Multiplex quantitative polymerase chain reaction assay for the identification and quantitation of major vaginal lactobacilli

Lactobacilli play a key role in promoting vaginal health. Depletion of these bacteria is associated with bacterial vaginosis (BV), the most common vaginal disorder. Here we describe the development and laboratory validation of a novel single-tube multiplex TaqMan quantitative polymerase chain reaction (qPCR) assay for the identification and quantitative assessment of the four major vaginal Lactobacillus species: L. crispatus, L. jensenii, L. gasseri, and L. iners. The assay utility was evaluated by the analysis of lactobacilli in non-cultured clinical vaginal swab specimens collected from BV patients and healthy individuals. As confirmed by the assay, L. crispatus, L. jensenii, and to a lesser extent L. gasseri, are common in the vagina of healthy women, whereas L. iners dominance is associated with BV. The major assay limitation was preferential detection of dominant Lactobacillus species in samples with mixed lactobacilli resulting in lower sensitivity for minor species. The multiplex qPCR assay described here is an advance in the detection and quantitation of the major vaginal lactobacilli, potentially facilitating the molecular diagnosis of BV and post-therapy restoration of the vaginal microflora.     

Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus

Identifying Lactobacillus species using only phenotypic and genotypic (16S rDNA sequence analysis) techniques yields inaccurate results. The objective of this study was to develop species-specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting to distinguish species within the closely related Lactobacillus plantarum group. One of these primers, OPD-3, produced a species-specific band that was found only in the tested Lactobacillus pentosus. This specific fragment was isolated from agarose gel and ligated into a vector for DNA sequencing. A pair of primers, SpOPD3Lpen-F1/R1, that were highly specific sequence-characterized-amplified-regions (SCARs) were designed according to the nucleotide sequences of the specific RAPD marker. These primers were used for PCR analysis of the template DNA of the Lactobacillus strains, and a single 542 bp species-specific band was found only in L. pentosus. Using PCR, a novel species-specific primer pair is shown to rapidly, accurately and effectively distinguish L. pentosus from other species in the L. plantarum group of probiotic bacteria.     

Development of a Microarray-Based Tool To Characterize Vaginal Bacterial Fluctuations and Application to a Novel Antibiotic Treatment for Bacterial Vaginosis

The healthy vaginal microbiota is generally dominated by lactobacilli that confer antimicrobial protection and play a crucial role in health. Bacterial vaginosis (BV) is the most prevalent lower genital tract infection in women in reproductive age and is characterized by a shift in the relative abundances of Lactobacillus spp. to a greater abundance of strictly anaerobic bacteria. In this study, we designed a new phylogenetic microarray-based tool (VaginArray) that includes 17 probe sets specific for the most representative bacterial groups of the human vaginal ecosystem. This tool was implemented using the ligase detection reaction-universal array (LDR-UA) approach. The entire probe set properly recognized the specific targets and showed an overall sensitivity of 6 to 12 ng per probe. The VaginArray was applied to assess the efficacy of rifaximin vaginal tablets for the treatment of BV, analyzing the vaginal bacterial communities of 22 BV-affected women treated with rifaximin vaginal tablets at a dosage of 25 mg/day for 5 days. Our results showed the ability of rifaximin to reduce the growth of various BV-related bacteria (Atopobium vaginae, Prevotella, Megasphaera, Mobiluncus, and Sneathia spp.), with the highest antibiotic susceptibility for A. vaginae and Sneathia spp. Moreover, we observed an increase of Lactobacillus crispatus levels in the subset of women who maintained remission after 1 month of therapy, opening new perspectives for the treatment of BV.     

High-Throughput Sequencing Reveals the Incomplete,Short-Term Recovery of Infant Gut Microbiota following Parenteral Antibiotic Treatment with Ampicillin and Gentamicin

The infant gut microbiota undergoes dramatic changes during the first 2 years of life. The acquisition and development of this population can be influenced by numerous factors, and antibiotic treatment has been suggested as one of the most significant. Despite this, however, there have been relatively few studies which have investigated the short-term recovery of the infant gut microbiota following antibiotic treatment. The aim of this study was to use high-throughput sequencing (employing both 16S rRNA and rpoB-specific primers) and quantitative PCR to compare the gut microbiota of nine infants who underwent parenteral antibiotic treatment with ampicillin and gentamicin (within 48 h of birth), 4 and 8 weeks after the conclusion of treatment, relative to that of nine matched healthy controls. The investigation revealed that the gut microbiota of the antibiotic-treated infants had significantly higher proportions of Proteobacteria (P = 0.0049) and significantly lower proportions of Actinobacteria (P = 0.00001) (and the associated genus Bifidobacterium [P = 0.0132]) as well as the genus Lactobacillus (P = 0.0182) than the untreated controls 4 weeks after the cessation of treatment. By week 8, the Proteobacteria levels remained significantly higher in the treated infants (P = 0.0049), but the Actinobacteria, Bifidobacterium, and Lactobacillus levels had recovered and were similar to those in the control samples. Despite this recovery of total Bifidobacterium numbers, rpoB-targeted pyrosequencing revealed that the number of different Bifidobacterium species present in the antibiotic-treated infants was reduced. It is thus apparent that the combined use of ampicillin and gentamicin in early life can have significant effects on the evolution of the infant gut microbiota, the long-term health implications of which remain unknown.     

甲氧西林耐药溶血性葡萄球菌的RAPD分型研究

目的建立随机扩增多态性DNA技术（RAPD）对甲氧西林耐药溶血性葡萄球菌（MRSH）的分型方法.方法收集本院2002年4月至2003年4月临床分离的MRSH 81株.共设计了5组引物进行RAPD,电泳条带采用SPSS 13.0软件分析,根据树状图分型.结果除引物H12和M13组无法扩增出条带外,其余的4种引物均可以将81株MRSH进行分型.其中引物ERIC2将其分为13型,而引物ERIC1R将其分为8型.两种引物均可以辨别出主要流行株.结论 RAPD技术采用引物ERIC2对MRSH分型具有快速、敏感、稳定性高等特点.在有适合引物的条件下,RAPD可以作为判断MRSH医院感染流行的初筛工具.     

Application of the SNaPshot minisequencing assay to species identification in the Lactobacillus casei group

This study used group-specific PCR combined with SNaPshot minisequencing for species identification within the Lactobacillus casei group. The L. casei group-specific PCR primer pair was designed using the rpoA gene sequence. A SNaPshot minisequencing assay using dnaK as a target gene was developed, and five SNP primers were designed by analysing the conserved regions of the dnaK sequences. The specificity of the minisequencing assay was evaluated using 63 strains of L. casei group species. The results showed that the group-specific PCR could assign Lactobacillus strains into the L. casei group, and the SNaPshot minisequencing assay was able to unambiguously and simultaneously discriminate strains belonging to the species L. casei, Lactobacillus paracasei, and Lactobacillus rhamnosus. In conclusion, we have successfully developed a rapid, accurate and cost-effective assay for species identification of members of the L. casei group.     

口服Cocktail A乳酸菌制剂对人肠道乳酸菌群影响的研究

目的评价口服Cocktail A乳酸菌制剂后人肠道乳酸菌群的变化。方法用需氧和厌氧定量培养方法分离并鉴定烧伤病房中30例腹泻患者给予Cocktail A乳酸菌制剂治疗前后肠道中的细菌,并以正常健康体检者的粪便培养鉴定结果作为对照。同时对未鉴定出的细菌用16S rRNA基因进行鉴定。结果腹泻患者肠道中的菌群与健康体检者肠道中的菌群相比,乳酸菌的数量和种类少,而口服乳酸菌制剂治疗后肠道中的植物乳杆菌、明串珠球菌、戊糖片球菌、副酪蛋白乳杆菌大量增加。结论口服Cocktail A乳酸菌制剂能调节肠道的菌群失调。     

Vaginal microflora in healthy women with Gardnerella vaginalis

In order to find the vaginal prevalence of Gardnerella vaginalis in a normal female population, we determined the incidence of G. vaginalis in relation to that of other bacterial genera and species in the vagina. Two-hundred and thirty-nine healthy women were the subjects of this study. Vaginal discharge was collected and bacteriological studies were performed. The mean total aerobe count in the G. vaginalis-positive group was 9.02 log₁₀ colony-forming units (CFU)/g, which was significantly higher (P < 0.0001) than that (6.80 log₁₀ CFU/g) in the G. vaginalis-negative group. In contrast, there was no difference in the mean total anaerobe count between the two groups of subjects (8.82 and 8.24 log₁₀ CFU/g, respectively in the case of including Lactobacillus species count). Also, the mean pH level of vaginal secretion in the G. vaginalis-positive group was 4.58, which was significantly higher (P < 0.005) than that (4.10) in the G. vaginalis-negative group. Aerobes were isolated at equal incidence in the two groups. Anaerobes were isolated at a significantly higher rate in the G. vaginalis-positive group (P < 0.005) than in the G. vaginalis-negative group. The mean count of Lactobacillus species was significantly higher (P < 0.0001) in the G. vaginalis-negative group than in the positive counterpart (7.02 vs 8.66). Elevation of vaginal pH, an increase in the anaerobe count, and decreases in the Lactobacillus species count could be good predictors of the prevalence of bacterial vaginosis in healthy women. Received: May 14, 1999 / Accepted: June 26, 2000     

Characteristics and potential diagnostic ability of vaginal microflora in patients with aerobic vaginitis using 16S Ribosomal RNA sequencing

PurposeWe aimed to explore the possible function of guiding dominant microflora for aerobic vaginitis (AV) women patients in China to know its diagnostic ability for the further clinical application.MethodThe characteristics and community compositions of AV vaginal microbiota was evaluated by high-throughput 16S ribosomal RNA (16S rRNA) sequencing after the vaginal microecology evaluations.ResultsThe increased colonization of Streptococcus agalactiae and Streptococcus anginosus and the absence of Lactobacillus are typical manifestations of AV patients. In addition, Lactobacillus were the major correlated bacteria with control groups of Modified Donders’ Score under 3. Most of the significant KEGG pathways were classified into metabolism pathway, where the two bacteria behaved their own metabolism characteristics.ConclusionThe increased colonizations of Streptococcus agalactiae and Streptococcus anginosus and the absences of Lactobacillus are the typical manifestations of AV.