Resistance of streptococci to macrolide antibiotics is caused by target-site modification or drug efflux. The phenotypic expression of target-site modification can be inducible or constitutive. The prevalence of the three phenotypes among Belgian erythromycin-resistant Group A streptococci (GAS) and Streptococcus pneumoniae isolates was surveyed, their MICs for seven antibiotics were determined and the clonality of the isolates was explored. Of the 2014 GAS isolates tested 131(6.5%) were erythromycin resistant (MIC > 1 mg/L): 110 (84.0%) showed the M-resistance phenotype whereas the remaining 21 strains (16.0%) were constitutively resistant. No inducibly resistant strains were detected. Of 100 S. pneumoniae isolates, 33 were erythromycin resistant (MIC > 1 mg/L). In contrast to the GAS isolates, only 9.1% of the 33 erythromycin-resistant S. pneumoniae isolates showed the M-resistance phenotype. The presence of mefA/E and ermB genes in the M-resistant and constitutively and inducibly resistant strains, respectively, was confirmed by PCR analysis. Genomic analysis based on pulsed-field gel electrophoresis (PFGE) using the restriction enzyme SfiI, revealed 54 different PFGE patterns among the 131 erythromycin-resistant GAS isolates, of which an M6 clone represented 16.0% of the strains; all other clones, exhibiting different M-types, represented <7% of the strains. The S. pneumoniae isolates also appeared to be polyclonally based, as determined by arbitrarily primed PCR. The macrolides miocamycin and rovamycin, the lincosamide clindamycin and the ketolide HMR 3647 showed excellent activity against the M-resistant GAS and S. pneumoniae strains.

Thirty-two strains originally identified as Lactobacillus acidophilus and L. gasseri were screened for their taxonomic homogeneity by SDS-PAGE of whole-cell proteins. After numerical comparison of the resulting protein electrophoretic fingerprints, two well-delineated clusters were detected. The majority of the strains grouped in one electrophoretic cluster, which contained the type strain of L. acidophilus and corresponds to DNA group A1 of Johnson, J. L., Phelps, C. F., Cummins, C. S., London, J. & Gasser, F. (1980; Znternational Journalof Systematic Bacteriology 30, 53-68). Another cluster corresponded to DNA group B. It contained two subclusters, which agreed perfectly with DNA subgroups B l (L. gasseri) and B2 (A. johnsonii), respectively. The 23s rRNA genes were partially sequenced and 23s-rRNA-targeted oligonucleotide probes were designed for identification of DNA groups Al, B1 and B2. Probe Lbg reacted with all strains of electrophoretic cluster B1 (L. gasseri), probe Lbj hybridized with strains of cluster B2 (L. johnsonii) and probe Lba with strains of cluster A1 (authentic L. acidophilus). The probes were successfully used for the identification of strains belonging to the respective species. The phylogenetic relationship of a representative of L. johnsonii was determined by comparative sequence analysis of the 16s rRNA genes. It is very closely related to L. gasseri.

The increasing problems encountered with enterococcal nosocomial infections and the intrinsic and acquired resistance of the enterococci to different antimicrobial compounds highlight the need for a rapid identification technique. Enterococcus fueculis is readily identified by biochemical tests, but species differentiation within the Enterococcus faecium and Enterococcus gullinurum species groups is less well established. In the present study, 66 strains representing the most prevalent human enterococci were used to develop a PCR-based species-specific identification protocol. Whole-cell protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used as a reference method for species identification. In addition, the genomic SmuI macro-restriction fragment distribution of all of the strains was examined by pulsed-field gel electrophoresis (PFGE). Oligonucleotide D11344-primed PCR was as discriminative as whole-cell protein analysis and resulted in more easily interpreted band patterns. This PCR-based technique allowed identification of clinical isolates by visual examination of the DNA profiles obtained. The inability of both methods to discriminate between Enterococcus cusseliflavus and Enterococcus fluvescens brought into question the species status of E. fluvescens. PFGE did not result in species-discriminative DNA bands or band patterns, but proved to be superior for interpretation of interstrain relationships.The incidence of nosocomial enterococcal infections has risen to an alarming extent (12). Ample examples of enterococcal endocarditis, bacteremia, urinary tract infection, and neonatal sepsis have been reported (33). Enterococcus faecium and Enterococcus faecalis are part of the intestinal and fecal flora of humans and animals (7-9, 25), yet they are notorious organisms in human and veterinary medicine for their inherent and acquired resistance to multiple antibiotics (2, 17).Because of naturally occurring differences in susceptibility, in some cases species level identification is very important in determining the appropriate antibiotic therapy (12, 25), while strain-specific characterization of the bacteria has great value for epidemiologic surveillance (25). However, the phenotypic schemes currently used to identify enterococcal species fail to distinguish several species, especially species in the Enterococcus gallinarum and E. faecium species groups (6). Alternative identification protocols based on penicillin-binding protein profiles (45), bacteriolytic pattern analysis (29), and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins (35) have been developed. The development of new methods involving various DNA-based typing techniques has opened new perspectives for identification of clinical isolates on both the strain level and the species level. In the present study we evaluated two of these DNAbased typing techniques, namely, PCR-based DNA typing in which random or repetitive sequences are used as primers and pulsed-field gel electr...

The taxonomic positions of five enteroadherent bacterial pig isolates, showing phenotypic characteristics most similar to those of Enterococcus durans and Enterococcus hirae, were investigated in a polyphasic study that included 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determinations, whole-cell protein fingerprinting, D11344-primed PCR typing and an extensive examination of phenotypic properties. The results demonstrated that the organisms represent a new species in the Enterococcus faecium species group, for which the name Enterococcus villorum sp. nov. is proposed. The type strain is LMG 12287 T (l CCM 4887 T ).

Using a set of 33 well-defined extended-spectrum ␤-lactamase (ESBL)-producing strains of Escherichia coli and Klebsiella pneumoniae, we compared three screening methods for ESBL detection: (i) a double-disk synergy test, (ii) a three-dimensional test (both the double-disk synergy test and the three-dimensional test were performed with ceftriaxone, ceftazidime, aztreonam, and cefepime), and (iii) the Etest ESBL screen (AB Biodisk, Solna, Sweden), based on the recognition of a reduction in the ceftazidime MIC in the presence of clavulanic acid. In the double-disk test, all four indicator antibiotics scored equally and 31 of the 33 reference strains were recognized. In the three-dimensional test, ceftriaxone was the only satisfactory indicator and 30 ESBL-positive strains were detected by this antibiotic. Both systems produced two false-positive results with cefepime. With the Etest ESBL screen, 15 of 16 TEM-related and 11 of 16 SHV-related ESBL-producing strains scored positive. In 10 cases the clavulanic acid on one end of the strip interfered with the MIC determination for ceftazidime, which was read on the opposite end. This MIC had to be determined with an extra ceftazidimeonly strip. No false-positive results were noted. Eighty-six blood isolates of E. coli and Klebsiella species were screened for ESBL expression by the double-disk and three-dimensional tests, both with ceftriaxone. Six strains with suspicious antibiogram phenotypes also gave positive results by the double-disk test. One E. coli strain remained undetected by the three-dimensional test. Identification of the enzymes suspected of being ESBLs by isoelectric focusing (all strains) and DNA sequencing (1 strain) confirmed the screening test results except for one Klebsiella oxytoca strain, which proved to be a hyperproducer of its chromosomal enzyme and which also had a negative Etest score. The five true ESBL producers were all confirmed by the Etest ESBL screen. Pulsed-field gel electrophoresis proved that the E. coli strains were unrelated, but that two of the three K. pneumoniae strains were closely related.