AFLP typing of Staphylococcus epidermidis in multiple sequential blood cultures.

AFLP is a novel high-resolution PCR-based DNA fingerprinting method generating complex banding patterns that can be used for comparative analysis. In the present study, the applicability of AFLP in fingerprinting Staphylococcus epidermidis isolates was investigated. The criteria considered were stability of patterns, reproducibility, discriminatory capacity and consistency with epidemiological context. Repeated testing of strains and investigation of subcultures showed that AFLP patterns were reproducible and stable with an intrastrain similarity of S > or = 94% as determined by analysis of digitized patterns. Fifteen unrelated strains were heterogeneous, with a level ranging from 78-93%. The applicability of AFLP in epidemiological studies of S. epidermidis was tested on 11 sets of four blood isolates each, from 11 patients with suspected septicaemia. Nine sets had indistinguishable or highly similar AFLP patterns for each isolate per set, while two sets had heterogeneous patterns. These results show that AFLP has high discriminatory power for strain identification in S. epidermidis.

Identification of multiresistant Staphylococcus epidermidis in neonates of a secondary care hospital using pulsed field gel electrophoresis and quantitative antibiogram typing.

AIMS: To determine the diversity of types of Staphylococcus epidermidis in a neonatal care unit of a secondary care hospital in the Netherlands. METHODS: In a prospective study, specimens from nose, ear, axilla, umbilicus, and groin were taken from patients twice a week during a period of up to two weeks. All isolates were typed by both pulsed field gel electrophoresis (PFGE) and antibiogram analysis. RESULTS: Fifty three S epidermidis isolates from 15 of 24 patients were obtained in one to four surveys. Fourteen isolates from six patients had a common PFGE pattern and were of one multiresistant antibiogram type. The remaining 39 isolates were allocated to 24 sporadic PFGE types and were more susceptible to antibiotics. Colonisation with the multiresistant strain correlated with a long period of stay and with the use of specific antibiotics. The multiresistant isolates were related closely to isolates of S epidermidis found in a recent study in a teaching hospital in the vicinity of the secondary care hospital. CONCLUSION: Repeated sampling and the use of two typing methods allowed the identification of two closely related multiresistant S epidermidis strains in two hospitals in the same area.

Biofilm production by Staphylococcus epidermidis isolates associated with catheter related bacteremia.

The mean biofilm production of 22 Staphylococcus epidermidis isolates associated with catheter related bacteremia was significantly higher than that of 32 nose isolates from healthy individuals. This difference was due to seven catheter related isolates. These findings do not show a clear association between biofilm production and virulence.

Susceptibility of Streptococcus pyogenes to azithromycin, clarithromycin, erythromycin and roxithromycin in vitro.

The susceptibility of 180 clinical isolates of Streptococcus pyrogenes from six regions of The Netherlands to the macrolide antibiotics azithromycin, clarithromycin, erythromycin and roxithromycin was analysed. The results of a microbroth MIC method, the E-test method and a disk diffusion assay were compared, and the MBC determined. In addition, the susceptibility to erythromycin of 436 clinical isolates of S. pyogenes from the Leiden region was determined. The microbroth MIC90s of azithromycin, clarithromycin, erythromycin and roxithromycin for group A streptococci were < or = 0.5 mg/L. Erythromycin had the lowest MIC90 (0.09 mg/L). The MIC data obtained with the E-test method suggested that clarithromycin and erythromycin had slightly higher anti-streptococcal activity than azithromycin and roxithromycin in vitro. MICs obtained with the E-test were lower than those found with the microbroth method. Only minor discrepancies were observed among the three methods. The MBC50 for both clarithromycin and erythromycin was 0.75 mg/L and 5.0 mg/L for azithromycin and roxithromycin. None of the 180 strains and two of the collection of 436 strains (0.5%) were resistant to erythromycin and the other macrolides tested; MICs ranged from 1 to 16 mg/L. The erythromycin-resistant strains showed an inducible type of macrolide-lincosamide-streptogramin B (MLS) resistance.

Diagnosis of conjunctivitis in primary care: comparison of two different culture procedures.

BACKGROUND: In general practice, infectious conjunctivitis is a common and mostly (64%) self-limiting disorder. In case of an aberrant course or severe symptoms, a general practitioner may take a culture. Direct inoculation is considered the reference standard, but usually a swab is sent to a laboratory. OBJECTIVES: To compare the diagnostic performance of the swab, transported by surface mail with direct inoculation. METHODS: 19 general practitioners took two samples of the conjunctiva from 88 patients with symptoms suggestive of infectious conjunctivitis by rolling a cotton swab across the conjunctiva of the lower fornix. One swab was used to inoculate three agar plates directly, while the other was sent in a Stuart medium to the laboratory and inoculated at the time of arrival. The numbers of positive cultures of both methods were compared. RESULTS: A pathogen was found in 31 of 88 samples (35% (95% CI 26 to 46)). Surprisingly, the number of positive cultures was higher for the Stuart medium (27/88) than for direct inoculation (23/88). The difference was 4.5% (90% CI 0 to 12, p = 0.388; one-sided McNemar test for paired proportions). In five of the 19 samples that were positive in both tests, the cultured pathogens were different. CONCLUSIONS: The Stuart medium detected more bacteria than direct inoculation. The lower 90% CI, testing non-inferiority at p = 0.05, indicates that it is unlikely that the Stuart medium misses any positive cultures compared with direct inoculation.

Changing susceptibilities of coagulase-negative staphylococci to teicoplanin in a teaching hospital.

The susceptibility of two collections of coagulase-negative staphylococci (CNS) isolated from clinical specimens for teicoplanin and vancomycin were compared. They comprised 91 and 101 isolates, collected in 1985 and 1994 respectively, from different departments of a teaching hospital. MICs of vancomycin and teicoplanin were determined by a modified Etest method. Additionally, a disc diffusion test was performed for teicoplanin. All isolates were susceptible to vancomycin (MIC < or = 4 mg/L). Two of the 91 isolates collected in 1985 were intermediate to teicoplanin (MIC between 8 and 32 mg/L), whereas in 1994 the number of intermediate isolates was 20 out of 101 (P < 0.01). The correlation between MICs, as determined by the modified Etest assay, and disc diffusion zones was poor (r = -0.35). Results show that resistance to teicoplanin in CNS has increased in the study hospital over a period of 9 years. This increase is likely to be correlated with the introduction of teicoplanin. Furthermore, a disc diffusion method does not appear to be the first method of choice for detection of strains of CNS with diminished susceptibility to teicoplanin.

Performance of phenotypic and genotypic methods to determine the clinical relevance of serial blood isolates of staphylococcus epidermidis in patients with septicemia.

Five typing methods, including biotyping (API ID32; BioMérieux, Marcy l'Etoile, France), quantitative antibiogram typing based on actual zone sizes, plasmid typing, randomly amplified polymorphic DNA (RAPD) analysis (with primer M13 and primer set ERIC-2-1026), and pulsed-field gel electrophoresis (PFGE), were compared with a previously performed method of DNA fingerprinting by AFLP (amplified fragment length polymorphism analysis) for their performance in the typing of blood isolates of Staphylococcus epidermidis. Sixteen epidemiologically unrelated strains and 11 sets of four blood culture isolates from 11 patients with septicemia were used. The stabilities and reproducibilities of the patterns, the discriminatory capacities of the methods, and the ability to apply the methods to blood culture isolates were used as performance criteria. All strains tested were typeable by each method, and the patterns were stable and reproducible. The numbers of different types within the collection of 16 epidemiologically different isolates were 5 by biotyping, 14 by antibiogram typing, 4 by plasmid typing, 9 by the RAPD assay (combination of results with primer M13 and primer set ERIC-2-1026), and 16 by PFGE. Within the 11 sets of four blood culture isolates the types found by quantitative antibiogram typing, plasmid typing, and PFGE were unique for each set, whereas by biotyping and RAPD analysis some types were observed in more than one set. The results of biotyping did not correspond with the results of the other methods or the results of AFLP. For 6 of the 11 sets, the results of all methods except those of biotyping corresponded completely. Quantitative antibiogram typing, PFGE, and AFLP proved to be the most accurate of the six typing methods tested.