Cystitis: symptomatology in women with suspected uncomplicated urinary tract infection.

BACKGROUND: Although cystitis in women is very common in general practice, its evolution in symptoms has not been clearly studied. Qualitative research has pointed to other than the classic symptomatology. METHODS: This was a prospective observational study of the symptomatology at presentation and the evolution of the symptoms in treated women with suspected uncomplicated urinary tract infection (UTI). Women consulting their general practitioner (GP) for dysuria, urgency, or frequency produced a urine sample (for bacteriologic processing) and kept a diary until the end of the symptoms. Exclusion criteria included complaints >1 week, fever, vaginal discharge, and known pathology. RESULTS: Of the 300 asked to participate, 148 (49%) returned the diary. Although none of the patients developed acute pyelonephritis, a substantial number of the women had such complaints as feeling feverish (33% in culture-positive group, 38% in culture-negative group), back pains (44% vs. 56%), and feeling weak and tired (71% vs. 65%). Differences between the culture-positive and culture-negative groups were not statistically significant except for the duration of symptoms, which was shorter in the culture-positive group (4 vs. 6 days). More severe symptoms at inclusion were correlated with a longer duration of these symptoms. CONCLUSIONS: The spectrum of complaints in women with suspected uncomplicated UTI is broad and comprises a number of symptoms usually associated with an upper UTI. The occurrence of these symptoms should not automatically prompt GPs to prescribe broad-spectrum antibiotics. Moreover, the duration of symptoms exceeding the recommended duration of antibiotic therapy does not indicate therapy failure and, thus, the need for changing antibiotic therapy.

Destructive otomastoiditis by MRSA from porcine origin.

A 63-year-old female pig farmer was referred to our department with a protracted course of otomastoiditis with destruction of the tympanic roof and cerebrospinal fluid leakage. The patient underwent a cortical mastoidectomy with closure of a large dural defect. Cultures of the middle ear effusion yielded a methicillin-resistant Staphylococcus aureus (MRSA), which upon further analysis was found to be from porcine origin. To our knowledge, this is the first report of a complicated case of otomastoiditis caused by a pig-type MRSA.

Evolution of bacterial susceptibility pattern of Escherichia coli in uncomplicated urinary tract infections in a country with high antibiotic consumption: a comparison of two surveys with a 10 year interval.

OBJECTIVES: For the empirical treatment of cystitis, clinicians are often guided by susceptibility data taken from urinary samples that sent to regional microbiological laboratories, which are not representatives for uncomplicated urinary tract infections (UTIs). To offer adequate recommendations, the distribution and susceptibility pattern of uropathogens in uncomplicated UTIs in women were compared with those obtained 10 years ago in our uropathogen surveillance in a primary healthcare setting. METHODS: Sixty-six general practitioners in the region of the city of Ghent were asked to inoculate a dipslide with midstream urine from every adult female patient with complaints suggestive for cystitis, during a period of 1 year. The dipslides were further processed in a central microbiological laboratory, where counting, identification and susceptibility testing were performed. RESULTS: Three hundred specimens were collected, of which 187 (62.3%) yielded a positive culture of 10(5) cfu/mL. In the age group of 18-54 years, Escherichia coli was the most frequently isolated uropathogen (77.5%), followed by Staphylococcus saprophyticus (13.5%) and Proteus spp. (2.7%). There were no statistically significant differences when compared with the data from 1996. In 2006, susceptibility of E. coli to nitrofurantoin was 100%, to quinolones 100%, to ampicillin 62.8% and to co-trimoxazole 86%, compared with 99.3%, 99.3%, 73.2% and 83.3%, respectively, in 1996 (no statistically significant differences). CONCLUSIONS: Over a period of 10 years, a systematic surveillance of uropathogens in female patients with uncomplicated UTI in general practice could not demonstrate a significant change in species distribution or antimicrobial susceptibility.

Health implications of antimicrobial resistance for patients with acute kidney injury and bloodstream infection.

Studies have produced conflicting findings on outcomes for patients with antimicrobial-resistant infection. This study evaluated whether infection with an antimicrobial-resistant organism affects outcome in critically ill patients with acute kidney injury treated with renal replacement therapy and whose clinical course is complicated with a nosocomial bloodstream infection. We found that infection with an antimicrobial-resistant organism did not adversely affect clinical outcome in this specific cohort, which already has a high mortality rate.

Infection risk related to the use of medicinal leeches.

OBJECTIVE: To assess the incidence of postoperative wound infections related to treatment with medicinal leeches at Ghent University Hospital. METHOD: A 2-year retrospective analysis of bacteriologic culture results of soft tissue infections in patients treated with medicinal leeches. RESULTS: Cultures of suspected wound infections were taken and susceptibility testing of isolates was performed on 17 of 47 patients (36.2%). Aeromonas was frequently isolated (18.5%). CONCLUSIONS: A high incidence of infection during and after application of medicinal leeches, despite their external decontamination, necessitates an antibiotic prophylaxis. In particular Aeromonas must be covered, as soft tissue infections with these bacteria can give serious complications. The prophylactic antibiotic should cover the most frequent isolated species taking into account the importance of Aeromonas and the susceptibility pattern. Based on the results, fluoroquinolones seem to be a good choice. The authors believe that practical recommendations to hospital pharmacists on prophylaxis during Hirudo medicinalis treatment, might enhance the safety of it's use by reducing the number of infections.

Combined use of Pastorex Staph-Plus and either of two new chromogenic agars, MRSA ID and CHROMagar MRSA, for detection of methicillin-resistant Staphylococcus aureus.

We describe the search toward a fast and reliable strategy to detect and confirm the presence of methicillin-resistant Staphylococcus aureus (MRSA) in screening samples. First, we evaluated the sensitivities and specificities of oxacillin resistance screening agar (ORSA) with enrichment (tryptic soy broth [TSB] and ORSA [TSB-ORSA]) and without enrichment (ORSA), MRSA ID (MRSA_ID) plates, and CHROMagar MRSA (C_MRSA) plates, all of which were inoculated with equal volumes of a suspension made by emulsifying screening swabs. Whereas the sensitivities after 48 h were similar for all media tested (77% for MRSA_ID and ORSA; 73% for C_MRSA and ORSA after enrichment [TSB-ORSA]), the specificities of MRSA_ID (98% after 24 h and 94% after 48 h) and C_MRSA (98% after 24 h and 90% after 48 h) were superior to the specificities of ORSAs (92% after 24 h and 83% after 48 h) and TSB-ORSA (86% after 24 h and 81% after 48 h). Subsequently, the performance of the Pastorex Staph-Plus agglutination test with presumptive MRSA isolates taken directly from chromogenic agars (direct_Pastorex agglutination) was compared to that of the Pastorex Staph-Plus agglutination test with isolates from blood agar subcultures (conventional_Pastorex agglutination). When the direct_Pastorex agglutination test on MRSA_ID plates was combined with Gram staining, the direct_Pastorex agglutination test with samples from MRSA_ID plates was as reliable as the conventional_Pastorex agglutination test with samples from blood agar subcultures from MRSA_ID plates. In contrast, the direct_Pastorex agglutination test with samples from C_MRSA plates gave false-negative results. Finally, we calculated the processing times of the four different strategies, namely, (i) enrichment in TSB supplemented with NaCl, subsequent culture on ORSA, and the conventional_Pastorex agglutination test; (ii) direct inoculation of ORSA combined with conventional_Pastorex agglutination test; (iii) direct inoculation of MRSA_ID plates combined with Gram staining and the direct_Pastorex agglutination test; and (iv) direct inoculation of C_MRSA plates combined with Gram staining and the direct_Pastorex agglutination test. We concluded that the use of MRSA_ID in combination with Gram staining and the direct_Pastorex agglutination test is faster and more specific than the other strategies tested.

Streptococcus cristatus isolated from a resected heart valve and blood cultures: case reports and application of phenotypic and genotypic techniques for identification.

BACKGROUND: Viridans streptococci are known as an important cause of endocarditis, but at present no cases of endocarditis caused by Streptococcus cristatus have been published, probably because phenotypic identification of viridans streptococci is tedious. Using tDNA-PCR, it is possible to identify to species level and to differentiate between species of the viridans group.

Outcome in bacteremia associated with nosocomial pneumonia and the impact of pathogen prediction by tracheal surveillance cultures.

OBJECTIVE: To assess whether pathogen prediction in bacteremia associated with nosocomial pneumonia (NP) by tracheal surveillance cultures improves adequacy of early antibiotic therapy and impacts mortality. DESIGN AND SETTING: A retrospective observational study of a prospectively gathered cohort. This cohort included all adult patients admitted to the ICU of a tertiary care hospital from 1992 through 2001 and who developed bacteremia associated with NP. MEASUREMENTS AND MAIN RESULTS: 128 episodes of bacteremia associated with NP were identified. In 110 episodes a tracheal surveillance culture 48-96h prior to bacteremia was available: this culture predicted the pathogen in 67 episodes (61%). Overall rates of appropriate empiric antibiotic therapy within 24 and 48h were 62 and 87%, respectively. Pathogen prediction was associated with a significantly higher rate of appropriate antibiotic therapy within 24h (71 vs 45%; p=0.01), but not within 48h (91 vs 82%; p=0.15). Crude in-hospital mortality was 50%. Pathogen prediction was associated with increased survival in univariate (OR 0.43; CI 0.19-0.93; p=0.04) and multivariate analysis (OR 0.32; CI 0.12-0.82; p=0.02). Multivariate analysis further identified age (OR 1.04; CI 1.01-1.07; p=0.02), increasing APACHEII score (OR 1.08; CI 1.02-1.15; p=0.01), and methicillin-resistant Staphylococcus aureus (OR 5.90; CI 1.36-25.36; p=0.01) and Pseudomonas aeruginosa (OR 3.30; CI 1.04-10.4; p=0.04) as independent risk factors for mortality. CONCLUSION: Pathogen prediction in bacteremia associated with NP by tracheal surveillance cultures is associated with a higher rate of adequate empiric antibiotic therapy within 24[Symbol: see text]h and with increased survival.

Antibiotic susceptibility of Atopobium vaginae.

BACKGROUND: Previous studies have indicated that a recently described anaerobic bacterium, Atopobium vaginae is associated with bacterial vaginosis (BV). Thus far the four isolates of this fastidious micro-organism were found to be highly resistant to metronidazole and susceptible for clindamycin, two antibiotics preferred for the treatment of BV. METHODS: Nine strains of Atopobium vaginae, four strains of Gardnerella vaginalis, two strains of Lactobacillus iners and one strain each of Bifidobacterium breve, B. longum, L. crispatus, L. gasseri and L. jensenii were tested against 15 antimicrobial agents using the Etest. RESULTS: All nine strains of A. vaginae were highly resistant to nalidixic acid and colistin while being inhibited by low concentrations of clindamycin (range: < 0.016 microg/ml), rifampicin (< 0.002 microg/ml), azithromycin (< 0.016-0.32 microg/ml), penicillin (0.008-0.25 microg/ml), ampicillin (< 0.016-0.94 microg/ml), ciprofloxacin (0.023-0.25 microg/ml) and linezolid (0.016-0.125 microg/ml). We found a variable susceptibility for metronidazole, ranging from 2 to more than 256 microg/ml. The four G. vaginalis strains were also susceptible for clindamycin (< 0.016-0.047 microg/ml) and three strains were susceptible to less than 1 microg/ml of metronidazole. All lactobacilli were resistant to metronidazole (> 256 microg/ml) but susceptible to clindamycin (0.023-0.125 microg/ml). CONCLUSION: Clindamycin has higher activity against G. vaginalis and A. vaginae than metronidazole, but not all A. vaginae isolates are metronidazole resistant, as seemed to be a straightforward conclusion from previous studies on a more limited number of strains.

Antimicrobial resistance in nosocomial bloodstream infection associated with pneumonia and the value of systematic surveillance cultures in an adult intensive care unit.

OBJECTIVE: To study the occurrence of multiple-drug-resistant pathogens in nosocomial bloodstream infection associated with pneumonia. To evaluate prediction of multiple drug resistance by systematic surveillance cultures. DESIGN: A retrospective study of a prospectively gathered cohort. SETTING: Fifty-four-bed adult medical-surgical intensive care unit of a tertiary hospital. PATIENTS: One hundred twelve intensive care unit patients with nosocomial bloodstream infection associated with pneumonia from 1992 through 2001. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Concordance of blood cultures with prior surveillance culture was assessed. Surveillance cultures were taken routinely as thrice weekly urinary cultures and oral swabs, once weekly anal swabs, and thrice weekly tracheal aspirates in intubated patients. Tracheal surveillance cultures from 48 to 96 hrs before bloodstream infection and surveillance cultures from any site during the same intensive care unit episode but >or=48 hrs before bloodstream infection were evaluated separately. Forty-four bloodstream infections (39%) were caused by a multiple-drug-resistant pathogen. Multiple-drug-resistant pathogens were predicted by tracheal surveillance culture in 70% (concordant); in 15%, tracheal surveillance culture grew a multiple-drug-resistant pathogen not found in blood cultures (discordant). Multiple-drug-resistant pathogens were predicted by any surveillance culture in 88%, but these surveillance cultures grew additional multiple-drug-resistant pathogens not causing bloodstream infection in up to 46% of patients. In 86% of bloodstream infections, early (i.e., within 48 hrs) antibiotic therapy was appropriate. Patients were divided into four risk categories for multiple-drug-resistant bloodstream infection based on length of prior intensive care unit stay and prior antibiotic exposure. In patients with two risk factors, knowledge of surveillance cultures increased appropriateness of early antibiotic therapy from 75-79% to 90% (p<.05) while limiting use of broad-spectrum antibiotics such as antipseudomonal betalactams, fluoroquinolones, and carbapenems. CONCLUSIONS: In our intensive care unit, tracheal surveillance culture predicted multiple-drug-resistant etiology of bloodstream infection associated with pneumonia in 70% of patients but yielded discordant resistant pathogens in 15%. In the subgroup of patients with two risk factors for multiple-drug-resistant infection, incorporating results of surveillance cultures moderately contributed to adequacy of early antibiotic therapy while limiting antibiotic consumption.

Comparison between Gram stain and culture for the characterization of vaginal microflora: definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora.

BACKGROUND: The microbiological diagnosis of bacterial vaginosis is usually made using Nugent's criteria, a useful but rather laborious scoring system based on counting bacterial cell types on Gram stained slides of vaginal smears. Ison and Hay have simplified the score system to three categories and added a fourth category for microflora with a predominance of the Streptococcus cell type. Because in the Nugent system several cell types are not taken into account for a final score, we carried out a detailed assessment of the composition of the vaginal microflora in relation to standard Gram stain in order the improve the diagnostic value of the Gram stain. To this purpose we compared Gram stain based categorization of vaginal smears with i) species specific PCR for the detection of Gardnerella vaginalis and Atopobium vaginae and with ii) tDNA-PCR for the identification of most cultivable species. RESULTS: A total of 515 samples were obtained from 197 pregnant women, of which 403 (78.3%) were categorized as grade I microflora, 46 (8.9%) as grade II, 22 (4.3%) as grade III and 8 (1.6%) as grade IV, according to the criteria of Ison and Hay. Another 36 samples (7.0%) were assigned to the new category 'grade I-like', because of the presence of diphtheroid bacilli cell types. We found that 52.7% of the grade I-like samples contained Bifidobacterium spp. while L. crispatus was present in only 2.8% of the samples and G. vaginalis and A. vaginae were virtually absent; in addition, the species diversity of this category was similar to that of grade II specimens.Based on the presence of different Lactobacillus cell types, grade I specimens were further characterized as grade Ia (40.2%), grade Iab (14.9%) and grade Ib (44.9%). We found that this classification was supported by the finding that L. crispatus was cultured from respectively 87.0% and 76.7% of grade Ia and Iab specimens while this species was present in only 13.3% of grade Ib specimens, a category in which L. gasseri and L. iners were predominant. CONCLUSION: Further refinement of Gram stain based grading of vaginal smears is possible by distinguishing additional classes within grade I smears (Ia, Iab and Ib) and by adding a separate category, designated grade I-like. A strong correlation was found between grade Ia and the presence of L. crispatus and between grade I-like and the presence of bifidobacteria. This refinement of Gram stain based scoring of vaginal smears may be helpful to improve the interpretation of the clinical data in future studies, such as the understanding of response to treatment and recurrence of bacterial vaginosis in some women, and the relationship between bacterial vaginosis and preterm birth.

Colonization status and appropriate antibiotic therapy for nosocomial bacteremia caused by antibiotic-resistant gram-negative bacteria in an intensive care unit.

OBJECTIVE: Timely initiation of antibiotic therapy is crucial for severe infection. Appropriate antibiotic therapy is often delayed for nosocomial infections caused by antibiotic-resistant bacteria. The relationship between knowledge of colonization caused by antibiotic-resistant gram-negative bacteria (ABR-GNB) and rate of appropriate initial antibiotic therapy for subsequent bacteremia was evaluated. DESIGN: Retrospective cohort study. SETTING: Fifty-four-bed intensive care unit (ICU) of a university hospital. In this unit, colonization surveillance is performed through routine site-specific surveillance cultures (urine, mouth, trachea, and anus). Additional cultures are performed when presumed clinically relevant. PATIENTS: ICU patients with nosocomial bacteremia caused by ABR-GNB. RESULTS: Infectious and microbiological characteristics and rates of appropriate antibiotic therapy were compared between patients with and without colonization prior to bacteremia. Prior colonization was defined as the presence (detected > or = 2 days before the onset of bacteremia) of the same ABR-GNB in colonization and subsequent blood cultures. During the study period, 157 episodes of bacteremia caused by ABR-GNB were suitable for evaluation. One hundred seventeen episodes of bacteremia (74.5%) were preceded by colonization. Appropriate empiric antibiotic therapy (started within 24 hours) was administered for 74.4% of these episodes versus 55.0% of the episodes that occurred without prior colonization. Appropriate therapy was administered within 48 hours for all episodes preceded by colonization versus 90.0% of episodes without prior colonization. CONCLUSION: Knowledge of colonization status prior to infection is associated with higher rates of appropriate therapy for patients with bacteremia caused by ABR-GNB.

Prevalence of Helicobacter pullorum among patients with gastrointestinal disease and clinically healthy persons.

Feces from 531 patients with gastroenteritis and from 100 clinically healthy individuals were tested for Helicobacter pullorum by use of PCR. Samples positive by PCR were qualified for isolation. H. pullorum DNA was demonstrated to be present in feces from 4.3% of patients with gastrointestinal disease but also in feces from 4.0% of clinically healthy persons. One strain was isolated from one patient with gastrointestinal disease.

Shared genotypes of Achromobacter xylosoxidans strains isolated from patients at a cystic fibrosis rehabilitation center.

During a study examining transmission of Pseudomonas aeruginosa among 76 cystic fibrosis patients in a rehabilitation center, where patients stay in close contact during prolonged periods, several clusters of patients carrying genotypically identical P. aeruginosa, as well as two clusters of 4 and 10 patients, respectively, colonized with genotypically identical Achromobacter xylosoxidans strains, were discovered.

An interlaboratory comparison of ITS2-PCR for the identification of yeasts, using the ABI Prism 310 and CEQ8000 capillary electrophoresis systems.

BACKGROUND: Currently, most laboratories identify yeasts routinely on the basis of morphology and biochemical reactivity. This approach has quite often limited discriminatory power and may require long incubation periods. Due to the increase of fungal infections and due to specific antifungal resistence patterns for different species, accurate and rapid identification has become more important. Several molecular techniques have been described for fast and reliable identification of yeast isolates, but interlaboratory exchangeability of identification schemes of molecular techniques has hardly been studied. Here, we compared amplified ITS2 fragment length determination by an ABI Prism 310 (Applied Biosystems, Foster City, Ca.) capillary electrophoresis system with that obtained by a CEQ8000 (Beckman Coulter, Fullerton, Ca.) capillary electrophoresis system. RESULTS: Although ITS2 size estimations on both systems differed and separate libraries had to be constructed for each system, both approaches had the same discriminatory power with regard to the 44 reference strains, identical identifications were obtained for 39/ 40 clinical isolates in both laboratories and strains from 51 samples were correctly identified using CEQ8000, when compared to phenotypic identification. CONCLUSION: Identification of yeasts with ITS2-PCR followed by fragment analysis can be carried out on different capillary electrophoresis systems with comparable discriminatory power.

Extended-Spectrum beta-lactamase (ESBL) producing Enterobacter aerogenes phenotypically misidentified as Klebsiella pneumoniae or K. terrigena.

BACKGROUND: Enterobacter aerogenes and Klebsiella pneumoniae are common isolates in clinical microbiology and important as producers of extended spectrum beta-lactamases (ESBL). The discrimination between both species, which is routinely based on biochemical characteristics, is generally accepted to be straightforward. Here we report that genotypically unrelated strains of E. aerogenes can be misidentified as K. pneumoniae by routine laboratories using standard biochemical identification and using identification automates. RESULTS: Ten clinical isolates, identified as K. pneumoniae or K. terrigena with the routinely used biochemical tests and with API-20E, were identified as E. aerogenes by tDNA-PCR - an identification that was confirmed by 16S rRNA gene sequencing for five of these isolates. Misidentification also occurred when using the automated identification systems Vitek 2 and Phoenix, and was due to delayed positivity for ornithine decarboxylase and motility. Subculture and prolonged incubation resulted in positive results for ornithine decarboxylase and for motility. It could be shown by RAPD-analysis that the E. aerogenes strains belonged to different genotypes. CONCLUSIONS: Clinical E. aerogenes isolates can be easily misidentified as Klebsiella due to delayed positivity for ornithine decarboxylase and motility. The phenomenon may be widespread, since it was shown to occur among genotypically unrelated strains from different hospitals and different isolation dates. A useful clue for correct identification is the presence of an inducible beta-lactamase, which is highly unusual for K. pneumoniae. In several instances, the use of genotypic techniques like tDNA-PCR may circumvent problems of phenotypic identification.

Bacteremic infection with Pantoea ananatis.

A 73-year-old man was hospitalized for dyspnea and bilateral ankle edema. During his hospital stay he presented anal hemorrhage and developed a high fever after colonoscopy. A set of aerobic and anaerobic blood culture bottles yielded a pure culture of gram-negative rods, susceptible to all antibiotics tested. The API20E code was 1005133, resulting in a very good identification as Pantoea sp. Subsequent sequencing of the 16S rRNA gene revealed a final identification as Pantoea ananatis. The patient was given intravenous and oral therapy with piperacillin-tazobactam and ofloxacin and recovered completely from his infection.

Antibiotic susceptibility of acute otitis media pathogens in otitis-prone Belgian children.

UNLABELLED: A regional surveillance study was carried out in children with recurrent acute otitis media (AOM) to determine the antimicrobial susceptibility of three common AOM pathogens. Susceptibility to relevant antimicrobial agents was determined on 149 Streptococcus pneumoniae, 246 Haemophilus influenzae and 119 Moraxella catarrhalis strains isolated between January 1999 and January 2002, either from the nasopharynx or middle ear of 74 children with recurrent AOM, the majority (77%) being otitis-prone. Overall pneumococcal resistance to penicillin was 9.4% (6.7% penicillin-intermediate resistant, 2.7% penicillin-resistant), whereas cotrimoxazole and erythromycin resistance accounted for 25.5% and 38.9% respectively. The prevalence of antimicrobial-non-susceptible S. pneumoniae was the highest in middle ear isolates (P<0.05) and in otitis-prone children (P<0.01). Moreover, otitis-prone children harboured significantly more pneumococci resistant to at least two antimicrobial agents (24.3% versus 7.4%; P<0.01). No patient age related variation was observed. Five serogroups (6, 19, 23, 14 and 9) covered by the 7-valent pneumococcal conjugate vaccine, constituted most of the antibiotic resistant pneumococci. Among nasopharyngeal and middle ear H. influenzae isolates, 17.1% were resistant to ampicillin and 16.3% to cotrimoxazole. For M. catarrhalis, 92.4% of all isolates was ampicillin-resistant. CONCLUSION: This study confirms international and national differences in antimicrobial susceptibility profiles of three acute otitis media pathogens with relatively favourable antibiotic resistance rates in Belgian children with frequent acute otitis media. This "at risk" population of otitis-prone children is shown to harbour more antimicrobial resistant and multidrug resistant pneumococci. If antimicrobial therapy in this group of children is indicated, high dose amoxicillin is recommended whereas the use of macrolides is obsolete.

Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis.

BACKGROUND: The pathogenesis of bacterial vaginosis remains largely elusive, although some microorganisms, including Gardnerella vaginalis, are suspected of playing a role in the etiology of this disorder. Recently culture-independent analysis of microbial ecosystems has proven its efficacy in characterizing the diversity of bacterial populations. Here, we report on the results obtained by combining culture and PCR-based methods to characterize the normal and disturbed vaginal microflora. RESULTS: A total of 150 vaginal swab samples from healthy women (115 pregnant and 35 non-pregnant) were categorized on the basis of Gram stain of direct smear as grade I (n = 112), grade II (n = 26), grade III (n = 9) or grade IV (n = 3). The composition of the vaginal microbial community of eight of these vaginal swabs (three grade I, two grade II and three grade III), all from non-pregnant women, were studied by culture and by cloning of the 16S rRNA genes obtained after direct amplification. Forty-six cultured isolates were identified by tDNA-PCR, 854 cloned 16S rRNA gene fragments were analysed of which 156 by sequencing, yielding a total of 38 species, including 9 presumptively novel species with at least five species that have not been isolated previously from vaginal samples. Interestingly, cloning revealed that Atopobium vaginae was abundant in four out of the five non-grade I specimens. Finally, species specific PCR for A. vaginae and Gardnerella vaginalis pointed to a statistically significant co-occurrence of both species in the bacterial vaginosis samples. CONCLUSIONS: Although historically the literature regarding bacterial vaginosis has largely focused on G. vaginalis in particular, several findings of this study--like the abundance of A. vaginae in disturbed vaginal microflora and the presence of several novel species--indicate that much is to be learned about the composition of the vaginal microflora and its relation to the etiology of BV.