reasons for this apparent anomaly include the following: the DNAs of these organisms being more difficult to extract from clinical samples, PCR bias of the DNA template of nondominant organisms, PCR primer bias, and differences between the copy numbers of the 16S rRNA genes in different organisms (15).
PCR-DGGE showed a greater diversity of bacteria to be present than did culture-based methods and also showed that a change occurred in the composition of the microbiota in those patients from whom samples were taken on different occasions. This is of significant interest because it shows the occurrence of microbial succession in chronic rhinosinusitis without regression of disease symptoms. This could have important implications for prescribing antibiotics for these patients, since some bacteria initially predominant within the microbiota could differ in their antibiotic susceptibilities compared to those that subsequently adopt a leading role within the consortium. Such population swings could lead to nonoptimum antibiotic treatments being administered and consequent protraction of the disease process for the patient. Chronic rhinosinusitis is a complex disease that is influenced by anatomical, immunological, and microbiological factors. The present study has for the first time utilized PCR-DGGE, a non-culture-based technique, to investigate the microbiology of chronic rhinosinusitis. The picture that has emerged is of a polymicrobial disease involving a dynamic succession of bacterial contributors and with some very preliminary evidence for a potential role in the microbial consortium of certain uncultured bacteria not previously associated with humans.
REFERENCES
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.
Brook, I., E. H. Frazier, and P. A. Foote. 1997. Microbiology of chronic maxillary sinusitis: comparison between specimens obtained by sinus endoscopy and by surgical drainage. J. Med. Microbiol. 46:430-432.
Burton, J. P., P. A. Cadieux, and G. Reid. 2003. Improved understanding of the bacterial vaginal microbiota of women before and after probiotic instillation. Appl. Environ. Microbiol. 69:97-101.
Burton, J. P., and G. Reid. 2002. Evaluation of the bacterial vaginal flora of 20 postmenopausal women by direct (Nugent score) and molecular (polymerase chain reaction and denaturing gradient gel electrophoresis) techniques. J. Infect. Dis. 186:1770-1780.
Chan, J., and J. Hadley. 2001. The microbiology of chronic rhinosinusitis: results of a community surveillance study. Ear Nose Throat J. 80:143-145.
Jiang, R. S., C. Y. Hsu, and J. W. Jang. 1998. Bacteriology of the maxillary and ethmoid sinuses in chronic sinusitis. J. Laryngol. Otol. 112:845-848.
Kiratisin, P., L. Li, P. R. Murray, and S. H. Fischer. 2005. Use of housekeeping gene sequencing for species identification of viridans streptococci. Diagn. Microbiol. Infect. Dis. 51:297-301.
Klossek, J. M., L. Dubreuil, H. Richet, B. Richet, and P. Beutter. 1998. Bacteriology of chronic purulent secretions in chronic rhinosinusitis. J. Laryngol. Otol. 112:1162-1166.
Klossek, J. M., L. Dubreuil, H. Richet, B. Richet, A. Sedallian, and P. Beutter. 1996. Bacteriology of the adult middle meatus. J. Laryngol. Otol. 110:847-849.
Millar, M. R., C. J. Linton, A. Cade, D. Glancy, M. Hall, and H. Jalal. 1996. Application of 16S rRNA gene PCR to study bowel flora of preterm infants with and without necrotizing enterocolitis. J. Clin. Microbiol. 34:2506-2510.
Ramadan, H. H., J. A. Sanclement, and J. G. Thomas. 2005. Chronic rhinosinusitis and biofilms. Otolaryngol. Head Neck Surg. 132:414-417.
Rocas, I. N., J. F. Siqueira, Jr., M. C. Aboim, and A. S. Rosado. 2004. Denaturing gradient gel electrophoresis analysis of bacterial communities associated with failed endodontic treatment. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 98:741-749.
Schabereiter-Gurtner, C., S. Maca, S. Kaminsky, S. Rolleke, W. Lubitz, and T. Barisani-Asenbauer. 2002. Investigation of an anaerobic microbial community associated with a corneal ulcer by denaturing gradient gel electrophoresis and 16S rDNA sequence analysis. Diagn. Microbiol. Infect. Dis. 43:193-199.
Takao, A., H. Nagamune, and N. Maeda. 2004. Identification of the anginosus group within the genus Streptococcus using polymerase chain reaction. FEMS Microbiol. Lett. 233:83-89.
von Wintzingerode, F., U. B. Gobel, and E. Stackebrandt. 1997. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev. 21:213-229.
Walter, J., C. Hertel, G. W. Tannock, C. M. Lis, K. Munro, and W. P. Hammes. 2001. Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 67:2578-2585.
Walter, J., G. W. Tannock, A. Tilsala-Timisjarvi, S. Rodtong, D. M. Loach, K. Munro, and T. Alatossava. 2000. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl. Environ. Microbiol. 66:297-303.
Wilson, K. H., R. B. Blitchington, and R. C. Greene. 1990. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J. Clin. Microbiol. 28:1942-1946.
Yildirim, A., C. Oh, H. Erdem, and T. Kunt. 2004. Bacteriology in patients with chronic sinusitis who have been medically and surgically treated. Ear Nose Throat J. 83:836-838.
Zoetendal, E. G., A. von Wright, T. Vilpponen-Salmela, K. Ben-Amor, A. D. Akkermans, and W. M. de Vos. 2002. Mucosa-associated bacteria in the human gastrointestinal tract are uniformly distributed along the colon and differ from the community recovered from feces. Appl. Environ. Microbiol. 68:3401-3407.(Daniel A. Power, Jeremy P)
BLIS Technologies Limited, Center for Innovation, University of Otago, Dunedin, New Zealand
Otolaryngology and Head and Neck Surgery, Department of Medical and Surgical Sciences, University of Otago, Dunedin, New Zealand
ABSTRACT
Middle meatus aspirates from patients with chronic rhinosinusitis were analyzed by bacterial culture, denaturing gradient gel electrophoresis (DGGE), and antibiotic sensitivity techniques. DGGE detected a greater bacterial diversity than culture methods. Although resistance to antibiotics was low, there was evidence of changes in the composition of the bacterial microbiota over time, and the presence of noncultured bacteria was demonstrated.
CASE REPORT
Using a glass canula, aspirates were taken from the middle meatus of six patients who had previously undergone surgery for chronic rhinosinusitis. These patients had continued infection after surgery despite ongoing nasal toilet and antibiotic treatment. The antibiotics prescribed to the patients within the 2 months prior to the aspirates being taken are listed in Table 1. Four of the patients had a second aspirate taken 2 to 3 months after the first sample. The aspirates were first suspended in 2 ml phosphate-buffered saline and then thoroughly homogenized by being vortexed before being divided into two equal volumes for use in culture-based and DNA-based analyses. For the isolation of staphylococci, mannitol salt agar (Difco, Sparks, MD) was incubated at 37°C aerobically. Total bacterial populations were isolated on chocolate agar (Columbia agar base [Difco] with 5% heat-lysed defibrinated sheep blood [New Zealand Venous Supplies, Tuakau, New Zealand]) by incubation at 37°C either aerobically, in air supplemented with 5% CO2, or in an anaerobic atmosphere (85% N2, 10% H2, 5% CO2). For the isolation of Streptococcus pneumoniae, sheep blood agar (Columbia agar base containing 5% defibrinated sheep blood) with 5 μg/ml gentamicin (Sigma, St. Louis, MO) was used with anaerobic incubation at 37°C. Representative bacterial isolates were then identified by sequencing of the 16S rRNA gene according to the method of Wilson et al. (18). Where isolates were identified as belonging to the anginosus group of streptococci, multiplex PCR according to the protocol of Takao et al. (14) was used to differentiate the subgroups. The antibiotic sensitivities of the isolates were tested using Etest Strips (AB BIODISK, Solna, Sweden) according to the manufacturer's instructions.
The DNA for the non-culture-based analysis was extracted as previously described (3). The extracted DNA was then used as a template for PCR-denaturing gradient gel electrophoresis (DGGE) according to the protocol of Walter et al. (17), except that a 40 to 55% denaturing gradient was used in a CBS Scientific (Del Mar, CA) denaturing gradient gel electrophoresis system (model DGGE-2001). DNA fragments of interest were excised from the gel and sequenced. The nucleotide sequences were analyzed using SeqMan software (version 5.51; DNASTAR, Inc., Madison, WI) and compared against sequences in the GenBank DNA database using the BLAST algorithm (1).
For each of the samples tested by culture, at least one bacterial colony type was detected, and two of the samples yielded two different bacterial species. The bacteria present included S. pneumoniae (n = 4), Staphylococcus aureus (n = 4), anginosus group streptococci (n = 3), Staphylococcus epidermidis (n = 1), and Haemophilus influenzae (n = 1) (Table 1). All three anginosus group streptococci were subsequently identified as Staphylococcus constellatus subsp. constellatus using the group-specific multiplex PCR. When tested against antibiotics commonly prescribed in New Zealand for upper respiratory tract infections, most of the isolates were sensitive (Table 1). However, four showed decreased sensitivity or resistance to at least one of the tested antibiotics. None of the isolates displayed resistance to amoxicillin. At least one isolate showed either resistance or decreased sensitivity to erythromycin, cefaclor, or trimethoprim-sulfamethoxazole.
PCR-DGGE detected three or more bacterial types (Fig. 1) in every sample. For the patients who had aspirates taken on two occasions, there was a clear difference between the profiles in the two samples. When major fragments (indicated in Fig. 1) were sequenced, a variety of bacteria were identified (Table 2). The most common identification was that of streptococci belonging to the mitis-sanguinis group. The potential pathogens S. aureus and H. influenzae were both detected by PCR-DGGE and by culture. Other bacteria of interest detected included the potential oral pathogens Porphyromonas, Fusobacterium, and Prevotella. In addition, two DNA fragments were identified as corresponding to bacteria previously identified as noncultured, high-G+C bacteria from aquatic or soil environments.
Discussion. The bacterial species isolated in the culture-based part of this study were consistent with those recovered in previous studies of the microbiology of infectious rhinosinusitis (2, 5, 6, 8, 9, 19). Although most appeared sensitive to amoxicillin, erythromycin, cefaclor, and trimethoprim-sulfamethoxazole, 4 of the 13 representative isolates tested showed resistance or decreased sensitivity to at least one of these antibiotics. Only patient 5 yielded an isolate that was resistant to a recently prescribed antibiotic. In this case, an H. influenzae isolate resistant to erythromycin was recovered from a patient who had received ongoing treatment with the antibiotic. Failure of antibiotics to resolve the infection could be due to other confounding factors, such as the chronic inflammatory process that characterizes this disease or perhaps its being a biofilm infection, which has been suggested by Ramadan et al. (11) to interfere with the action of antibiotics.
PCR-DGGE has previously been used for analysis of the human microbiota of the gastrointestinal tract (10, 16, 20), vagina (4), oral cavity (12), and eye (13). However, this is the first report of the application of this technique to samples from the sinuses. The results of the PCR-DGGE analysis show a greater variety of bacterial species to be present in all of the samples than was detected by direct culture. This indicates that there may be some bacterial species involved in the infection, or that are commensal organisms of the sinuses, not regularly detected by standard culture methods. This could be due to their being present in smaller numbers, being difficult to culture, or possibly being outcompeted in culture by more dominant bacterial species. The bacterial types not detected so readily in culture included H. influenzae and the relatively slow-growing oral pathogen Prevotella sp. In two cases, putative environmental bacteria were also shown to be present. It is unclear whether these uncultured bacteria are commensals or are actively involved in the disease process or whether they exist as transient inhabitants of the sinuses. The detection of these bacteria by PCR-DGGE suggests that they must be present in substantial numbers (greater than 1% of the population), and thus, the potential for their direct involvement in the disease process appears to merit further investigation. The inability to distinguish between the various members of the mitis-sanguinis group of streptococci by PCR-DGGE is due to the high level of similarity within their 16S rRNA genes (7). This makes differentiation between common pathogens, such as S. pneumoniae, and nonpathogenic members of this group quite difficult.
Although staphylococci were readily isolated by culture-based methods, there was only one specimen in which they were identified by PCR-DGGE. A previous study involving PCR-DGGE (4) also failed to detect staphylococci in specimens in which they appeared dominant when tested by other means. Possible reasons for this apparent anomaly include the following: the DNAs of these organisms being more difficult to extract from clinical samples, PCR bias of the DNA template of nondominant organisms, PCR primer bias, and differences between the copy numbers of the 16S rRNA genes in different organisms (15).
PCR-DGGE showed a greater diversity of bacteria to be present than did culture-based methods and also showed that a change occurred in the composition of the microbiota in those patients from whom samples were taken on different occasions. This is of significant interest because it shows the occurrence of microbial succession in chronic rhinosinusitis without regression of disease symptoms. This could have important implications for prescribing antibiotics for these patients, since some bacteria initially predominant within the microbiota could differ in their antibiotic susceptibilities compared to those that subsequently adopt a leading role within the consortium. Such population swings could lead to nonoptimum antibiotic treatments being administered and consequent protraction of the disease process for the patient. Chronic rhinosinusitis is a complex disease that is influenced by anatomical, immunological, and microbiological factors. The present study has for the first time utilized PCR-DGGE, a non-culture-based technique, to investigate the microbiology of chronic rhinosinusitis. The picture that has emerged is of a polymicrobial disease involving a dynamic succession of bacterial contributors and with some very preliminary evidence for a potential role in the microbial consortium of certain uncultured bacteria not previously associated with humans.
REFERENCES
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.
Brook, I., E. H. Frazier, and P. A. Foote. 1997. Microbiology of chronic maxillary sinusitis: comparison between specimens obtained by sinus endoscopy and by surgical drainage. J. Med. Microbiol. 46:430-432.
Burton, J. P., P. A. Cadieux, and G. Reid. 2003. Improved understanding of the bacterial vaginal microbiota of women before and after probiotic instillation. Appl. Environ. Microbiol. 69:97-101.
Burton, J. P., and G. Reid. 2002. Evaluation of the bacterial vaginal flora of 20 postmenopausal women by direct (Nugent score) and molecular (polymerase chain reaction and denaturing gradient gel electrophoresis) techniques. J. Infect. Dis. 186:1770-1780.
Chan, J., and J. Hadley. 2001. The microbiology of chronic rhinosinusitis: results of a community surveillance study. Ear Nose Throat J. 80:143-145.
Jiang, R. S., C. Y. Hsu, and J. W. Jang. 1998. Bacteriology of the maxillary and ethmoid sinuses in chronic sinusitis. J. Laryngol. Otol. 112:845-848.
Kiratisin, P., L. Li, P. R. Murray, and S. H. Fischer. 2005. Use of housekeeping gene sequencing for species identification of viridans streptococci. Diagn. Microbiol. Infect. Dis. 51:297-301.
Klossek, J. M., L. Dubreuil, H. Richet, B. Richet, and P. Beutter. 1998. Bacteriology of chronic purulent secretions in chronic rhinosinusitis. J. Laryngol. Otol. 112:1162-1166.
Klossek, J. M., L. Dubreuil, H. Richet, B. Richet, A. Sedallian, and P. Beutter. 1996. Bacteriology of the adult middle meatus. J. Laryngol. Otol. 110:847-849.
Millar, M. R., C. J. Linton, A. Cade, D. Glancy, M. Hall, and H. Jalal. 1996. Application of 16S rRNA gene PCR to study bowel flora of preterm infants with and without necrotizing enterocolitis. J. Clin. Microbiol. 34:2506-2510.
Ramadan, H. H., J. A. Sanclement, and J. G. Thomas. 2005. Chronic rhinosinusitis and biofilms. Otolaryngol. Head Neck Surg. 132:414-417.
Rocas, I. N., J. F. Siqueira, Jr., M. C. Aboim, and A. S. Rosado. 2004. Denaturing gradient gel electrophoresis analysis of bacterial communities associated with failed endodontic treatment. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 98:741-749.
Schabereiter-Gurtner, C., S. Maca, S. Kaminsky, S. Rolleke, W. Lubitz, and T. Barisani-Asenbauer. 2002. Investigation of an anaerobic microbial community associated with a corneal ulcer by denaturing gradient gel electrophoresis and 16S rDNA sequence analysis. Diagn. Microbiol. Infect. Dis. 43:193-199.
Takao, A., H. Nagamune, and N. Maeda. 2004. Identification of the anginosus group within the genus Streptococcus using polymerase chain reaction. FEMS Microbiol. Lett. 233:83-89.
von Wintzingerode, F., U. B. Gobel, and E. Stackebrandt. 1997. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev. 21:213-229.
Walter, J., C. Hertel, G. W. Tannock, C. M. Lis, K. Munro, and W. P. Hammes. 2001. Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 67:2578-2585.
Walter, J., G. W. Tannock, A. Tilsala-Timisjarvi, S. Rodtong, D. M. Loach, K. Munro, and T. Alatossava. 2000. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl. Environ. Microbiol. 66:297-303.
Wilson, K. H., R. B. Blitchington, and R. C. Greene. 1990. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J. Clin. Microbiol. 28:1942-1946.
Yildirim, A., C. Oh, H. Erdem, and T. Kunt. 2004. Bacteriology in patients with chronic sinusitis who have been medically and surgically treated. Ear Nose Throat J. 83:836-838.
Zoetendal, E. G., A. von Wright, T. Vilpponen-Salmela, K. Ben-Amor, A. D. Akkermans, and W. M. de Vos. 2002. Mucosa-associated bacteria in the human gastrointestinal tract are uniformly distributed along the colon and differ from the community recovered from feces. Appl. Environ. Microbiol. 68:3401-3407.(Daniel A. Power, Jeremy P)