A study was conducted to evaluate the new VITEK 2 system (bioMérieux) for identification and antibiotic susceptibility testing of gram-positive cocci. Clinical isolates of Staphylococcus aureus (n ‫؍‬ 100), coagulasenegative staphylococci (CNS) (n ‫؍‬ 100), Enterococcus spp. (n ‫؍‬ 89), Streptococcus agalactiae (n ‫؍‬ 29), and Streptococcus pneumoniae (n ‫؍‬ 66) were examined with the ID-GPC identification card and with the AST-P515 (for staphylococci), AST-P516 (for enterococci and S. agalactiae) and AST-P506 (for pneumococci) susceptibility cards. The identification comparison methods were the API Staph for staphylococci and the API 20 Strep for streptococci and enterococci; for antimicrobial susceptibility testing, the agar dilution method according to the procedure of the National Committee for Clinical Laboratory Standards (NCCLS) was used. The VITEK 2 system correctly identified to the species level (only one choice or after simple supplementary tests) 99% of S. aureus, 96.5% of S. agalactiae, 96.9% of S. pneumoniae, 92.7% of Enterococcus faecalis, 91.3% of Staphylococcus haemolyticus, and 88% of Staphylococcus epidermidis but was least able to identify Enterococcus faecium (71.4% correct). More than 90% of gram-positive cocci were identified within 3 h. According to the NCCLS breakpoints, antimicrobial susceptibility testing with the VITEK 2 system gave 96% correct category agreement, 0.82% very major errors, 0.17% major errors, and 2.7% minor errors. Antimicrobial susceptibility testing showed category agreement from 94 to 100% for S. aureus, from 90 to 100% for CNS, from 91 to 100% for enterococci, from 96 to 100% for S. agalactiae, and from 91 to 100% for S. pneumoniae. Microorganism-antibiotic combinations that gave very major errors were CNS-erythromycin, CNS-oxacillin, enterococci-teicoplanin, and enterococci-high-concentration gentamicin. Major errors were observed for CNS-oxacillin and S. agalactiaetetracycline combinations. In conclusion the results of this study indicate that the VITEK 2 system represents an accurate and acceptable means for performing identification and antibiotic susceptibility tests with medically relevant gram-positive cocci.Automation in clinical microbiology is still in a very early stage of development compared with the level of automation that has been achieved in clinical chemistry, hematology, and immunology laboratories.In the last 20 years, a variety of automated systems for the identification and antimicrobial susceptibility testing (AST) of microorganisms has been developed based on automated interpretation of the results of biochemical tests or using microdilution trays following overnight incubation and photometric determination of growth (6,18,20,21). Advances in technology that may provide rapid bacterial identification and AST are now recognized as having both clinical and financial benefits (2).The VITEK system originated in the 1970s as an automated system for identification and AST and has evolved today into the VITEK 2 system, which automatically performs ...

A total of 121 reference and clinical strains of both slowly and rapidly growing mycobacteria belonging to 54 species were studied for restriction fragment length polymorphism of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein. Restriction digests were separated by 10% polyacrylamide gel electrophoresis (PAGE). By including a size standard in each sample, the restriction fragment profile was calculated using a computer-aided comparison program. An algorithm describing these 54 species (including 22 species not previously described) is proposed. We found that this assay based on 10% PAGE provided a more precise estimate than that based on agarose gel electrophoresis of the real size of restriction fragments as deduced from the sequence analysis and allowed identification of mycobacteria whose PCR-restriction fragment length polymorphism analysis patterns were unequivocally identified by fragments shorter than 60 bp.Mycobacteria other than Mycobacterium tuberculosis (MOTT) are increasingly recognized as causing human infections (31). Conventional biochemical methods and phenotypic tests for species differentiation are laborious and time-consuming and frequently require specialized testing that is beyond the capacity of clinical laboratories. Genotypic methods for the identification of mycobacteria have been developed in recent years (3,10,23,29). These molecular methods are gaining increasing importance because they yield rapid and, in most cases, unequivocal results.In 1992 Plikaytis et al. (15) developed a method for differentiating among slowly growing Mycobacterium species by PCR and restriction fragment length polymorphism analysis (PRA). A similar approach was used by Telenti et al. (27) for rapid identification of mycobacteria to species level based on evaluation of the gene coding for the 65-kDa heat shock protein (22) by PCR and restriction enzyme analysis. Subsequently, this approach was used for the taxonomic separation of rapidly growing mycobacteria (18, 24), for routine identification of mycobacteria (1,4,7,9,11,12,23,26), and for identifying Mycobacterium leprae (16,25) and Mycobacterium kansasii subspecies (17).In all these studies the algorithm describing the mycobacteria species is based on the use of two restriction enzymes (BstEII and HaeIII) and separation of the restriction fragments on an agarose gel. PRA patterns are then interpreted by converting the running distance in electrophoresis to apparent molecular size (in base pairs). Difficulties in PRA interpretation may stem from similarities in a number of band sizes that are critical for discrimination of species and are not sufficiently resolved by agarose-based gel electrophoresis.In view of the application of PRA-based identification of mycobacteria in our diagnostic laboratory and its application to an increasing number of different species, we conducted the present study in order to propose an algorithm based on 10% polyacrylamide gel electrophoresis (PAGE) of restriction digests to improve the resolu...

A rapid method for the detection of methicillin resistance in staphylococci was developed. The method was based on the polymerase chain reaction (PCR) and primers that targeted the internal region of the coding frame of the mec gene. The amplification reaction was carried out with crude cell lysates as a source of target DNA and provided data in less than 5 h. Seventy-four isolates of coagulase-negative staphylococci were tested by PCR, DNA hybridization with a probe derived from the mec gene, and an agar dilution susceptibility assay.PCR results showed a 100% correlation with the susceptibility assay carried out with high inocula (108 CFU) and incubation at 32°C for 48 h. PCR was more sensitive and specific than DNA hybridization in detecting methicillin resistance in coagulase-negative staphylococci. The former technique identified the mec gene in all the strains which were phenotypically resistant but which did not hybridize with the probe. Identification of methicillin-resistant strains by PCR offers a very specific, sensitive, and rapid alternative to traditional susceptibility tests and DNA hybridization as a guide for the treatment of infections caused by staphylococci.Several groups of investigators have described DNA probes that can be successfully used to detect methicillinresistant staphylococci (1,5,12).These probes are specific fragments generated by restriction endonuclease digestion (1, 5) or are polymerase chain reaction (PCR)-generated fragments (12) of the gene which, in both coagulase-positive and -negative staphylococci, encodes for the synthesis of a new penicillin-binding protein (PBP 2a) which mediates clinically relevant resistance to all beta-lactams (2,3,7,8,14,18,21,23).Hybridization of the probes with the chromosomal DNA target, immobilized on a nitrocellulose filter, has shown that the presence of the PBP 2a gene in both Staphylococcus aureus and coagulase-negative staphylococci correlates in almost every instance with the phenotypic expression of methicillin resistance detected by in vitro susceptibility tests (1,12).These studies have provided the basis for introducing tests for genetic identification of methicillin resistance in clinical microbiology laboratories. These tests may help to overcome the numerous problems posed by the well-known heterogeneous expression of methicillin resistance, which makes routine susceptibility tests unreliable in detecting this resistance (3,4,15,17,19 Lysis procedure. A number of colonies obtained after overnight growth on tryptic soy agar (TSA; Difco) or mannitol salt agar (MSA; Difco) were suspended in 1 ml of water to a final density of 1 unit at an optical density at 600 nm, which corresponded to 109 CFU/ml. The suspension was centrifuged, and the pellet was resuspended in 100 p1l of 50 mM Tris (pH 8) containing 50 mM NaCl, 25% sucrose, 10 ,ug of lysostaphin (Sigma Chemical Co., St. Louis, Mo.), and 100 pug of lysozyme (Sigma). After incubation at 37°C for 60

Five ampicillin-resistant clinical isolates of Enterococcus faecium were analyzed for a correlation between overproduction of the low-affinity penicillin-binding protein (PBP 5) and the level of ampicillin resistance.Comparison was made with one susceptible clinical isolate and its ampicillin-resistant derivative obtained in the laboratory by selection with increasing concentrations of penicillin. Overproduction of the low-affinity PBP relative to the susceptible isolate was noted in moderately resistant strains (MIC, 32 ,ug/ml) but not in highly resistant strains (MIC, 128 ,ug/ml). Polyclonal antibodies specifically reacting with the low-affinity PBP of Enterococcus hirae, Enterococcusfaecalis, and Enterococcusfaecium (M. Ligozzi, M. Aldegheri, S. C. Predari, and R. Fontana, FEMS Microbiol. Lett. 83:335-340, 1991) were used to determine the amount of this PBP in the E. faecium isolates. In all strains, the antibody preparation reacted with a membrane protein of the same molecular mass as PBP 5. The amount of this protein was very small in the susceptible strain but large in all of the resistant strains. These results suggest that the highly resistant strains also overproduced the low-affinity PBP, which, compared with PBP 5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability.Despite several reports of enterococcal strains producing a P-lactamase (16,17,19), the main pathway of penicillin resistance development in these microorganisms still remains modification of penicillin-binding proteins (PBPs), in particular, overproduction of a low-affinity PBP which is a normal component of the PBP pattern of these bacteria (9, 10). The mechanism by which overproduction of PBP 5 confers resistance has been ascribed to the ability of this PBP to substitute the functions of the other PBPs (3,4,7,8,10). This proposal stems from the finding that ATCC 9790 mutants overproducing the low-affinity PBP grow normally in the presence of penicillin concentrations which saturate all PBPs but not PBP 5, which, in these conditions, is apparently the only active PBP. In addition, it has been shown that, in ATCC 9790 derivatives which carry a temperature-sensitive defect in the essential PBPs 1, 2, and 3 associated with a temperature-* Corresponding author. sensitive cell division, a second mutation causing overproduction of PBP 5 allows normal growth at the nonpermissive temperature (4).The recent cloning of the E. hirae PBP 5 gene (pbp5) (5) has provided insights into the genetic mechanism of penicillin resistance development (14). It has been shown that, in resistant mutants, PBP 5 overproduction is associated with a deletion in a genetic element, located 1 kb upstream of pbp5, which negatively controls PBP 5 synthesis (14). Hypersusceptibility to penicillin is associated with a point mutation inpbp5, which causes premature termination of translation. E. hirae pbpS does not hybridize with DNA of other enterococcal species, suggesting that the genes which code for low-affinity PBPs are not str...

Enterococcus hirae ATCC 9790 produces a penicillin-binding protein (PBP5) of low penicillin affinity which under certain conditions can take over the functions of all the other PBPs. The 7.1-kb EcoRI fragment containing the pbp5 gene of this strain and of two mutants, of which one (E. hirae R40) overproduces PBP5 and the other (E. hirae Revl4) does not produce PBP5, was cloned in pUC18 and sequenced. In (4,7,12,23,30). These PBPs share several properties: they can be produced by the cells in large amounts, have similar molecular masses (about 75,000 Da), have an unusually low affinity for beta-lactam antibiotics, and play an important role in the mechanism of resistance to these antibiotics in enterococci and staphylococci.In enterococci, the low-affinity PBPs either are components of the normal PBP pattern or are generated by mutations in genes of the normal PBPs (1,12,21,32,33), whereas in staphylococci their synthesis is due to the acquisition of an additional gene (mec) which is practically identical in Staphylococcus aureus and several coagulase-negative species (22,27,29).In contrast to the evident and well-documented role of the low-affinity PBPs in the mechanism of resistance of grampositive cocci to beta-lactam antibiotics, very little is known about the role these proteins play in cell physiology. The most accepted hypothesis is that these proteins are multifunctional PBPs which are not essential for cell growth, at least under laboratory conditions, and can synthesize peptidoglycans under conditions which impair the activity of the other PBPs (4-6, 12, 13). Such an alternative system of peptidoglycan synthesis would represent an interesting and novel aspect of bacterial physiology.Previous studies of the low-affinity PBP of Enterococcus hirae ATCC 9790 (PBP5) based on mutants with a cell division defect and mutants with altered synthesis (either overproduction or lack) of PBP5 have elucidated certain aspects of the role of low-affinity PBPs in cell physiology (5, * Corresponding author. 6, 12, 13). Recently, the E. hirae PBP5-encoding gene (pbp5) has been cloned and sequenced (9), thus making possible investigations into the function of this PBP by a genetic approach.In this study, we sequenced the 7.1-kb EcoRI fragment, containing thepbp5 gene, cloned from chromosomal DNA of the wild-type E. hirae ATCC 9790, the PBP5 overproducer mutant E. hirae R40, and the PBP5 nonproducer mutant E. hirae Revl4. We found that the overproducer phenotype was associated with an 87-bp deletion in a genetic element, which we calledpsr (PBP5 synthesis repressor), located 1 kb upstream of the pbp5 gene, whereas the lack of PBP5 synthesis in E. hirae Revl4 was found to be caused by a nucleotide substitution in the coding frame of the protein which converted the 42nd codon, TCA, to the stop codon, TAA. MATERIALS AND METHODSBacterial strains and plasmids. The relevant properties of E. hirae strains and the recombinant plasmids constructed in this study are listed in Table 1. E. hirae strains were grown in brain heart infu...