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In Staphylococcus aureus, mecA and femA are the genetic determinants of methicillin resistance. By using a multiplex PCR strategy, 310-and 686-bp regions of the mecA and femA genes, respectively, were coamplified to identify susceptible (lacking mecA) and resistant (mecA ؉) staphylococci and to differentiate S. aureus (femA ؉) from coagulase-negative staphylococci (lacking femA). A third staphylococcal genomic sequence, corresponding to IS431 and spanning 444 bp, was used as a PCR control. One hundred sixty-five staphylococcal strains were tested. All 72 methicillin-resistant strains were found to be mecA ؉ , and 92 of the 93 susceptible isolates lacked mecA. Only one coagulase-negative Staphylococcus isolate carrying the mecA gene was highly susceptible to oxacillin. The femA determinant was a unique feature of S. aureus; it was found in 100% of the S. aureus strains tested but was undetectable in all of the coagulase-negative staphylococci tested. The possibility of directly detecting the mecA and femA genes in blood samples was also investigated. After two amplification steps, a sensitivity of 50 microorganisms per ml of freshly collected spiked blood was achieved. In conclusion, coamplification of mecA and femA determinants proved to be very reliable both for rapid detection of methicillin resistance and differential diagnosis between S. aureus and other staphylococci. This technique, which can be successfully performed with blood samples, could be a useful tool in the diagnosis and treatment monitoring of staphylococcal infections.

Susceptibilities to 11 antimicrobial agents were determined by Etest for 93 Nocardia isolates from clinical specimens and 15 type strains belonging to different Nocardia spp. All isolates were susceptible to trimethoprim-sulphamethoxazole, amikacin and linezolid, but susceptibilities of the various Nocardia spp. to beta-lactams, aminoglycosides, ciprofloxacin and clarithromycin varied markedly. Overall, there was a good correlation between the drug resistance patterns and the species identification established by conventional phenotypic tests and 16S rDNA sequencing. Among the different species encountered, Nocardia farcinica and Nocardia brasiliensis displayed the most multiresistant profiles, with resistance to imipenem occurring mainly among isolates of N. brasiliensis and Nocardia abscessus. The species variability in susceptibility profiles and the numerous recent taxonomic changes means that in-vitro susceptibility tests may be a complementary tool for the identification of Nocardia isolates from human clinical specimens. Further studies on a larger number of species from more diverse geographical sources, including species that are found less commonly among clinical isolates, are required to validate and extend the results.

The intra-and interspecies genetic relationships of 58 strains representing all currently known species of the genus Yersinia were examined by multilocus sequence typing (MLST), using sequence data from 16S RNA, glnA, gyrB, recA, and Y The genus Yersinia is a member of the gamma subdivision of Proteobacteria (60), and it is grouped in the family Enterobacteriaceae, based on biochemical tests and DNA-DNA similarity studies (10). Yersiniae have undergone extensive diversification during the course of their evolution, with one Yersinia species (Yersinia pestis) becoming the deadliest bacterium ever known in human history, and other species (e.g., Y. aldovae) diverging into completely nonpathogenic organisms (46, 55). Extensive research has been conducted to characterize 3 (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) of the 11 currently recognized species of Yersinia; however, the remaining 8 "Y. enterocolitica-like" species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. aldovae) have been only moderately studied because they were not clearly recognized as human pathogens (55). This situation has resulted in a marked paucity of information pertaining to the phylogenetic interrelationships of all 11 species within the genus and to the mechanisms responsible for their serological and genetic divergences.Initial studies examining the relatedness among Yersinia species used serotyping and other phenotypic characteristics, such as biochemical properties (biotyping), susceptibility to antibiotics (13), and phage typing (7, 44). Brenner et al. (11) subsequently introduced DNA hybridization techniques to classify Y. enterocolitica-like species and to determine the genetic relatedness among them. However, the methodology was limited because it did not provide information required to determine evolutionary relationships among yersiniae, and it was prone to yield potentially misleading results influenced by the level of gene acquisition and loss (39). Therefore, several molecular typing methodologies, such as plasmid profile analysis (22, 50), restriction fragment length polymorphism of chromosomal DNA (9), ribotyping (40, 42), sequence analysis of the 16S RNA gene (31), pulsed-field gel electrophoresis (PFGE) (43), and variable-number tandem repeat analysis (2) were subsequently applied for typing of yersiniae during epidemiological investigations and for determining genetic relatedness among Yersinia strains. Most of these approaches have good or excellent discriminatory power (e.g., PFGE was proposed as the "gold standard" for typing of Yersinia strains [34]) and are well suited for short-term epidemiological studies. However, they are less suited for long-term epidemiological studies and for determining evolutionary traits of, and phylogenetic relationships among, various strains or species (18). A relatively recently developed approach called multilocus enzyme electrophoresis (MLEE) addressed some of the shortcomings of the above-mentioned meth...

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The identification of Nocardia species, usually based on biochemical tests together with phenotypic in vitro susceptibility and resistance patterns, is a difficult and lengthy process owing to the slow growth and limited reactivity of these bacteria. In this study, a panel of 153 clinical and reference strains of Nocardia spp., altogether representing 19 different species, were characterized by matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS). As reference methods for species identification, fulllength 16S rRNA gene sequencing and phenotypical biochemical and enzymatic tests were used. In a first step, a complementary homemade reference database was established by the analysis of 110 Nocardia isolates (pretreated with 30 min of boiling and extraction) in the MALDI BioTyper software according to the manufacturer's recommendations for microflex measurement (Bruker Daltonik GmbH, Leipzig, Germany), generating a dendrogram with species-specific cluster patterns. In a second step, the MALDI BioTyper database and the generated database were challenged with 43 blind-coded clinical isolates of Nocardia spp. Following addition of the homemade database in the BioTyper software, MALDI-TOF MS provided reliable identification to the species level for five species of which more than a single isolate was analyzed. Correct identification was achieved for 38 of the 43 isolates (88%), including 34 strains identified to the species level and 4 strains identified to the genus level according to the manufacturer's log score specifications. These data suggest that MALDI-TOF MS has potential for use as a rapid (<1 h) and reliable method for the identification of Nocardia species without any substantial costs for consumables.

The gene encoding the heat-stable enterotoxin (yst) was cloned from the chromosome of Yersinia enterocolitica W1024 (serotype 0:9), and the nucleotide sequence was determined. The yst gene encodes a 71-amino-acid polypeptide. The C-terminal 30 amino acids of the predicted protein exactly correspond to the amino acid sequence of the toxin extracted from culture supernatants (T. Takao, N. Tominaga, and Y. Shimonishi, Biochem. Biophys. Res. Commun. 125:845-851, 1984). The N-terminal 18 amino acids have the properties of a signal sequence. The central 22 residues are removed during or after the secretion process. This organization in three domains (Pre, Pro, and mature Yst) resembles that of the enterotoxin STa of Escherichia coli. The degree of conservation between the E. coli and Y. enterocolitica toxins is much lower in the Pre and the Pro domains than in the mature proteins. The mature toxin of Y. enterocolitica is much larger than that of E. coli, but the active domain appears to be highly conserved. The yst gene of Y. enterocolitica introduced in E. coli K-12 directed the secretion of an active toxin. The cloned yst gene was used as an epidemiological probe among a collection of 174 strains representative of all Yersinia species except Yersinia pestis and numerous Y. enterocolitica subgroups. In Y. enterocolitica, there was a clear-cut difference between pathogenic and nonpathogenic strains: 89 of 89 pathogenic and none of 51 nonpathogenic strains contained yst-homologous DNA, suggesting that Yst is involved in pathogenesis. Among the other Yersinia species, only four strains of Yersinia kristensenii had DNA homologous to yst.