Jean Freney

Staphylococcus lugdunensis sp. nov. and Staphylococcus schleiferi sp. nov., Two Species from Human Clinical Specimens

Deoxyribonucleic acid relatedness studies (S1 nuclease method) showed that 23 unidentified Staphylococcus strains form two homogeneous genomic species related 1 to 9% to 24 type strains representing known Staphylococcus species. These new species were named Staphylococcus lugdunensis and Staphylococcus schleiferi. Strains of S. lugdunensis were susceptible to novobiocin, produced a fibrinogen affinity factor, and failed to produce coagulase, heat-stable nuclease, and staphylokinase. S. lugdunensis strains differed from S. hominis (the phenotypically closest species) by production of ornithine decarboxylase and the fibrinogen affinity factor. The guanine-plus-cytosine content of the deoxyribonucleic acid was 32 mol%. The type strain is N860297 (= ATCC 43809). Strains of S. schleiferi were susceptible to novobiocin, produced a heat-stable nuclease and a fibrinogen affinity factor, and failed to produce coagulase and staphylokinase. S. schleiferi strains differed from S. aureus by production of an antigenically different heat-stable nuclease and the lack of pigmentation, free coagulase, protein A, and β-ribitol teichoic acid. The guanine-plus-cytosine content of the deoxyribonucleic acid was 37 mol%. The type strain is N850274 (= ATCC 43808).

Identification of Staphylococcus species by 16S-23S rDNA intergenic spacer PCR analysis

To investigate whether 16S-23S rDNA (rDNA) spacer region length polymorphisms are suitable for the identification of Staphylococcus strains, the 16S-23S rDNA intergenic spacer region lengths of 221 strains belonging to 31 species were studied by using a PCR-based method. Each species presented a specific 16S-23S pattern made of 1-8 fragments ranging from 104-771 bp, with the exception of the species Staphylococcus warnei, Staphylococcus caprae and Staphylococcus piscifermentans, which presented larger or smaller fragments. Very few species showed more than one pattern, Staphylococcus saprophyticus subsp. saprophyticus and Staphylococcus aureus being the most heterogeneous species (five different patterns for eight strains). Five clinical strains that could not be identified at the species level by phenotypical tests were finally identified using this method. Discrimination between some species that showed close patterns (Staphylococcus aureus/Staphylococcus chromogenes/Staphylococcus equorum, Staphylococcus aureus/staphylococcus intermedius, Staphylococcus delphini/Staphylococcus felis, Staphylococcus gallinarum, Staphylococcus delphini/Staphylococcus felis, Staphylococcus vitulus/Staphylococcus auricularis) was further achieved after Dral digestion of the PCR products. Although it does not allow discrimination of subspecies, the use of 16S-23S spacer region length data determined by PCR-mediated amplification is suitable for the identification of the 31 Staphylococcus species tested in this study. The method is rapid, easy and may be a useful tool for the identification of Staphylococcus species in the clinical microbiology laboratory.

Clinical impact of rapid oxacillin susceptibility testing using a PCR assay in Staphylococcus aureus bactaeremia

OBJECTIVES The aim of this work was to establish the clinical impact of rapid oxacillin susceptibility testing in nosocomial Staphylococcus aureus bacteraemia. METHODS This study was performed in 145 critically ill patients infected by S. aureus. Patients were randomly assigned to one of two groups: patients for whom susceptibility testing was performed using a rapid same day multiplex PCR assay for detection of the staphylococcal mecA (mean delay of response: 6 h) and those for whom testing was accomplished using traditional overnight techniques (21 h). RESULTS The results of this study showed no significant difference between the two groups in terms of age, Simplified Acute Physiologic Score, severity of infection, severity of underlying disease and clinical outcome (control vs. PCR): unfavourable outcome of infection, 12.32 vs. 12.5%; 95% CI for the difference = -11.49 to 11.09 (P = 0.975); unfavourable general outcome, 16.43 vs. 20.83%; 95% CI for the difference = -17.35 to 8.50 (P = 0.497). For the oxacillin-susceptible S. aureus bactaeraemia, results were: unfavourable outcome of infection = 13.04 vs. 11.11%; 95% CI for the difference = -11.38 to 16.18 (P = 0.767); unfavourable general outcome = 13.04 vs. 20.37%; 95% CI for the difference = -22.12 to 8.07 (P = 0.331). CONCLUSION This study seemed to demonstrate that rapid oxacillin susceptibility testing using a PCR assay did not have a major impact on the care and outcome of patients with S. aureus bactaeremia.

Improvement of the identification of staphylococci isolated from bovine mammary infections using molecular methods

Fifty-six Staphylococcus strains isolated from cases of bovine mammary infections were identified by using phenotypic and genotypic methods. Twenty-eight strains (50%) were identified at the species level according to their phenotypic characteristics, whereas the remaining 28 strains presented atypical or unreliable profiles. A combination of phenotypic and genotypic methods allowed the 56 strains studied to be classified. Internal transcribed spacer-polymerase chain reaction (ITS-PCR) based on the polymorphism of the 16S-23S rDNA spacer region appeared as a rapid and reliable method for the classification of bovine staphylococcal isolates at the species and subspecies levels.

Susceptibilities to antibiotics and antiseptics of new species of the family Enterobacteriaceae.

One hundred and sixty-nine strains of new species of the family Enterobacteriaceae, isolated mainly from the environment, were tested to determine their susceptibilities to 13 antibiotics and 4 antiseptics or disinfectants. All the species were susceptible to aminoglycosides, doxycycline, and trimethoprim but were resistant to chloramphenicol. Susceptibility to beta-lactams varied more among the strains. However, all the strains were cefotaxime susceptible, apart from some Buttiauxella agrestis strains for which MICs were greater than 256 micrograms/ml. The antiseptic MBCs were similar to those published elsewhere for species of the Enterobacteriaceae of clinical origin. No resistance to chlorhexidine was observed. On the other hand, the environmental strains presented a greater resistance to active chlorine than did the reference strains.

Difficulties in identifying Klebsiella strains of clinical origin.

Two hundred and four strains of Gram-negative bacteria of clinical origin, initially identified as Klebsiella using the API 20 E system, and 10 reference strains were further analysed with the API 20 EC test system and the API 50 CH, API 50 AO, API 50 AA assimilation systems. Four clusters corresponding to the species Klebsiella pneumoniae subsp. pneumoniae, K. oxytoca, K. planticola, and K. terrigena were formed after numerical analysis of 155 selected tests and the 26 most discriminating tests were determined. A comparison was made between conventional identification using the API 20 E system and the results of the numerical analysis. The conventional method resulted in incorrect identification of 13% of the strains tested, especially for the new species: K. planticola and K. terrigena. After numerical analysis, 17 out of 204 strains (8.3%) of clinical origin were identified as K. planticola. Only 1 strain of clinical origin was identified as K. terrigena, and 1 strain as K. ornithinolytica.

In-Vitro Study of Bacterial Adherence to Different Types of Intraocular Lenses

ABSTRACT The aim of this study was to determine the adherence of Staphylococcus epidermidis to intraocular lenses made of five different biomaterials: polymethylmethacrylate (PMMA), heparinized PMMA, silicone, hydrophilic acrylic, and hydrogel. The extent of bacterial binding was measured by counting. The results were compared using a one-factor variance analysis. Adherence was weakest on hydrogel and strongest on the silicone polymer. Bacterial adherence to the implant surface must therefore depend on the hydrophobicity or hydrophilicity of the biomaterial.

Legionella taurinensis sp. nov., a new species antigenically similar to Legionella spiritensis.

A group of 42 Legionella-like organisms reacting specifically with Legionella spiritensis serogroup 1 antisera were collected throughout Europe by the Centre National de Référence (French National Reference Centre) for Legionella. This group of isolates differed somewhat from L. spiritensis in terms of biochemical reactions, ubiquinone content and protein profile. The latter two analyses revealed that one of these L. spiritensis-like isolates, Turin I no. 1T, was highly related, but not identical to any of the red autofluorescent species of Legionella. In fact, this strain was the first of these particular isolates recognized to emit a red autofluorescence when exposed to UV light. Profile analysis of randomly amplified polymorphic DNA established that the red autofluorescent L. spiritensis-like isolates constituted a homogeneous group distinct from Legionella rubrilucens and Legionella erythra. DNA-DNA hybridization studies involving the use of S1 nuclease confirmed that the indicated group of isolates are a new species of Legionella, for which the name Legionella taurinensis is proposed with strain Turin I no. 1T (deposited as ATCC 700508T) as the type strain.

Laboratory diagnosis of oxacillin resistance in Staphylococcus aureus by a multiplex-polymerase chain reaction assay

A polymerase chain reaction (PCR) test was developed in which the mecA gene responsible for the intrinsic resistance to oxacillin in Staphylococcus aureus and the gyrA gene, always present in this species, were amplified in one operation. Among the 468 clinical isolates tested, the results obtained for 454 of the isolates (97%) were consistent with those of MIC determination. Discrepant results were noted for strains with low-level oxacillin resistance (MICs, 4-8 micrograms/ml) and mecA gene negative. For these strains, susceptibility to oxacillin was restored in the presence of a beta-lactamase inhibitor, which suggested a resistance by penicillinase hyperproduction. In contrast, all of the high-level resistant strains (MICs, > 8 micrograms/ml) carried the mecA gene. The presence of this gene has frequently been associated with resistance to gentamicin, tetracycline, erythromycin, lincomycin, and pefloxacin. The PCR assay described in this study can be accomplished with ease and total confidence in the clinical microbiologic laboratory for a rapid and effective establishment of antistaphylococcal chemotherapy.

Genomic Diversity of Several Corynebacterium Species Identified by Amplification of the 16s-23s rRNA Gene Spacer Regions

In order to investigate whether 16S–23S ribosomal DNA (rDNA) spacer region length polymorphisms are suitable identification of Corynebacterium strains at the species level, the 16S–23S rDNA intergenic spacer region strains belonging to 11 Corynebacterium species were studied by a PCR-based method. The lengths 16S–23S rDNA spacer regions varied from 394 to 585 bp, fragment lengths which are similar to those described for other genera. A single PCR profile was obtained for each of the following species: Corynebacterium renale, Corynebacterium urealyticum, Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium pseudodiphtheriticum, and Corynebacterium kutscheri. In contrast, two and three PCR patterns were detected for Corynebacterium minutissimum, Corynebacterium striatum, Corynebacterium amycolatum, and Corynebacterium jeikeium, suggesting that genomic heterogeneity occurs in these four species. The 16S–23S rDNA spacer region length polymorphisms allowed us to discriminate among C. minutissimum, C. striatum, and C. amycolatum, three species that are frequently isolated and misidentified in clinical laboratories. Type strain Corynebacterium xerosis ATCC 373, which exhibited a PCR pattern similar to that of C. amycolatum strains classified in PCR group I, could nevertheless be discriminated from PCR group II (C. amycolatum) strains, as Well as minutissimum and C. striatum strains. Type strain C. xerosis ATCC 373 and C. amycolatum strains classified PCR group I could not be distinguished from strains belonging to C. diphtheriae, C. ulcerans, and C. pseudodiphtheriticum. The lipophilic species C. urealyticum and C. jeikeium, which are frequently encountered in clinical specimens, could be clearly distinguished from each other by this method. The use of 16S–23S spacer region length data determined by PCR-mediated amplification is suitable for identification of several Corynebacterium species. This rapid and easy method may be a useful identification tool for clinical microbiologists.