Gerda Verschraegen

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

BackgroundThe 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.ResultsA 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.ConclusionsAlthough 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.

Role of coagulase-negative staphylococci in human disease.

Coagulase-negative staphylococci (CNS) are normal inhabitants of human skin and mucous membranes. They have long been dismissed as culture contaminants, but now the potentially important role of CNS as pathogens and their increasing incidence has been recognized. Approximately 55-75% of nosocomial isolates is methicillin resistant. CNS were the first organisms in which glycopeptide resistance was recognized. In the immunocompetent host, CNS endocarditis and urinary tract infections with Staphylococcus saprophyticus are the most common CNS infections. Other patients are usually immunocompromised, with indwelling or implanted foreign bodies. CNS account for approximately 30% of all nosocomial blood stream infections. The majority of these concern catheter-related sepsis. Other important infections due to CNS include central nervous system shunt infections, endophthalmitis, surgical site infections, peritonitis in patients with continuous ambulatory peritoneal dialysis and foreign body infections. CNS are rarely associated with mastitis in humans. Staphylococcus lugdunensis is more pathogenic than other CNS as it expresses several potential virulence factors. The distinction between clinically significant, pathogenic and contaminating isolates is a major problem. Several studies show clonal intra and inter hospital spread of Staphylococcus epidermidis strains which suggests that infection control measures may be necessary for multiresistant CNS isolates as for methicillin resistant Staphylococcus aureus. As a result of medical progress, mainly due to the use of invasive and indwelling medical devices, CNS are now a major cause of nosocomial and health-care related infections.

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.

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 ≥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.

Application and Evaluation of the Interlaboratory Reproducibility of tRNA Intergenic Length Polymorphism Analysis (tDNA-PCR) for Identification of Streptococcus Species

ABSTRACT The discriminatory power, speed, and interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) combined with capillary electrophoresis was evaluated for the identification of streptococci. This method was carried out in three different laboratories under highly standardized conditions for 54 strains belonging to 18 different species. It was concluded that interlaboratory reproducibility of tDNA fingerprints produced by means of capillary electrophoresis was sufficiently high to permit the exchange between different laboratories and the construction of common libraries which can be consulted for comparison with fingerprints obtained independently in separate laboratories. In a second step, 17 other species were included in the study and examined in one of the participating laboratories. All Streptococcus species studied, except S. mitis, S. oralis, S. parasanguinis, S. pneumoniae, S. thermophilus, and S. vestibularis, showed distinguishable tDNA fingerprints. A database of well-characterized strains was constructed to enable computer-aided identification of unknown streptococcal isolates.

The national prevalence survey of nosocomial infections in Belgium, 1984

A national one-day prevalence survey of nosocomial infections was carried out in March 1984 in 106 Belgian acute-care hospitals involving 8723 patients of whom 6130 had undergone surgery. Three infections were studied: surgical wound infection, bacteraemia and urinary-tract infection. One or more of these three infections was recorded in 9.3% of all patients and in 11.8% of surgical patients. Prevalences increased with increasing duration of hospital stay and with higher ages, but the association of HAI with age was no longer significant after correction for duration of hospital stay. Prevalences varied considerably in different specialties. After adjustment for age and duration of stay, there was no association between perioperative antibiotic prophylaxis and the prevalence of the infections studied, but bias due to selection of higher risk patients in the antibiotic group was probable. Larger hospitals had a higher overall prevalence, but populations differed according to the size of the hospital. Bacteraemia was strongly associated with the presence of an intravenous catheter, and urinary-tract infection with a urinary catheter.

Identification of cultured isolates of clinically important yeast species using fluorescent fragment length analysis of the amplified internally transcribed rRNA spacer 2 region

BackgroundThe number of patients with yeast infection has increased during the last years. Also the variety of species of clinical importance has increased. Correct species identification is often important for efficient therapy, but is currently mostly based on phenotypic features and is sometimes time-consuming and depends largely on the expertise of technicians. Therefore, we evaluated the feasibility of PCR-based amplification of the internally transcribed spacer region 2 (ITS2), followed by fragment size analysis on the ABI Prism 310 for the identification of clinically important yeasts.ResultsA rapid DNA-extraction method, based on simple boiling-freezing was introduced. Of the 26 species tested, 22 could be identified unambiguously by scoring the length of the ITS2-region. No distinction could be made between the species Trichosporon asteroides and T. inkin or between T. mucoides and T. ovoides. The two varieties of Cryptococcus neoformans (var. neoformans and var. gattii) could be differentiated from each other due to a one bp length difference of the ITS2 fragment. The three Cryptococcus laurentii isolates were split into two groups according to their ITS2-fragment lengths, in correspondence with the phylogenetic groups described previously. Since the obtained fragment lengths compare well to those described previously and could be exchanged between two laboratories, an internationally usable library of ITS2 fragment lengths can be constructed.ConclusionsThe existing ITS2 size based library enables identification of most of the clinically important yeast species within 6 hours starting from a single colony and can be easily updated when new species are described. Data can be exchanged between laboratories.

Antibiotic susceptibility of Atopobium vaginae

BackgroundPrevious 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.MethodsNine 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.ResultsAll nine strains of A. vaginae were highly resistant to nalidixic acid and colistin while being inhibited by low concentrations of clindamycin (range: < 0.016 μg/ml), rifampicin (< 0.002 μg/ml), azithromycin (< 0.016 – 0.32 μg/ml), penicillin (0.008 – 0.25 μg/ml), ampicillin (< 0.016 – 0.94 μg/ml), ciprofloxacin (0.023 – 0.25 μg/ml) and linezolid (0.016 – 0.125 μg/ml). We found a variable susceptibility for metronidazole, ranging from 2 to more than 256 μg/ml. The four G. vaginalis strains were also susceptible for clindamycin (< 0.016 – 0.047 μg/ml) and three strains were susceptible to less than 1 μg/ml of metronidazole. All lactobacilli were resistant to metronidazole (> 256 μg/ml) but susceptible to clindamycin (0.023 – 0.125 μg/ml).ConclusionClindamycin 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.

Bacteremic Infection with Pantoea ananatis

ABSTRACT 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.

Comparison of Five Genotypic Techniques for Identification of Optochin-Resistant Pneumococcus-Like Isolates

ABSTRACT Three PCR techniques (amplification of the psaA, ply, and lytA genes) and a commercial kit (AccuProbe [GenProbe, San Diego, Calif.], based on hybridization with the 16S rRNA gene), all four of which claimed to be specific for Streptococcus pneumoniae, were used to identify 49 alpha-hemolytic streptococcal isolates suspected of being pneumococci. The definite phenotypic identification of these organisms as S. pneumoniae was difficult when optochin susceptibility and the presence of a capsule were taken as markers. Furthermore, RsaI digestion of the amplified 16S rRNA gene was applied. All 49 strains were optochin resistant. Eleven of these were encapsulated and were identified as pneumococci by all tests. Twenty of the 38 unencapsulated strains were unambiguously identified as nonpneumococci by all tests. The identities of another 18 unencapsulated strains remained inconclusive due to highly variable reactions for all phenotypic and genotypic techniques applied. The AccuProbe test was positive for seven strains for which the results of the other tests were inconclusive. RsaI restriction of the amplified 16S rRNA gene confirmed the AccuProbe result for all strains, while the result of the psaA-specific PCR was in concordance with encapsulation for all strains. The results presented here indicate that identification problems continue to exist for some strains, despite the application of genotypic and phenotypic tests in combination. We found the psaA-specific PCR to be the genotypic technique best suited for the identification of genuine pneumococci and optochin-resistant pneumococci.