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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...