- Research article
- Open Access
Rapid species-level identification of vaginal and oral lactobacilli using MALDI-TOF MS analysis and 16S rDNA sequencing
© Anderson et al.; licensee BioMed Central Ltd. 2014
- Received: 23 July 2014
- Accepted: 25 November 2014
- Published: 14 December 2014
Lactobacillus represents a large genus with different implications for the human host. Specific lactobacilli are considered to maintain vaginal health and to protect from urogenital infection. The presence of Lactobacillus species in carious lesions on the other hand is associated with progressive caries. Despite their clinical significance, species-level identification of lactobacilli still poses difficulties and mostly involves a combination of different phenotypic and genotypic methods. This study evaluated rapid MALDI-TOF MS analysis of vaginal and oral Lactobacillus isolates in comparison to 16S rDNA analysis.
Both methods were used to analyze 77 vaginal and 21 oral Lactobacillus isolates. The concordance of both methods was at 96% with five samples discordantly identified. Fifteen different Lactobacillus species were found in the vaginal samples, primarily L. iners, L. crispatus, L. jensenii and L. gasseri. In the oral samples 11 different species were identified, mostly L. salivarius, L. gasseri, L. rhamnosus and L. paracasei. Overall, the species found belonged to six different phylogenetic groups. For several samples, MALDI-TOF MS analysis only yielded scores indicating genus-level identification. However, in most cases the species found agreed with the 16S rDNA analysis result.
MALDI-TOF MS analysis proved to be a reliable and fast tool to identify lactobacilli to the species level. Even though some results were ambiguous while 16S rDNA sequencing yielded confident species identification, accuracy can be improved by extending reference databases. Thus, mass spectra analysis provides a suitable method to facilitate monitoring clinically relevant Lactobacillus species.
- Bacterial Vaginosis
- Carious Lesion
- Lactobacillus Species
- Vaginal Flora
Lactobacilli, gram-positive, highly acidogenic and acid tolerant rods, represent a genus with over 180 known species so far, with lactic acid as their primary fermentation end product ,. The human body hosts various Lactobacillus species in different anatomic regions entailing different interactions with the host: e.g. the oral cavity, the intestines and the female genital tract -.
For the latter region lactobacilli play a significant role in the balance of the microbial flora. The healthy female vaginal flora is characterized by different species of lactobacilli and is mostly dominated by one of the following species: Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii,-. Within the last decade, culture-independent studies, involving several ethnic groups, revealed differences in vaginal microbiota as well as the absence of lactobacilli in healthy study participants, particularly Hispanic and black women, complicating the understanding of a healthy versus an unhealthy vaginal flora -. Despite these recent discoveries, it is still acknowledged that these Lactobacillus taxa constitute the predominant genus in the healthy vaginal flora, primarily considering Caucasian women -.
The vaginal lactobacilli play a significant role in protecting their habitat from unwanted invaders. Next to the competition with other species for nutrient supply and adherence to the vaginal epithelium, the production of lactic acid, hydrogen peroxide (H2O2), bacteriocins and other antimicrobial substances inhibit the vaginal colonization by more pathogenic microorganisms . Consequently, these species help to prevent the shift of the vaginal microbial composition towards high concentrations of mainly anaerobic bacteria such as Gardnerella vaginalis, Prevotella spp. or Atopobium vaginalis which can lead to a condition known as bacterial vaginosis (BV) ,. Increased bacterial counts for L. iners have also been associated with the beginning stages of BV ,. The latter represents the most prevalent vaginal syndrome with a prevalence between 20-50% for fertile, premenopausal women , correlating with an increased risk of various infections. Urinary tract infections, sexually transmitted diseases, late miscarriage and preterm birth are the most frequent among these -. As a result, the maintenance of a healthy vaginal flora is considered very important. To date, many efforts have been made to develop administrable probiotic Lactobacillus species to reduce the recurrence of infections and promote a healthy vaginal flora ,. Some of these probiotic lactobacilli have shown promising results in treating and preventing BV as well as preventing urinary tract infections -.
In addition, high counts of Lactobacillus species are not only present in the vaginal and intestinal flora, but also in saliva, the supragingival biofilm and advanced carious lesions ,. In the oral biofilm in particular, the bacterial count of Lactobacillus species increases when the pH in the oral cavity decreases following carbohydrate metabolism .
Caries is characterized by demineralization of tooth enamel in the presence of high concentrations of organic acids that are produced by different species in the oral biofilm . Commonly initial carious lesions develop under the influence of cariogenic bacteria, e.g. mutans streptococci and with the progression of a carious lesion the typical microflora shifts towards more lactobacilli and Bifidobacterium spp. as well as other acidogenic and aciduric species . Indeed, various Lactobacillus species have been reported in carious lesions . Lactobacillus fermentum has been found in childhood caries , and Lactobacillus casei/paracasei as well as Lactobacillus rhamnosus have been reported in root caries of elderly patients . Molecular and culture analysis of advanced carious dentine revealed the presence of Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus delbrueckii, Lactobacillus ultunensis, L. crispatus, L. fermentum, Lactobacillus salivarius, Lactobacillus casei/rhamnosus and other related species ,.
Despite the scientific consensus about the significance of the genus Lactobacillus for the human host, its species identification still poses several difficulties. Phenotypic detection criteria proved to be unreliable , e.g. in distinguishing between Lactobacillus acidophilus and closely related species ,,,. It was not until molecular identification techniques had been applied that six distinct vaginal Lactobacillus species have been identified that had been classified as L. acidophilus on the basis of phenotypic characteristics before. Up to now, L. iners, as a very common vaginal species, was only described and classified in 1999 with 16S rRNA gene sequencing . L. iners can indicate an abnormal flora or the tendency towards BV, e.g. during pregnancy. In general, clear species identification has only been possible using culture-independent methods not based on phenotypic criteria.
Precise species identification is indispensable in light of the findings that different Lactobacillus species can have very diverse effects on the host concerning pathogenesis of BV or triggering health-promoting mechanisms, e.g. stabilizing a healthy vaginal flora. Also tools for correct species identification are needed to further elucidate oral pathogenic processes, e.g. the development of advanced caries.
MALDI-TOF-MS analysis is a method which has been used for microbial species identification in routine laboratories for the last decade -. It holds the potential to provide a fast method for species identification of Lactobacillus isolates and would therefore facilitate beneficial monitoring of the vaginal flora. Yet the use of MALDI-TOF MS analysis has not been reported for the species identification of vaginal lactobacilli so far. 16S rDNA-sequencing has been used for routine clinical diagnostics of bacterial species in infectious diseases as well ,, and provides a highly discriminative comparison method.
To date there is no study comparing MALDI-TOF MS analysis and 16S rDNA-sequencing for the identification of Lactobacillus species, isolated from clinical vaginal and oral samples. Therefore this study aimed to assess the capability of MALDI-TOF MS analysis for accurate species identification and the applicability of this method.
A total of 77 lactobacilli isolates gained from vaginal samples were used. The vaginal samples were obtained within a gynaecological examination with a speculum using sterile swabs (Sarstedt, Nümbrecht, Germany) that were placed in transport medium. The age range of the subjects was 20–69 (with only 1 subject beyond the reproductive age). All subjects gave their written informed consent to the study protocol. In addition, 21 lactobacilli isolates were collected from supragingival plaque samples and saliva samples. The oral samples were taken from unstimulated saliva and supragingival plaque of healthy male and female volunteers (aged 23–45) that had given their written informed consent to the study protocol. Prior to sampling, a detailed clinical oral examination was performed by a dentist, DMFT values were collected and saliva flow rates were measured at 1.2 ± 0.2. The preconditions for the volunteers were: Healthy dental status, i.e. low DMFT values (decayed, missing, filled teeth) of 0–5, non-smokers, no severe systemic disease, no diseases of the salivary glands, no pregnancy or lactation, no use of antibiotics or local antimicrobial mouth rinses (e.g. Chlorhexidine) within the last month and no participation in another study. The supragingival plaque samples were treated for 30 s in an ultrasonic bath on ice, and dilution series of the samples were prepared as described by Al-Ahmad .
Additionally, 10 Lactobacillus reference strains from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) were tested.
All bacterial strains and isolates were cultivated on Columbia blood agar plates (CB; Oxoid, Wesel, Germany), Yeast-cysteine blood agar plates (HCB-agar; Becton-Dickinson, Nürnberg, Germany) and MRS agar plates (deMan Rogosa and Sharpe agar; Merck, Darmstadt, Germany). Cultivation was performed aerobically on CB and MRS agar for up to 7 days to capture slow-growing lactobacilli and anaerobically on HCB agar for 3 days (anaerobic chamber, GENbox BioMérieux, Nürtingen, Germany).
MALDI-TOF sample preparation and MS analysis
Without a detailed extraction step, bacteria from single colonies were used for MALDI-TOF analysis in a MALDI Biotyper Microflex LT (Bruker Daltonik, Bremen, Germany). For the acquisition of the mass spectra the authors followed the manufacturer’s recommendations. The mass spectra were acquired within less than 5 minutes including the sample preparation. The BioTyper 3.0 software compared the obtained spectra with a reference database containing 3740 reference spectra (representing 319 genera and 1,946 species) and expressed the resulting similarity value as a log score. A score of ≥ 2.000 indicated identification on the species level, a score of ≥ 1.700 indicated identification on the genus level whereas any score under 1.700 meant no significant similarity of the obtained spectrum with any database entry. If the results were questionable, the procedure was repeated.
DNA-extraction, genotypic analysis with 16S rDNA sequencing and phylogenetic analysis
Material of pure cultures was used to extract total bacterial DNA. The colony was placed in 60 μl PBS buffer and 240 μl lysis buffer B (1% Triton X-100 (Sigma-Aldrich, Taufkirchen, Germany), 0.5% Tween 20 (Merck, Darmstadt, Germany), 10 mM TrisHCl (AppliChem, Darmstadt, Germany), 1 mM EDTA (SERVA, Heidelberg, Germany), in ultrapure water (Merck, Darmstadt, Germany), pH 8.0) were added. The solution was heated and incubated at 95 °C for 15 min. Then the lysed cells were centrifuged at 14.000 rpm for 5 min and the supernatant was transferred to a new 1.5 ml vial and used for PCR.
A 1233 bp fragment of the 16S rDNA was amplified using the following primers:
TP16U1: AGAGTTTGATCMTGGCTCAG and
RT16U6: ATTGTAGCACGTGTGTNGCCC .
The reaction mixture of 50 μl contained 25 μl PCR-Mastermix (Qiagen, Hilden, Germany, including PCR Buffer, deoxyribonucleoside triphosphates and HotStar Taq DNA-Polymerase), 2.5 mM MgCl2 (Qiagen, Hilden, Germany) and 300nM of each primer. The PCR cycling conditions consisted of an initial denaturation step at 94°C for 2 min, followed by 30 cycles with denaturation at 92°C for 1 min, annealing at 55 °C at 1 min and extension at 72°C for 1.5 min, with a final extension step at 72°C for 5 min. A no-template control and a positive control were included in each set of PCR reactions. Amplicons were analyzed by electrophoresis on a 1.5% agarose gel and positive reactions were used for sequencing.
The 16S rDNA-PCR products were purified before sequencing, using GFX PCR DNA and Gel Band Purification Kit (GE-Healthcare, Munich, Germany). Subsequently, Cycle Sequencing was performed on a 3130 Genetic Analyzer (Applied Biosystems, Life Technologies GmbH, Darmstadt, Germany) applying the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany).
The sequence data obtained was visually proofread and edited and then compared to the public sequence databases, Genbank, EMBL and DDBJ using the BLAST program. The program was run through the server hosted by the National Center for Biotechnology Information NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) ,. Sequences with a ≥ 98% match to a database sequence were considered to be of the same species as the one with the highest similarity and score bits. Additionally, all 16S rDNA sequences were compared with the database RIDOM (Ribosomal Differentiation of Microorganisms, Ridom GmbH, Münster, Germany, accessed through the website www.ridomrdna.de).
The 16 s-rDNA sequences obtained were used for further comparative sequence analysis and phylogenetic analysis using the tools implemented in the software package ARB . To implement the obtained sequences the reference dataset SSURef 111 SILVA NR 98 04 08 12 from the SILVA project  was used. Alignments were performed using the SINA Aligner plugin . After manual correction of the alignment, a phylogenetic tree was constructed with the ARB Neighbor joining method applying the Jukes-Cantor correction and with the Maximum Parsimony and Maximum likelihood methods. For the Neighbor joining tree, bootstrapping was calculated based on 1000 replicates. Partial sequences were added without allowing changes of the tree topology by use of the ARB “parsimony interactive” method.
The partial 16S rDNA sequences supporting the results were deposited in the NCBI Genbank database under the accession numbers KM250382-250423.
The data of the phylogenetic analysis are available from the Dryad Digital Repository: http://doi.org/10.5061/dryad.78vs2.
Lactobacilli collection strains
Reference strains tested with MALDI-TOF MS analysis
Culture collection number*
BioTyper log score
Identification of vaginal and oral isolates with MALDI-TOF MS analysis and 16S rDNA analysis
Identification of lactobacilli isolates with MALDI-TOF MS and 16S rDNA-analysis: Vaginal isolates
Discordantly identified vaginal and oral isolates
BioTyper log score
Identification of lactobacilli isolates with MALDI-TOF MS and 16S rDNA-analysis: Oral isolates
Overall, the oral isolates represented 11 different species with L. salivarius, L. gasseri, L. rhamnosus and L. paracasei identified in more than one sample.
Comparison of genotypic and MALDI-TOF analysis for vaginal and oral isolates
In this study MALDI-TOF MS analysis was compared with 16S rDNA sequencing to identify 98 lactobacilli isolates from vaginal and oral samples on the species level with the aim of evaluating the potential of mass spectra analysis as a specific and fast detection method. The two methods yielded 96% concordant results. Combining isolates from different clinically relevant sources further affirms the validity of the method. Other studies report similar percentage concordance comparing mass spectrometry with different genotypic methods, e.g. Callaway et al. 93%  and Angelakis et al. 92% , or somewhat lower values, e.g. Duskova et al. 74%  and Dec et al. 88.5% .
The MALDI-TOF MS analysis demonstrated accurate species-level identification for those Lactobacillus species that are of importance for vaginal health as well as those that can indicate the beginning stages of BV. Equally, species associated with progressive caries were identified correctly. Even though MALDI-TOF MS analysis has been used to analyze bacterial species from clinical specimens for nearly two decades ,, it has not been thoroughly evaluated for vaginal lactobacilli. To date the method has been applied to identify lactobacilli originating from food to the species level ,,, and recently attempts have been made to even differentiate subspecies of Lactobacillus brevis that are known to cause beer spoilage . Furthermore, Callaway  has reported successful species identification of lactobacilli from carious dentine.
16S rDNA sequencing, which was applied as the comparison method, is regarded as an established method for exact species identification and is also applied for clinical diagnostic purposes . Given the known difficulties of exact species identification of lactobacilli by means of phenotypic methods ,,, studies on the taxonomy of lactobacilli or on Lactobacillus species identification have been performed using molecular methods based on 16S sequence differences, such as Amplified Ribosomal DNA Restriction Analysis (ARDRA) -, Ribotyping ,, 16S rDNA sequencing and others ,,,. Identifying lactobacilli employing 16S rDNA sequencing has shown to be a highly discriminative method and has revealed new species, e.g. L. iners. Nevertheless, it is not an infallible method and it has failed to differentiate between L. casei and L. paracasei in our study, due to the substantial similarities of their 16S rDNA genes. In some cases, Lactobacillus species can only be correctly identified combining several genotypic methods .
Overall, our study demonstrated a high discriminatory power of the mass spectra analysis for the identification of Lactobacillus species that are relevant indicators for vaginal health, e.g. L. crispatus, L. gasseri and L. jensenii as well as L. iners, a species that is frequently detected in vaginal flora exhibiting a tendency towards BV ,. Before the application of molecular techniques L. crispatus, L. jensenii and L. gasseri, all belonging to the L. acidophilus-group had not been differentiated as distinct species in the past . In our study, all these species were correctly identified by MALDI-TOF MS analysis. Mass spectra analysis proved equally suitable for the detection of Lactobacillus species associated with different forms of caries. The species found most frequently in saliva and supragingival plaque samples in this study, namely L. salivarius, L. gasseri, L. rhamnosus and L. paracasei concur with those species found in carious lesions ,.
Apart from the high accuracy of the method, the rapidity of the analysis is particularly advantageous for the application in clinical laboratories. After obtaining the bacterial isolates the acquisition of the mass spectra, including the sample preparation, takes less than five minutes compared to several hours or even several days by molecular or phenotypic analysis depending on the respective method. Thus the biochemical identification of lactobacilli with the commercial API 50 CHL system (Biomérieux, Marcy l’ Etoile, France) includes fifty reactions and takes an additional 48 h of incubation time after obtaining pure cultures.
Most of the differing results with the two compared methods can be explained: Two samples, identified as L. gasseri with 16S rDNA analysis were identified as L. crispatus and L. jensenii with MALDI-TOF analysis respectively. For both samples, the BioTyper log score was below 2.0, indicating uncertain species identification. Also, L. gasseri, L. jensenii and L. crispatus are all phylogenetically closely related species. The same applies to one oral sample that was identified as L. helveticus genotypically and as L. ultunensis / L. acidophilus by mass spectrometry. Both species are closely related and again the BioTyper log score was below 2.0. The phylogenetic analysis affirms the sequence similarity with L. helveticus.
One of the main weaknesses of the mass spectrometry method revealed by our experiments was the frequent occurrence of MALDI-BioTyper log scores between 1.68 and 1.99. Values between 1.7 and 2.0 indicate correct genus identification but only presumptive species identification. Yet from 19 samples showing log scores < 2.0, the vast majority of the presumed species by the BioTyper software (16 samples) agreed with the results achieved by 16S rDNA sequencing, suggesting that despite the low log score the MALDI-TOF MS identification on the species level was correct. This outcome of the mass spectrometry might be improved by extending the BioTyper reference database, as Duskova  has stated before. Evidently, the MALDI BioTyper database and corresponding databases of other MALDI-TOF devices will be continuously expanded with the increase of its applications.
Apart from the above mentioned minor drawback, our study demonstrates the excellent applicability of mass spectra analysis to identify lactobacilli to the species level. The MALDI-TOF MS analysis, e.g. using the commercially available MALDI BioTyper system, provides a fast as well as cost-effective method, which proves to be especially relevant considering the labor intensity of genotypic and phenotypic methods characteristic for Lactobacillus species identification. Accurate species identification for clinically relevant species in relation to vaginal health, BV and caries can be achieved in a short time once isolates are obtained. Since it has been shown in recent years that different Lactobacillus species have different protective functions for the vaginal flora, specific methods for species-level identification are necessary and profitable for monitoring vaginal health during pregnancy and other stages. MALDI-TOF MS analysis can also facilitate analysis of lactobacilli involved in pathological processes, e. g. progressive caries. Beyond that scope, mass spectra analysis can also help to analyze Lactobacillus strains that hold the potential for probiotic application.
This study was approved by the Ethics Committee of the Albert Ludwigs-University Freiburg (42/08).
Andreas Clad posthumous.
The authors would like to thank Heike Wölk and Annette Wittmer for their excellent technical support as well as Dr. Michael Schmid for helpful, informal ARB support.
- Balashov SV, Mordechai E, Adelson ME, Sobel JD, Gygax SE: Multiplex quantitative polymerase chain reaction assay for the identification and quantitation of major vaginal lactobacilli. Diagn Microbiol Infect Dis. 2014, 78: 321-327. 10.1016/j.diagmicrobio.2013.08.004.View ArticlePubMedGoogle Scholar
- Tannock GW: A special fondness for lactobacilli. Appl Environ Microbiol. 2004, 70: 3189-3194. 10.1128/AEM.70.6.3189-3194.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Pavlova SI, Kilic AO, Kilic SS, So JS, Nader-Macias ME, Simoes JA, Tao L: Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences. J Appl Microbiol. 2002, 92: 451-459. 10.1046/j.1365-2672.2002.01547.x.View ArticlePubMedGoogle Scholar
- Munson MA, Banerjee A, Watson TF, Wade WG: Molecular analysis of the microflora associated with dental caries. J Clin Microbiol. 2004, 42: 3023-3029. 10.1128/JCM.42.7.3023-3029.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- O’Callaghan J, O’Toole PW: Lactobacillus: host-microbe relationships. Curr Top Microbiol Immunol. 2013, 358: 119-154.PubMedGoogle Scholar
- Redondo-Lopez V, Cook RL, Sobel JD: Emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora. Rev Infect Dis. 1990, 12: 856-872. 10.1093/clinids/12.5.856.View ArticlePubMedGoogle Scholar
- Reid G, McGroarty JA, Tomeczek L, Bruce AW: Identification and plasmid profiles of Lactobacillus species from the vagina of 100 healthy women. FEMS Immunol Med Microbiol. 1996, 15: 23-26. 10.1111/j.1574-695X.1996.tb00354.x.View ArticlePubMedGoogle Scholar
- Antonio MA, Rabe LK, Hillier SL: Colonization of the rectum by Lactobacillus species and decreased risk of bacterial vaginosis. J Infect Dis. 2005, 192: 394-398. 10.1086/430926.View ArticlePubMedGoogle Scholar
- Vasquez A, Jakobsson T, Ahrne S, Forsum U, Molin G: Vaginal lactobacillus flora of healthy Swedish women. J Clin Microbiol. 2002, 40: 2746-2749. 10.1128/JCM.40.8.2746-2749.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou X, Bent SJ, Schneider MG, Davis CC, Islam MR, Forney LJ: Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods. Microbiology. 2004, 150: 2565-2573. 10.1099/mic.0.26905-0.View ArticlePubMedGoogle Scholar
- Zhou X, Brown CJ, Abdo Z, Davis CC, Hansmann MA, Joyce P, Foster JA, Forney LJ: Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women. Isme j. 2007, 1: 121-133. 10.1038/ismej.2007.12.View ArticlePubMedGoogle Scholar
- Linhares IM, Giraldo PC, Baracat EC: [New findings about vaginal bacterial flora]. Rev Assoc Med Bras. 2010, 56: 370-374. 10.1590/S0104-42302010000300026.View ArticlePubMedGoogle Scholar
- Ma B, Forney LJ, Ravel J: Vaginal microbiome: rethinking health and disease. Annu Rev Microbiol. 2012, 66: 371-389. 10.1146/annurev-micro-092611-150157.PubMed CentralView ArticlePubMedGoogle Scholar
- Lamont RF, Sobel JD, Akins RA, Hassan SS, Chaiworapongsa T, Kusanovic JP, Romero R: The vaginal microbiome: new information about genital tract flora using molecular based techniques. Bjog. 2011, 118: 533-549. 10.1111/j.1471-0528.2010.02840.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, McCulle SL, Karlebach S, Gorle R, Russell J, Tacket CO, Brotman RM, Davis CC, Ault K, Peralta L, Forney LJ: Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A. 2011, 108 (Suppl 1): 4680-4687. 10.1073/pnas.1002611107.PubMed CentralView ArticlePubMedGoogle Scholar
- Yoshimura K, Morotomi N, Fukuda K, Nakano M, Kashimura M, Hachisuga T, Taniguchi H: Intravaginal microbial flora by the 16S rRNA gene sequencing.Am J Obstet Gynecol 2011, 205:235.e231-239.,Google Scholar
- Nardis C, Mosca L, Mastromarino P: Vaginal microbiota and viral sexually transmitted diseases. Ann Ig. 2013, 25: 443-456.PubMedGoogle Scholar
- Pendharkar S, Magopane T, Larsson PG, de Bruyn G, Gray GE, Hammarstrom L, Marcotte H: Identification and characterisation of vaginal lactobacilli from South African women. BMC Infect Dis. 2013, 13: 43-10.1186/1471-2334-13-43.PubMed CentralView ArticlePubMedGoogle Scholar
- Boris S, Barbes C: Role played by lactobacilli in controlling the population of vaginal pathogens. Microbes Infect. 2000, 2: 543-546. 10.1016/S1286-4579(00)00313-0.View ArticlePubMedGoogle Scholar
- Fredricks DN, Fiedler TL, Marrazzo JM: Molecular identification of bacteria associated with bacterial vaginosis. N Engl J Med. 2005, 353: 1899-1911. 10.1056/NEJMoa043802.View ArticlePubMedGoogle Scholar
- Donders GG: Definition and classification of abnormal vaginal flora. Best Pract Res Clin Obstet Gynaecol. 2007, 21: 355-373. 10.1016/j.bpobgyn.2007.01.002.View ArticlePubMedGoogle Scholar
- Verstraelen H, Verhelst R, Claeys G, De Backer E, Temmerman M, Vaneechoutte M: Longitudinal analysis of the vaginal microflora in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microflora and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microflora. BMC Microbiol. 2009, 9: 116-10.1186/1471-2180-9-116.PubMed CentralView ArticlePubMedGoogle Scholar
- Allsworth JE, Peipert JF: Prevalence of bacterial vaginosis: 2001–2004 National Health and Nutrition Examination Survey data. Obstet Gynecol. 2007, 109: 114-120. 10.1097/01.AOG.0000247627.84791.91.View ArticlePubMedGoogle Scholar
- Peipert JF, Montagno AB, Cooper AS, Sung CJ: Bacterial vaginosis as a risk factor for upper genital tract infection. Am J Obstet Gynecol. 1997, 177: 1184-1187. 10.1016/S0002-9378(97)70038-3.View ArticlePubMedGoogle Scholar
- Martin HL, Richardson BA, Nyange PM, Lavreys L, Hillier SL, Chohan B, Mandaliya K, Ndinya-Achola JO, Bwayo J, Kreiss J: Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition. J Infect Dis. 1999, 180: 1863-1868. 10.1086/315127.View ArticlePubMedGoogle Scholar
- Pybus V, Onderdonk AB: Microbial interactions in the vaginal ecosystem, with emphasis on the pathogenesis of bacterial vaginosis. Microbes Infect. 1999, 1: 285-292. 10.1016/S1286-4579(99)80024-0.View ArticlePubMedGoogle Scholar
- Harmanli OH, Cheng GY, Nyirjesy P, Chatwani A, Gaughan JP: Urinary tract infections in women with bacterial vaginosis. Obstet Gynecol. 2000, 95: 710-712. 10.1016/S0029-7844(99)00632-8.View ArticlePubMedGoogle Scholar
- Wang Y, Chen C, Ai L, Zhou F, Zhou Z, Wang L, Zhang H, Chen W, Guo B: Complete genome sequence of the probiotic Lactobacillus plantarum ST-III. J Bacteriol. 2011, 193: 313-314. 10.1128/JB.01159-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Reid G: Probiotic and prebiotic applications for vaginal health. J AOAC Int. 2012, 95: 31-34. 10.5740/jaoacint.SGE_Reid.View ArticlePubMedGoogle Scholar
- Marelli G, Papaleo E, Ferrari A: Lactobacilli for prevention of urogenital infections: a review. Eur Rev Med Pharmacol Sci. 2004, 8: 87-95.PubMedGoogle Scholar
- Stapleton AE, Au-Yeung M, Hooton TM, Fredricks DN, Roberts PL, Czaja CA, Yarova-Yarovaya Y, Fiedler T, Cox M, Stamm WE: Randomized, placebo-controlled phase 2 trial of a Lactobacillus crispatus probiotic given intravaginally for prevention of recurrent urinary tract infection. Clin Infect Dis. 2011, 52: 1212-1217. 10.1093/cid/cir183.PubMed CentralView ArticlePubMedGoogle Scholar
- Mastromarino P, Vitali B, Mosca L: Bacterial vaginosis: a review on clinical trials with probiotics. New Microbiol. 2013, 36: 229-238.PubMedGoogle Scholar
- Verstraelen H, Swidsinski A: The biofilm in bacterial vaginosis: implications for epidemiology, diagnosis and treatment. Curr Opin Infect Dis. 2013, 26: 86-89. 10.1097/QCO.0b013e32835c20cd.View ArticlePubMedGoogle Scholar
- de Soet JJ, de Graaff J: Microbiology of carious lesions. Dent Update. 1998, 25: 319-324.PubMedGoogle Scholar
- Callaway A, Kostrzewa M, Willershausen B, Schmidt F, Thiede B, Kupper H, Kneist S: Identification of Lactobacilli from deep carious lesions by means of species-specific PCR and MALDI-TOF mass spectrometry. Clin Lab. 2013, 59: 1373-1379.PubMedGoogle Scholar
- Marsh PD, Bradshaw DJ: Physiological approaches to the control of oral biofilms. Adv Dent Res. 1997, 11: 176-185. 10.1177/08959374970110010901.View ArticlePubMedGoogle Scholar
- van Houte J: Role of micro-organisms in caries etiology. J Dent Res. 1994, 73: 672-681.PubMedGoogle Scholar
- Takahashi N, Nyvad B: The role of bacteria in the caries process: ecological perspectives. J Dent Res. 2011, 90: 294-303. 10.1177/0022034510379602.View ArticlePubMedGoogle Scholar
- Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, Galvin JL, Boches SK, Dewhirst FE, Griffen AL: Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol. 2002, 40: 1001-1009. 10.1128/JCM.40.3.1001-1009.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Tanner AC, Mathney JM, Kent RL, Chalmers NI, Hughes CV, Loo CY, Pradhan N, Kanasi E, Hwang J, Dahlan MA, Papadopolou E, Dewhirst FE: Cultivable anaerobic microbiota of severe early childhood caries. J Clin Microbiol. 2011, 49: 1464-1474. 10.1128/JCM.02427-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Preza D, Olsen I, Willumsen T, Boches SK, Cotton SL, Grinde B, Paster BJ: Microarray analysis of the microflora of root caries in elderly. Eur J Clin Microbiol Infect Dis. 2009, 28: 509-517. 10.1007/s10096-008-0662-8.PubMed CentralView ArticlePubMedGoogle Scholar
- Chhour KL, Nadkarni MA, Byun R, Martin FE, Jacques NA, Hunter N: Molecular analysis of microbial diversity in advanced caries. J Clin Microbiol. 2005, 43: 843-849. 10.1128/JCM.43.2.843-849.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Duskova M, Sedo O, Ksicova K, Zdrahal Z, Karpiskova R: Identification of lactobacilli isolated from food by genotypic methods and MALDI-TOF MS. Int J Food Microbiol. 2012, 159: 107-114. 10.1016/j.ijfoodmicro.2012.07.029.View ArticlePubMedGoogle Scholar
- Klein G, Pack A, Bonaparte C, Reuter G: Taxonomy and physiology of probiotic lactic acid bacteria. Int J Food Microbiol. 1998, 41: 103-125. 10.1016/S0168-1605(98)00049-X.View ArticlePubMedGoogle Scholar
- Antonio MA, Hawes SE, Hillier SL: The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. J Infect Dis. 1999, 180: 1950-1956. 10.1086/315109.View ArticlePubMedGoogle Scholar
- Eigner U, Holfelder M, Oberdorfer K, Betz-Wild U, Bertsch D, Fahr AM: Performance of a matrix-assisted laser desorption ionization-time-of-flight mass spectrometry system for the identification of bacterial isolates in the clinical routine laboratory. Clin Lab. 2009, 55: 289-296.PubMedGoogle Scholar
- Marklein G, Josten M, Klanke U, Muller E, Horre R, Maier T, Wenzel T, Kostrzewa M, Bierbaum G, Hoerauf A, Sahl HG: Matrix-assisted laser desorption ionization-time of flight mass spectrometry for fast and reliable identification of clinical yeast isolates. J Clin Microbiol. 2009, 47: 2912-2917. 10.1128/JCM.00389-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, Raoult D: Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis. 2009, 49: 543-551. 10.1086/600885.View ArticlePubMedGoogle Scholar
- Christner M, Rohde H, Wolters M, Sobottka I, Wegscheider K, Aepfelbacher M: Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting. J Clin Microbiol. 2010, 48: 1584-1591. 10.1128/JCM.01831-09.PubMed CentralView ArticlePubMedGoogle Scholar
- van Veen SQ, Claas EC, Kuijper EJ: High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. J Clin Microbiol. 2010, 48: 900-907. 10.1128/JCM.02071-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Harris KA, Hartley JC: Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology service. J Med Microbiol. 2003, 52: 685-691. 10.1099/jmm.0.05213-0.View ArticlePubMedGoogle Scholar
- Al-Ahmad A, Maier J, Follo M, Spitzmuller B, Wittmer A, Hellwig E, Hubner J, Jonas D: Food-borne enterococci integrate into oral biofilm: an in vivo study. J Endod. 2010, 36: 1812-1819. 10.1016/j.joen.2010.08.011.View ArticlePubMedGoogle Scholar
- Schuppler M, Mertens F, Schon G, Gobel UB: Molecular characterization of nocardioform actinomycetes in activated sludge by 16S rRNA analysis. Microbiology. 1995, 141 (Pt 2): 513-521. 10.1099/13500872-141-2-513.View ArticlePubMedGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410. 10.1016/S0022-2836(05)80360-2.View ArticlePubMedGoogle Scholar
- Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25: 3389-3402. 10.1093/nar/25.17.3389.PubMed CentralView ArticlePubMedGoogle Scholar
- Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar, Buchner A, Lai T, Steppi S, Jobb G, Förster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, König A, Liss T, Lüssmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, et al: ARB: a software environment for sequence data.Nucleic Acids Res 2004, 32:1363–1371.,Google Scholar
- Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glockner FO: The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013, 41: D590-596. 10.1093/nar/gks1219.PubMed CentralView ArticlePubMedGoogle Scholar
- Pruesse E, Peplies J, Glockner FO: SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics. 2012, 28: 1823-1829. 10.1093/bioinformatics/bts252.PubMed CentralView ArticlePubMedGoogle Scholar
- Canchaya C, Claesson MJ, Fitzgerald GF, van Sinderen D, O’Toole PW: Diversity of the genus Lactobacillus revealed by comparative genomics of five species. Microbiology. 2006, 152: 3185-3196. 10.1099/mic.0.29140-0.View ArticlePubMedGoogle Scholar
- Angelakis E, Million M, Henry M, Raoult D: Rapid and accurate bacterial identification in probiotics and yoghurts by MALDI-TOF mass spectrometry. J Food Sci. 2011, 76: M568-572. 10.1111/j.1750-3841.2011.02369.x.View ArticlePubMedGoogle Scholar
- Dec M, Urban-Chmiel R, Gnat S, Puchalski A, Wernicki A: Identification of Lactobacillus strains of goose origin using MALDI-TOF mass spectrometry and 16S-23S rDNA intergenic spacer PCR analysis. Res Microbiol. 2014, 165: 190-201. 10.1016/j.resmic.2014.02.003.View ArticlePubMedGoogle Scholar
- Mazzeo MF, Sorrentino A, Gaita M, Cacace G, Di Stasio M, Facchiano A, Comi G, Malorni A, Siciliano RA: Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the discrimination of food-borne microorganisms. Appl Environ Microbiol. 2006, 72: 1180-1189. 10.1128/AEM.72.2.1180-1189.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Claydon MA, Davey SN, Edwards-Jones V, Gordon DB: The rapid identification of intact microorganisms using mass spectrometry. Nat Biotechnol. 1996, 14: 1584-1586. 10.1038/nbt1196-1584.View ArticlePubMedGoogle Scholar
- Doan NT, Van Hoorde K, Cnockaert M, De Brandt E, Aerts M, Le Thanh B, Vandamme P: Validation of MALDI-TOF MS for rapid classification and identification of lactic acid bacteria, with a focus on isolates from traditional fermented foods in Northern Vietnam. Lett Appl Microbiol. 2012, 55: 265-273. 10.1111/j.1472-765X.2012.03287.x.View ArticlePubMedGoogle Scholar
- Kern CC, Vogel RF, Behr J: Differentiation of lactobacillus brevis strains using matrix-assisted-laser-desorption-ionization-time-of-flight mass spectrometry with respect to their beer spoilage potential. Food Microbiol. 2014, 40: 18-24. 10.1016/j.fm.2013.11.015.View ArticlePubMedGoogle Scholar
- Patel JB: 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Mol Diagn. 2001, 6: 313-321. 10.2165/00066982-200106040-00012.View ArticlePubMedGoogle Scholar
- Dimitonova SP, Bakalov BV, Aleksandrova-Georgieva RN, Danova ST: Phenotypic and molecular identification of lactobacilli isolated from vaginal secretions. J Microbiol Immunol Infect. 2008, 41: 469-477.PubMedGoogle Scholar
- Pal K, Szen O, Kiss A, Naar Z: Comparison and evaluation of molecular methods used for identification and discrimination of lactic acid bacteria. J Sci Food Agric. 2012, 92: 1931-1936. 10.1002/jsfa.5564.View ArticlePubMedGoogle Scholar
- Moreira JL, Mota RM, Horta MF, Teixeira SM, Neumann E, Nicoli JR, Nunes AC: Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling. BMC Microbiol. 2005, 5: 15-10.1186/1471-2180-5-15.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhong W, Millsap K, Bialkowska-Hobrzanska H, Reid G: Differentiation of lactobacillus species by molecular typing. Appl Environ Microbiol. 1998, 64: 2418-2423.PubMed CentralPubMedGoogle Scholar
- Giraffa G, Gatti M, Rossetti L, Senini L, Neviani E: Molecular diversity within Lactobacillus helveticus as revealed by genotypic characterization. Appl Environ Microbiol. 2000, 66: 1259-1265. 10.1128/AEM.66.4.1259-1265.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang R, Argimon S, Li Y, Gu H, Zhou X, Caufield PW: Determining the genetic diversity of lactobacilli from the oral cavity. J Microbiol Methods. 2010, 82: 163-169. 10.1016/j.mimet.2010.05.010.PubMed CentralView ArticlePubMedGoogle Scholar
- Falsen E, Pascual C, Sjoden B, Ohlen M, Collins MD: Phenotypic and phylogenetic characterization of a novel Lactobacillus species from human sources: description of Lactobacillus iners sp. nov. Int J Syst Bacteriol 1999, 49 Pt 1:217–221.View ArticlePubMedGoogle Scholar
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