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This study aimed to evaluate the accuracy of disk diffusion and Etest methods, compared to that of the broth dilution reference method for identifying beta-lactam susceptibilities of Penicillin-Resistant, Ampicillin-Susceptible Enterococcus faecalis (PRASEF) isolates. Fifty-nine PRASEF and 15 Penicillin-Susceptible, Ampicillin-Susceptible E. faecalis (PSASEF) clinical nonrepetitive isolates were evaluated. The effectiveness of five beta-lactams (ampicillin, amoxicillin, imipenem, penicillin, and piperacillin) was tested. All antimicrobial susceptibility tests were performed and interpreted according to the Clinical and Laboratory Standards Institute guidelines. Interpretative discrepancies, such as essential agreement, categorical agreement, and errors, were assessed. The acceptability was ≥ 90% for both categorical agreement and essential agreement. Etest proved to be an accurate method for testing beta-lactam susceptibilities of the emerging PRASEF isolates, disk diffusion presented poor performance, particularly for imipenem and piperacillin.

The aim of the present study was to verify whether penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) occurred in Brazil prior to the beginning of the 21st century, and to verify whether ampicillin susceptibility can predict susceptibility to other β-lactams in E. faecalis with this inconsistent phenotype. The presence of polymorphisms in the pbp4 gene and genetic diversity among the isolates were investigated. Of 21 PRASEF analyzed, 5 (23.8%) and 4 (19.0%) were imipenem and piperacillin resistant simultaneously by disk diffusion and broth dilution respectively, contradicting the current internationally accepted standards of susceptibility testing. Sequencing of pbp4 gene revealed an amino acid substitution (Asp-573→Glu) in all PRASEF isolates but not in the penicillin-susceptible, ampicillin-susceptible E. faecalis. Most PRASEF (90.5%) had related pulsed-field gel electrophoresis profiles, but were different from other PRASEF described to date. Results demonstrate that penicillin-resistant, ampicillin-susceptible phenotype was already a reality in the 1990s in E. faecalis isolates in different Brazilian states, and some of these isolates were also imipenem- and piperacillin-resistant; therefore, internationally accepted susceptibility criteria cannot be applied to these isolates. According to pbp4 gene sequencing, this study suggests that a specific amino acid substitution in pbp4 gene found in all PRASEF analyzed is associated with penicillin resistance., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Despite the spread of penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) isolates in diverse countries, the mechanisms leading to this unusual resistance phenotype have not yet been investigated. The aim of this study was to evaluate whether polymorphism in the pbp4 gene is associated with penicillin resistance in PRASEF isolates and to determine their genetic diversity. E. faecalis isolates were recovered from different clinical specimens of hospitalized patients from February 2006 to June 2010. The β-lactam minimal inhibitory concentrations (MICs) were determined by E-test®. The PCR-amplified pbp4 gene was sequenced with an automated sequencer. The genetic diversities of the isolates were established by PFGE (pulsed-field gel electrophoresis) and MLST (multilocus sequencing typing). Seventeen non-producing β-lactamase PRASEF and 10 penicillin-susceptible, ampicillin-susceptible E. faecalis (PSASEF) strains were analyzed. A single-amino-acid substitution (Asp-573→Glu) in the penicillin-binding domain was significantly found in all PRASEF isolates by sequencing of the pbp4 gene but not in the penicillin-susceptible isolates. In contrast to the PSASEF isolates, a majority of the PRASEFs had similar PFGE profiles. Six representative PRASEF isolates were resolved by MLST into ST9 and ST524 and belong to the globally dispersed clonal complex 9 (CC9). In conclusion, it appears quite likely that the amino acid alteration (Asp-573→Glu) found in the PBP4 of the Brazilian PRASEF isolates may account for their reduced susceptibility to penicillin, although other resistance mechanisms remain to be investigated., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Our findings demonstrated that the results obtained for ampicillin may accurately predict the in vitro susceptibility to amoxicillin but not to imipenem and piperacillin among isolates of Enterococcus faecalis resistant to penicillin but susceptible to ampicillin, which have emerged recently, in contrast to penicillin- and ampicillin-susceptible isolates.