Guido Lopes dos Santos Santiago

Identification and genotyping of bacteria from paired vaginal and rectal samples from pregnant women indicates similarity between vaginal and rectal microflora

BackgroundThe vaginal microflora is important for maintaining vaginal health and preventing infections of the reproductive tract. The rectum has been suggested as the major source for the colonisation of the vaginal econiche.MethodsTo establish whether the rectum can serve as a possible bacterial reservoir for colonisation of the vaginal econiche, we cultured vaginal and rectal specimens from pregnant women at 35-37 weeks of gestation, identified the isolates to the species level with tRNA intergenic length polymorphism analysis (tDNA-PCR) and genotyped the isolates for those subjects from which the same species was isolated simultaneously vaginally and rectally, by RAPD-analysis.One vaginal and one rectal swab were collected from a total of each of 132 pregnant women at 35-37 weeks of gestation. Swabs were cultured on Columbia CNA agar and MRS agar. For each subject 4 colonies were selected for each of both sites, i.e. 8 colonies in total.ResultsAmong the 844 isolates that could be identified by tDNA-PCR, a total of 63 bacterial species were present, 9 (14%) only vaginally, 26 (41%) only rectally, and 28 (44%) in both vagina and rectum. A total of 121 (91.6%) of 132 vaginal samples and 51 (38.6%) of 132 rectal samples were positive for lactobacilli. L. crispatus was the most frequently isolated Lactobacillus species from the vagina (40% of the subjects were positive), followed by L. jensenii (32%), L. gasseri (30%) and L. iners (11%). L. gasseri was the most frequently isolated Lactobacillus species from the rectum (15%), followed by L. jensenii (12%), L. crispatus (11%) and L. iners (2%).A total of 47 pregnant women carried the same species vaginally and rectally. This resulted in 50 vaginal/rectal pairs of the same species, for a total of eight different species. For 34 of the 50 species pairs (68%), isolates with the same genotype were present vaginally and rectally and a high level of genotypic diversity within species per subject was also established.ConclusionIt can be concluded that there is a certain degree of correspondence between the vaginal and rectal microflora, not only with regard to species composition but also with regard to strain identity between vaginal and rectal isolates.These results support the hypothesis that the rectal microflora serves as a reservoir for colonisation of the vaginal econiche.

Gardnerella vaginalis comprises three distinct genotypes of which only two produce sialidase

OBJECTIVE Sialidase and the presence of Gardnerella vaginalis have been proposed as biomarkers for bacterial vaginosis. Sialidase has been associated with adverse pregnancy outcome. We genotyped G vaginalis isolates, assessed the presence and diversity of sialidase-encoding genes, and determined the production of sialidase. STUDY DESIGN One hundred thirty-four G vaginalis isolates were genotyped by random amplified polymorphic deoxyribonucleic acid (RAPD) and a selection of 29 isolates with amplified ribosomal deoxyribonucleic acid restriction analysis (ARDRA). A G vaginalis sialidase quantitative polymerase chain reaction was developed, and the sialidase production was assessed with the filter spot test. RESULTS Three G vaginalis genotypes could be distinguished by both RAPD and ARDRA. Only 2 genotypes encoded and produced sialidase. CONCLUSION Three genotypes exist among G vaginalis isolates, and there is a clear link between genotype and sialidase production. A possible link between sialidase production and (symptomatic) bacterial vaginosis and biofilm production can be hypothesized.

Longitudinal Study of the Dynamics of Vaginal Microflora during Two Consecutive Menstrual Cycles

Background Although the vaginal microflora (VMF) has been well studied, information on the fluctuation of the different bacterial species throughout the menstrual cycle and the information on events preceding the presence of disturbed VMF is still very limited. Documenting the dynamics of the VMF during the menstrual cycle might provide better insights. In this study, we assessed the presence of different Lactobacillus species in relation to the BV associated species during the menstrual cycle, assessed the influence of the menstrual cycle on the different categories of vaginal microflora and assessed possible causes, such as menstruation and sexual intercourse, of VMF disturbance. To our knowledge, this is the first longitudinal study in which swabs and Gram stains were available for each day of two consecutive menstrual cycles, whereby 8 grades of VMF were distinguished by Gram stain analysis, and whereby the swabs were cultured every 7th day and identification of the bacterial isolates was carried out with a molecular technique. Methods Self-collected vaginal swabs were obtained daily from 17 non pregnant, menarchal volunteers, and used for daily Gram staining and weekly culture. Bacterial isolates were identified with tDNA-PCR and 16 S rRNA gene sequencing. Results Nine women presented with predominantly normal VMF and the 8 others had predominantly disturbed VMF. The overall VMF of each volunteer was characteristic and rather stable. Menses and antimicrobials were the major disturbing factors of the VMF. Disturbances were always accompanied by a rise in Gram positive cocci, which also appeared to be a significant group within the VMF in general. Conclusions We observed a huge interindividual variability of predominantly stable VMF types. The importance of Gram positive cocci in VMF is underestimated. L. crispatus was the species that was most negatively affected by the menses, whereas the presence of the other lactobacilli was less variable.

Longitudinal qPCR Study of the Dynamics of L. crispatus, L. iners, A. vaginae, (Sialidase Positive) G. vaginalis, and P. bivia in the Vagina

Background To obtain more detailed understanding of the causes of disturbance of the vaginal microflora (VMF), a longitudinal study was carried out for 17 women during two menstrual cycles. Methods Vaginal swabs were obtained daily from 17 non-pregnant, menarchal volunteers. For each woman, Gram stains were scored, the quantitative changes of 5 key vaginal species, i.e. Atopobium vaginae, Lactobacillus crispatus, L. iners, (sialidase positive) Gardnerella vaginalis and Prevotella bivia were quantified with qPCR and hydrogen-peroxide production was assessed on TMB+ agar. Results Women could be divided in 9 subjects with predominantly normal VMF (grades Ia, Ib and Iab, group N) and 8 with predominantly disturbed VMF (grades I-like, II, III and IV, group D). VMF was variable between women, but overall stable for most of the women. Menses were the strongest disturbing factor of the VMF. L. crispatus was present at log7–9 cells/ml in grade Ia, Iab and II VMF, but concentrations declined 100-fold during menses. L. crispatus below log7 cells/ml corresponded with poor H2O2-production. L. iners was present at log 10 cells/ml in grade Ib, II and III VMF. Sialidase negative G. vaginalis strains (average log5 cells/ml) were detected in grade I, I-like and IV VMF. In grade II VMF, predominantly a mixture of both sialidase negative and positive G. vaginalis strains (average log9 cells/ml) were present, and predominantly sialidase positive strains in grade III VMF. The presence of A. vaginae (average log9 cells/ml) coincided with grade II and III VMF. P. bivia (log4–8 cells/ml) was mostly present in grade III vaginal microflora. L. iners, G. vaginalis, A. vaginae and P. bivia all increased around menses for group N women, and as such L. iners was considered a member of disturbed VMF. Conclusions This qPCR-based study confirms largely the results of previous culture-based, microscopy-based and pyrosequencing-based studies.

In vivo evaluation of the vaginal distribution and retention of a multi-particulate pellet formulation

Non-disintegrating microcrystalline cellulose pellets (MCC) and disintegrating starch-based pellets were evaluated as new vaginal drug delivery forms and compared with a powder formulation. Pellets and powder were packed in a HPMC or hard gelatine capsule and vaginally administered to five series of five healthy volunteers. Distribution and retention of the multi-particulate formulation was monitored by colposcopy and swabbing. Capsule disintegration in the vagina was slow. MCC pellets clustered around the fornix 3h after administration, and after 24h only a few pellets were detected in the vaginal cavity. In contrast, starch-based pellets already started to disintegrate 6h after administration, resulting in a complete coverage of the vaginal mucosa after 24h in 8 out of 10 volunteers. The powder formulation had a better distribution after 6h, although after 24h almost no powder remained in the vagina. These results were confirmed by swabbing to determine the amount of riboflavin sodium phosphate (used as marker) distributed in the different vaginal regions.

Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients

BackgroundPseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR) for the detection of P. aeruginosa in respiratory samples of not chronically infected CF patients.ResultsIn this national study, we followed CF patients during periods between 1 to 15 months. For a total of 852 samples, 729 (86%) remained P. aeruginosa negative by both culture and qPCR, whereas 89 samples (10%) were positive by both culture and qPCR.Twenty-six samples were negative by culture but positive by qPCR, and 10 samples were positive by culture but remained negative by qPCR. Five of the 26 patients with a culture negative, qPCR positive sample became later P. aeruginosa positive both by culture and qPCR.ConclusionBased on the results of this study, it can be concluded that qPCR may have a predictive value for impending P. aeruginosa infection for only a limited number of patients.

Edwardsiella tarda sepsis in a live-stranded sperm whale (Physeter macrocephalus).

Whale strandings remain poorly understood, although bacterial infections have been suggested to contribute. We isolated Edwardsiella tarda from the blood of a stranded sperm whale. The pathogen was identified with MALDI-TOF MS, confirmed by 16S rRNA gene sequencing and quantified in blood by qPCR. We report the first case of sepsis in a sperm whale. The zoonotic potential of E. tarda and the possible role of bacterial infections in the enigmatic strandings of cetaceans are discussed.

A pilot study evaluating the safety of vaginal administration of a multi-particulate pellet formulation

AIM Quantitative evaluation of the effect caused by vaginal administration of gelatin capsules loaded with starch pellets and lyophilized powder, respectively, on vaginal pH and microflora. METHOD Administration of gelatin capsules loaded with fast-disintegrating starch pellets (group P) or lyophilized lactose/skimmed milk (group L) was compared to no intervention (group C) in a 3-way randomized, double-blinded, parallel study with 18 volunteers. Follow-up visits were at day 6 (immediately after administration), day 14 (pill stop), day 22 (after withdrawal bleeding) and day 35 (midcycle). Vaginal pH was measured and swabs were taken for Gram staining and culture to assess the presence of hydrogen peroxide-producing lactobacilli. Colposcopy was performed to assess the occurrence of adverse effects on the vaginal and ectocervical mucosa. RESULTS No severe adverse events occurred. For all women, vaginal pH and Gram stain were normal from screening until pill stop. Although immediately after withdrawal bleeding, 8 out of 18 women had an elevated pH, a disturbed microflora or lacked hydrogen peroxide-producing lactobacilli, all women had hydrogen peroxide-producing lactobacilli and a normal vaginal pH at midcycle, and all but two had a normal Gram stain. CONCLUSION No major differences could be observed between the groups, whereby all changes in pH and microflora could be ascribed to withdrawal bleeding, indicating that gelatin capsules, starch pellets and lyophilized powder are acceptable carrier materials for the vaginal delivery of probiotic strains.

Susceptibility testing of Atopobium vaginae for dequalinium chloride

BackgroundAtopobium vaginae and Gardnerella vaginalis are major markers for bacterial vaginosis. We aimed to determine the MIC and MBC range of the broad-spectrum anti-infective and antiseptic dequalinium chloride for 28 strains, belonging to 4 species of the genus Atopobium, i.e. A. vaginae, A. minutum, A. rimae and A. parvulum.MethodsThe MIC was determined with a broth microdilution assay.ResultsThe MIC and MBC for Atopobium spp. for dequalinium chloride ranged between < 0.0625 and 2 μg/ml.ConclusionsThis study demonstrated that dequalinium chloride inhibits and kills clinical isolates of A. vaginae at concentrations similar to those of clindamycin and lower than those of metronidazole.

Application of the antibiotic batumin for accurate and rapid identification of staphylococcal small colony variants

BackgroundStaphylococcus aureus is a major human pathogen causing significant morbidity and mortality. The S. aureus colonies in osteomyelitis, in patients with cystic fibrosis and patients with endoprosthesis rejection frequently have an atypical morphology, i.e. staphylococcal small-colony variants, which form a naturally occurring subpopulation of clinically important staphylococci. Identification of these small colony variants is difficult, because of the loss of typical phenotypic characteristics of these variants.We wanted to improve and simplify the diagnosis of staphylococcal infection using a diagnostic preparation, consisting of 5 μg batumin paper disks. Batumin possesses a unique selective activity against all studied Staphylococcus spp., whereas all other species tested thus far are batumin resistant. We assessed the efficacy of the batumin diagnostic preparation to identify staphylococcal small colony variants, isolated from osteomyelitis patients.FindingsWith the batumin diagnostic preparation, all 30 tested staphylococcal small-colony variants had a growth inhibition zone around the disk of minimum 25 mm, accordant with the inhibition zones of the parent strains, isolated from the same patients.ConclusionsThe batumin diagnostic preparation correctly identified the small-colony variants of S. aureus, S. haemolyticus and S. epidermidis as belonging to the genus Staphylococcus, which differ profoundly from parental strains and are difficult to identify with standard methods. Identification of staphylococcal small-colony variants with the batumin diagnostic preparation is technically simple and can facilitate practical laboratory work.