Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.

BMC Microbiology

Nuno Cerca³  Nuno Filipe Azevedo⁴  Ligia Rodrigues³  Maria João Vieira³  Freddy Haesebrouck¹  Mario Vaneechoutte²  Ana Henriques³  Débora Salgueiro³  Carina Almeida⁴  António Machado³ 

[1] Laboratory of Veterinary Bacteriology and Mycology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, B9820, Belgium;Laboratory of Bacteriology Research, Faculty Medicine & Health Sciences, University of Ghent, Ghent, B-9000, Belgium;IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga, 4710-057, Portugal;LEPAE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, rua Dr. Roberto Frias, Porto, 4200-465, Portugal

关键词: Bacterial vaginosis;    Gardnerella vaginalis;    Lactobacillus spp.;    Peptide Nucleic Acid Probe (PNA probe);    Fluorescence in situ Hybridization (FISH);   

Others  :  1143872
DOI  :  10.1186/1471-2180-13-82

 received in 2012-11-26, accepted in 2013-03-27,  发布年份 2013

【 摘 要 】

Background

Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples.

Results

Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH.

Conclusions

This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.

【 授权许可】

   
2013 Machado et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Spiegel CA: Bacterial vaginosis. Clin Microbiol Rev 1991, 4:485-502.
• [2]Turovskiy Y, Noll KS, Chikindas ML: The etiology of bacterial vaginosis. J Appl Microbiol 2011, 110:1105-1128.
• [3]Vitali B, Pugliese C, Biagi E, Candela M, Turroni S, Bellen G, Donders GG, Brigidi P: Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR. Appl Environ Microbiol 2007, 73:5731-5741.
• [4]Oakley BB, Fiedler TL, Marrazzo JM, Fredricks DN: Diversity of human vaginal bacterial communities and associations with clinically defined bacterial vaginosis. Appl Environ Microbiol 2008, 74:4898-4909.
• [5]Ling Z, Kong J, Liu F, Zhu H, Chen X, Wang Y, Li L, Nelson KE, Xia Y, Xiang C: Molecular analysis of the diversity of vaginal microbiota associated with bacterial vaginosis. BMC Genomics 2010, 11:488-503. BioMed Central Full Text
• [6]Fredricks DN, Fiedler TL, Marrazzo JM: Molecular identification of bacteria associated with bacterial vaginosis. N Engl J Med 2005, 353:1899-1911.
• [7]De Backer E, Verhelst R, Verstraelen H, Alqumber MA, Burton JP, Tagg JR, Temmerman M, Vannechoutte M: Quantitative determination by real-time PCR of four vaginal Lactobacillus species, Gardnerella vaginalis and Atopobium vaginae indicates an inverse relationship between L. gasseri and L. iners. BMC Microbiol 2007, 7:115. BioMed Central Full Text
• [8]Schwebke JR: New concepts in the etiology of bacterial vaginosis. Curr Infect Dis Rep 2009, 11:143-147.
• [9]Nugent R, Krohn M, Hillier S: Reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. J Clin Microbiol 1991, 29:297-301.
• [10]Swidsinski A, Mendling W, Loening-Baucke V, Ladhoff A, Swidsinski S, Hale LP, Lochs H: Adherent biofilms in bacterial vaginosis. Obstet Gynecol 2005, 106:1013-1023.
• [11]Rosenstein IJ, Fontaine EA, Morgan DJ, Sheehan M, Lamont RF, Taylor-Robinson D: Relationship between hydrogen peroxide-producing strains of lactobacilli and vaginosis-associated bacterial species in pregnant women. Eur J Clin Microbiol Infect Dis 1997, 16:517-522.
• [12]Patterson JL, Girerd PH, Karjane NW, Jefferson KK: Effect of biofilm phenotype on resistance of Gardnerella vaginalis to hydrogen peroxide and lactic acid. Am J Obstet Gynecol 2007, 197:170. e1–7
• [13]Patterson JL, Stull-Lane A, Girerd PH, Jefferson KK: Analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of Gardnerella vaginalis relative to other bacterial-vaginosis-associated anaerobes. Microbiol 2010, 156:392-399.
• [14]Nath J, Johnson KL: A review of fluorescence in situ hybridization (FISH): current status and future prospects. Biotech Histochem 2000, 75:54-78.
• [15]Justé A, Thommad B, Lievens B: Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes. Food Microbiol 2008, 25:745-761.
• [16]Demidov VV: PNA and LNA throw light on DNA. Trends Biotechnol 2003, 21:4-7.
• [17]Isacsson J, Cao H, Nordgren S, Svanvik N, Westman G, Kubista M, Sjöback R, Sehlstedt U: Rapid and specific detection of PCR products using light-up probes. Mol Cell Probes 2000, 14:321-328.
• [18]Rigby S, Procop GW, Haase G, Wilson D, Hall G, Kurtzman C, Oliveira K, Von Oy S, Hyldig-Nielsen JJ, Coull J, Stender H: Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles. J Clin Microbiol 2002, 40:2182-2186.
• [19]Stender H, Fiandaca M, Hyldig-Nielsen JJ, Coull J: PNA for rapid microbiology. J Microbiol Methods 2002, 48:1-17.
• [20]Oliveira K, Procop GW, Wilson D, Coull J, Stender H: Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes. J Clin Microbiol 2002, 40:247-251.
• [21]Wilson DA, Joyce MJ, Hall LS, Reller LB, Roberts GD, Hall GS, Alexander BD, Procop GW: Multicenter evaluation of a Candida albicans peptide nucleic acid fluorescent in situ hybridization probe for characterization of yeast isolates from blood cultures. J Clin Microbiol 2005, 43:2909-2912.
• [22]Lefmann M, Schweickert B, Buchholz P, Göbel B, Ulrichs T, Seiler P, Theegarten D, Moter A: Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections. J Clin Microbiol 2006, 44:3760-3767.
• [23]Peleg AY, Tilahun Y, Fiandaca MJ, D’Agata EMC, Venkataraman L, Moellering RC, Eliopoulos GM: Utility of peptide nucleic acid fluorescence in situ hybridization for rapid detection of Acinetobacter spp. and Pseudomonas aeruginosa. J Clin Microbiol 2009, 47:830-832.
• [24]Ashelford K, Weightman A, Fry J: PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database. Nucleic Acids Res 2002, 30:3481-3489.
• [25]Cole J, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM: The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 2009, 37:D141-D145.
• [26]Machado A, Almeida C, Carvalho A, Boyen F, Haesebrouck F, Rodrigues L, Cerca N, Azevedo NF: Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Lactobacillus spp. in Milk Samples. Int J of Food Microbiol 2013, 162:64-70.
• [27]Almeida C, Azevedo NF, Fernandes R, Keevil C, Vieira MJ: A fluorescence in situ hybridization method using a peptide nucleic acid probe for the identification of Salmonella spp. in a broad spectrum of samples. Appl Environ Microbiol 2010, 76:4476-4485.
• [28]Harmsen H, Elfferich P, Schut F, Welling G: A 16S rRNA-targeted probe for detection of lactobacilli and enterococci in faecal samples by fluorescent in situ hybridization. Microb Ecol Health D 1999, 11:3-12.
• [29]Meier H, Amann R, Ludwig W, Schleifer K: Specific oligonucleotide probes for in situ detection of a major group of gram-positive bacteria with low DNA G + C content. Syst Appl Microbiol 1999, 22:186-196.
• [30]Zijnge V, van Leeuwen MB, Degener JE, Abbas F, Thurnheer T, Gmur R, Harmsen HJ: Oral biofilm architecture on natural teeth. PLoS ONE 2010, 5:e9321.
• [31]Burton J, McCormick J, Cadieux P, Reid G: Digoxigenin-labelled peptide nucleic acid to detect lactobacilli PCR amplicons immobilized on membranes from denaturing gradient gel electrophoresis. Lett Appl Microbiol 2003, 36:145-149.
• [32]Fredricks DN, Fiedler TL, Thomas KK, Mitchell CM, Marrazzo JM: Changes in vaginal bacterial concentrations with intravaginal metronidazole therapy for bacterial vaginosis as assessed by quantitative PCR. J Clin Microbiol 2009, 47:721-726.
• [33]Sheiness D, Dix K, Watanabe S, Hillier SL: High levels of Gardnerella vaginalis detected with an oligonucleotide probe combined with elevated pH as a diagnostic indicator of bacterial vaginosis. J Clin Microbiol 1992, 30:642-648.
• [34]Lebeer S, Verhoeven T, Claes I, De Hertogh G, Vermeire S, Buyse J, Van Immerseel F, Vanderleyden J, De Keersmaecker SC: FISH analysis of Lactobacillus biofilms in the gastrointestinal tract of different hosts. Lett Appl Microbiol 2011, 52:220-226.
• [35]Olsen K, Henriksen M, Bisgaard M, Nielsen O, Christensen H: Investigation of chicken intestinal bacterial communities by 16S rRNA targeted fluorescence in situ hybridization. Antonie van Leeuwenhoek 2008, 94:423-437.
• [36]Quevedo B, Giertsen E, Zijnge V, Lüthi-Schaller H, Guggenheim B, Thurnheer T, Gmur R: Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms. BMC Microbiol 2011, 11:14. BioMed Central Full Text
• [37]Almeida C, Azevedo NF, Iversen C, Fanning S, Keevil C, Vieira MJ: Development and application of a novel peptide nucleic acid Probe for the specific detection of Chronobacter genomospecies (Enterobacter sakazakii) in powdered infant formula. Appl Environ Microbiol 2009, 75:2925-2930.
• [38]Byun R, Nadkarni MA, Chhour KL, Martin FE, Jacques NA, Hunter N: Quantitative analysis of diverse Lactobacillus species present in advanced dental caries. J Clin Microbiol 2004, 42:3128-3136.
• [39]McKechnie ML, Hillman R, Couldwell D, Kong F, Freedman E, Wang H, Gilbert GL: Simultaneous identification of 14 genital microorganisms in urine by use of a multiplex PCR-based reverse line blot assay. J Clin Microbiol 2009, 47:1871-1877.
• [40]Zozaya-Hinchliffe M, Lillis R, Martin DH, Ferris MJ: Quantitative PCR assessments of bacterial species in women with and without bacterial vaginosis. J Clin Microbiol 2010, 48:1812-1819.
• [41]Atassi F, Brassart D, Grob P, Graf F, Servin AL: Lactobacillus strains isolated from the vaginal microbiota of healthy women inhibit Prevotella bivia and Gardnerella vaginalis in coculture and cell culture. FEMS Immunol Med Microbiol 2006, 48:424-432.
• [42]Martín R, Sánchez B, Suárez JE, Urdaci MC: Characterization of the adherence properties of human Lactobacilli strains to be used as vaginal probiotics. FEMS Microbiol Lett 2012, 328:166-173.
• [43]Frick JS, Schenk K, Quitadamo M, Kahl F, Köberle M, Bohn E, Aepfelbacher M, Autenrieth IB: Lactobacillus fermentum attenuates the proinflammatory effect of Yersinia enterocolitica on human epithelial cells. Inflamm Bowel Dis 2007, 13:83-90.
• [44]Bernardeau M, Vernoux J, Gueguen M: Usefulness of epifluorescence for quantitative analysis of lactobacilli in probiotic feed. Appl Environ Microbiol 2001, 91:1103-1109.
• [45]Bernardeau M, Vernoux J, Henri-Dubernet S, Guéguen M: Safety assessment of dairy microorganisms: the Lactobacillus genus. Int J Food Microbiol 2008, 126:278-285.
• [46]Matte-Taillez O, Quénée P, Çibik R, Van Opstal J, Dessevre F, Firmesse O, Tailliez P: Detection and identification of lactic acid bacteria in milk and industrial starter culture with fluorescently labeled rRNA-targeted peptide nucleic acid probes. Lait 2001, 81:237-248.
• [47]Swidsinski A, Dorffel Y, Loening-Baucke V, Schilling J, Mendling W: Response of Gardnerella vaginalis biofilm to 5 days of moxi£oxacin treatment. FEMS Immunol Med Microbiol 2011, 61:41-46.
• [48]Munoa FJ, Pares R: Selective medium for isolation and enumeration of Bifidobacterium spp. Appl Environ Microbiol 1988, 54:1715-1718.
• [49]El Aila NA, Tency I, Saerens B, De Backer E, Cools P, dos Santos Santiago GL, Verstraelen H, Verhelst R, Temmerman M, Vaneechoutte M: Strong correspondence in bacterial loads between the vagina and rectum of pregnant women. Res Microbiol 2011, 162:506-513.
• [50]Gomes AMP, Malcata FX: Bifidobacterium spp. and Lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics. Trends Food Sci. Technol 1999, 10:139-157.
• [51]Verstraelen H, Verhelst R, Vaneechoutte M, Temmerman M: Group A streptococcal vaginitis: an unrecognized cause of vaginal symptoms in adult women. Arch Gynecol Obstet 2011, 284:95-8.
• [52]Cerqueira L, Azevedo NF, Almeida C, Jardim T, Keevil C, Vieira MJ: DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH). Int J Mol Sci 2008, 9:1944-1960.
• [53]Huys G, Vancanneyt M, D’Haene K, Falsen E, Wauters G, Vandamme P: Alloscardovia omnicolens gen. nov., sp. nov., from human clinical samples. Int J Syst Evol Microbiol 2007, 57:1442-1446.
• [54]Aroutcheva AA, Simoes JA, Behbakht K, Faro S: Gardnerella vaginalis isolated from patients with bacterial vaginosis and from patients with healthy vaginal ecosystems. Clin Infect Dis 2001, 33:1022-1027.
• [55]Briselden A, Hillier S: Longitudinal study of the biotypes of Gardnerella vaginalis. J Clin Microbiol 1990, 28:2761-2764.
• [56]Eren AM, Zozaya M, Taylor CM, Dowd SE, Martin DH, Ferris MJ: Exploring the diversity of Gardnerella vaginalis in the genitourinary tract microbiota of monogamous couples through subtle nucleotide variation. PLoS One 2011, 6:e26732-e26740.
• [57]Udayalaxmi J, Bhat GK, Kotigadde S: Biotypes and virulence factors of Gardnerella vaginalis isolated from cases of bacterial vaginosis. Indian J Med Microbiol 2011, 29:165-168.
• [58]Harwich MD, Alves JM, Buck GA, Strauss JF, Patterson JL, Oki AT, Girerd PH, Jefferson KK: Drawing the line between commensal and pathogenic Gardnerella vaginalis through genome analysis and virulence studies. BMC Genomics 2010, 11:375-386. BioMed Central Full Text
• [59]Witt A, Petricevic L, Kaufmann U, Gregor H, Kiss H: DNA hybridization test: rapid diagnostic tool for excluding bacterial vaginosis in pregnant women with symptoms suggestive of infection. J Clin Microbiol 2002, 40:3057-3059.
• [60]Berlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ, Telford WG, Yue S, Liu J, Cheung C, Chang W, Hirsch JD, Beechem JM, Haugland RP: Quantitative comparison of long-wavelength alexa fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. J Histochem Cytochem 2003, 51:1699-1712.