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...

A semiautomated, repetitive-sequence-based PCR (rep-PCR) instrument (DiversiLab system) was evaluated in comparison with pulsed-field gel electrophoresis (PFGE) to investigate an outbreak of Serratia marcescens infections in a neonatal intensive care unit (NICU). A selection of 36 epidemiologically related and 8 epidemiologically unrelated isolates was analyzed. Among the epidemiologically related isolates, PFGE identified five genetically unrelated patterns. Thirty-two isolates from patients and wet nurses showed the same PFGE profile (pattern A). Genetically unrelated PFGE patterns were found in one patient (pattern B), in two wet nurses (patterns C and D), and in an environmental isolate from the NICU (pattern G). Rep-PCR identified seven different patterns, three of which included the 32 isolates of PFGE type A. One or two band differences in isolates of these three types allowed isolates to be categorized as similar and included in a unique cluster. Isolates of different PFGE types were also of unrelated rep-PCR types. All of the epidemiologically unrelated isolates were of different PFGE and rep-PCR types. The level of discrimination exhibited by rep-PCR with the DiversiLab system allowed us to conclude that this method was able to identify genetic similarity in a spatio-temporal cluster of S. marcescens isolates.Concerns regarding the emergence of hospital-acquired infections, increasing antimicrobial resistance, and the increase in morbidity, mortality, and costs associated with these infections drive the need for refinement of molecular approaches to aid in the diagnosis and epidemiological analysis of nosocomial infections.Several methods based on DNA analysis are now available that provide information about the genetic relatedness of isolates of the same species (26). These DNA-based molecular methodologies include pulsed-field gel electrophoresis (PFGE), PCR-based typing methods, and multilocus sequence analysis.

A total of 78 isolates of Pseudomonas aeruginosa grouped according to the phenotype for ceftazidime and imipenem susceptibility/resistance were used to assess the accuracy of the Vitek 2 system in antimicrobial susceptibility testing. Comparisons were made with a MIC gradient test for piperacillin-tazobactam, ceftazidime, aztreonam, imipenem, meropenem, gentamicin, and ciprofloxacin. For the total of 546 isolate-antimicrobial combinations tested, the category agreement was 83.6%, with 2.0, 1.6, and 12.8% very major, major, and minor errors, respectively. Vitek 2 accuracy was influenced differently by the mechanism responsible for resistance, and interpretation of the results in relation to phenotype could improve the performance of the system.Broth microdilution and disk diffusion are the reference methods for antimicrobial susceptibility testing (AST) of Pseudomonas aeruginosa to broad-spectrum ␤-lactam antibiotics, whereas there has been longstanding concern about the accuracy of commercial automated systems (6,9,10,14). Since the automated systems are potentially capable of improving standardization, requiring less labor, and validating MIC results by providing expert systems, the poor performance they display in P. aeruginosa AST constitutes a major drawback for clinical microbiology laboratories. There is no obvious explanation for the discrepancy between the results obtained with automated systems and reference methods, but the importance of factors such as the phenotypic characteristics of the organisms and the underlying resistance mechanisms is well known (14). Several mechanisms may lead to resistance to broad-spectrum ␤-lactams: hyperproduction of broad-spectrum ␤-lactamases (including carbapenemases), decreased outer membrane permeability, and active efflux (5,13,17,18).In our study, we considered isolates with different phenotypes of susceptibility/resistance (each based on different biochemical mechanisms) to imipenem (Ipm) and ceftazidime (Caz) and evaluated the accuracy of a commonly used automated system, the Vitek 2 system (bioMérieux, Rome, Italy), for isolates belonging to each phenotype. Our intention was to produce information for reassessment of the ␤-lactam interpretative algorithms of this system for tests with P. aeruginosa.Seventy-eight isolates of P. aeruginosa selected from our collection on the basis of AST performed by an agar diffusion technique were used in this study. , and were unique pulsedfield gel electrophoresis types, without patient duplicates to minimize any clonal influence. In 2006, the distribution of these phenotypes among routine clinical isolates in our institution was 6.7% (group 1), 7.4% (group 2), 13.6% (group 3), and 67% (group 4). The remaining 5% were not named.Susceptibility to piperacillin-tazobactam (Tzp), Caz, aztreonam (Azt), Ipm, meropenem (Mer), gentamicin (Gen), and ciprofloxacin (Cip) was determined by the Vitek 2 system by using the AST-N022 card (bioMérieux, Rome, Italy) and a MIC gradient test validated for testing P. aeruginosa (2) (Etest; AB Bio...

The binding of ceftriaxone, a cephalosporin that exhibits high serum protein binding and prolonged serum half-life, to penicillin-binding proteins (PBPs) of Escherichia coli K12 in the presence of human serum albumin was compared with plasma concentrations of cefotaxime, a cephalosporin with low serum protein binding and a short serum half-life. Ceftriaxone concentrations equivalent to those maintained in plasma for 8 h after an intravenous infusion of 1 g saturated PBPs 2 and 3. Cefotaxime saturated both PBPs at concentrations equivalent to those maintained for 2 h, and PBP 3 only at concentrations maintained for 2-8 h. These results indicate that high serum protein binding does not impair the ability of ceftriaxone to inhibit essential PBPs, and explain the high in-vivo efficacy of the drug.

Penicillin‐binding protein (PBP) 5 of Enterococcus hirae ATCC 9790 belongs to the class of the high‐molecular mass, low‐affinity PBPs which have been correlated with penicillin resistance in most Enterococcus species. Polyclonal antibodies were raised against PBP 5 and used to detect immunologically related membrane proteins in E. faecium and E. faecalis strains. Several strains of both species were found to have a membrane protein of similar molecular mass to E. hirae PBP 5 which reacted with the antibodies. Some E. faecium strains did not react with antibodies but their derivatives with increased penicillin minimal inhibitory concentrations did. In some E. faecalis strains the lack of a PBP 5‐related protein was associated with failure to select stable penicillin‐resistant derivatives.