Spread of an unusual penicillin- and imipenem-resistant but ampicillin-susceptible phenotype among Enterococcus faecalis clinical isolates

Sir, Enterococci have emerged as pathogens related to serious nosocomial infections, particularly in hospitals where cephalosporins are extensively used. The treatment of enterococcal infections is difficult and usually requires the administration of a penicillin compound alone or, for severe infections, in synergic combination with an aminoglycoside, usually gentamicin in high concentrations. Enterococcus faecalis usually demonstrates susceptibility to penicillin, ampicillin, amoxicillin/clavulanate and imipenem, whereas Enterococcus faecium is commonly resistant to the above-mentioned β-lactam antibiotics. Enterococci exhibiting susceptibility to ampicillin are regularly cross-susceptible to penicillin and MICs of both antibiotics may be equal or more often MICs of ampicillin are one to two dilutions lower than those of penicillin although the NCCLS/CLSI breakpoints for both antibiotics versus enterococci are identical.1,2 In our hospital, recent data from the identification and susceptibility automated system have shown that a large percentage of our clinical E. faecalis isolates exhibit an unusual phenotype being penicillin-resistant but ampicillin-susceptible. The present report describes the spread of this phenotype among our E. faecalis population. During September 2003–December 2004, 287 E. faecalis isolates were consecutively collected from clinical materials of separate patients hospitalized at Hippokration University Hospital, Thessaloniki, Greece, a 1012-bed general hospital with medical, paediatric and surgical services, specialist intensive care units and a dialysis ward. Identification to the species level was performed with the automated Vitek 2 system (bioMerieux, Marcy l'Etoile, France) using the Vitek GPI card and in cases of the unusual phenotype the result was confirmed by using a conventional test scheme.3 The Vitek 2 system was also used to determine susceptibility to a range of antimicrobials. MICs of ampicillin, penicillin and imipenem were additionally determined in duplicate by an agar dilution method.3 The assay was performed by inoculating 104 cfu/spot onto cation supplemented Mueller–Hinton agar plates containing antibiotic dilutions in the range 0.015–128 mg/L. Interpretative criteria for susceptibility status were those of the NCCLS/CLSI.4E. faecalis ATCC 29212 was used for quality control. Cefinase discs (BBL Microbiology Systems, Cockeysville, MD, USA) were used in order to screen for possible β-lactamase production. PFGE of SmaI-digested genomic DNA of E. faecalis isolates was performed with a CHEF-DRIII system (Bio-Rad, Hemel Hempstead, UK). Banding patterns were compared visually and all loci were scored for the presence or absence of a band. Susceptibility testing by the Vitek system identified that as many as 90 (31.4%) of the E. faecalis isolates exhibited an unusual phenotype, being ampicillin-susceptible but penicillin-resistant; they were recovered from blood (18), bronchial secretions (5), exudates (9), intravenous catheters (13), urine (26) and wounds (19). Most of these isolates were also resistant to high levels of gentamicin and streptomycin (73 and 78 isolates, respectively), while two exhibited cross-resistance to vancomycin and teicoplanin. Ampicillin, penicillin and imipenem MIC determinations for the 90 E. faecalis isolates were repeated by the agar dilution method and their MICs ranged from 0.25 to 2 mg/L, 16 to 64 mg/L and 4 to 16 mg/L, respectively (Table 1). In particular, ampicillin MICs of 0.25 mg/L were detected in three isolates, ampicillin MICs of 0.5 mg/L were detected in 46 isolates, ampicillin MICs of 1 mg/L were detected in 33 isolates and ampicillin MICs of 2 mg/L were detected in 8 isolates; cross-resistance to penicillin was detected in all these isolates, while MICs of imipenem were elevated and in most cases revealed imipenem-intermediate or -resistant isolates. Similar results were also obtained when the MIC determinations were repeated by using Etest strips (AB Biodisk, Solna, Sweden) (data not shown). β-Lactamase production was not detected in any of the E. faecalis isolates using the cefinase disc test. PFGE analysis revealed that the 90 E. faecalis isolates belonged to six distinct genotypes. Fifty-two of the isolates belonged to one major genotype containing three subtypes. The remaining 38 isolates were grouped in five genotypes containing from 1 to 15 isolates each. Cumulative percentages of E. faecalis isolates (n = 90) inhibited by ampicillin, penicillin and imipenem at Hippokration University Hospital Cumulative percentages of E. faecalis isolates (n = 90) inhibited by ampicillin, penicillin and imipenem at Hippokration University Hospital The results of this study demonstrate a wide dissemination of penicillin- and imipenem-resistant but ampicillin-susceptible isolates among our clinical E. faecalis population. Spread of a particular clone was not found to be responsible for the substantial increase in the prevalence of such enterococcal isolates during the study period. It has been previously suggested that MICs of penicillin and ampicillin show good concordance and testing the susceptibilities of enterococcal isolates to penicillin or ampicillin might be also used as a method of predicting the in vitro activity of imipenem.2,5 This concordance was mostly detected among E. faecalis isolates and it has been postulated that if these results are confirmed, a therapeutic note in the guidelines might indicate that the in vitro results obtained for penicillin or ampicillin will accurately predict the in vitro susceptibility of imipenem. However, a few studies have reported that ampicillin-susceptible, imipenem-resistant E. faecium isolates might exist.6,7 In such isolates imipenem resistance was associated with hyperproduction of PBP5 that exhibited a selectively decreased affinity for imipenem.6 A decreased affinity of PBP4 for β-lactams was also detected in a collection of ampicillin- and imipenem-non-susceptible E. faecalis clinical isolates.8 The present study is the first one that demonstrates dissemination of penicillin- and imipenem-resistant but ampicillin-susceptible E. faecalis isolates in a hospital environment. Although occurrence of other resistance mechanisms cannot be excluded, alterations in PBPs that specifically influence affinity for penicillin and imipenem might be responsible for this observation. Since imipenem is clinically indicated only for enterococcal infections caused by E. faecalis, the increasing detection of these pathogens in our clinical setting necessitates the continuous evaluation of their antimicrobial susceptibility trends in order to identify substantial changes. Work is currently in progress, to gain some insight into the variation of the PBP domains conferring the unusual phenotype among enterococci. We have no conflicts to declare. 1Department of Clinical Microbiology, Hippokration University Hospital, Thessaloniki, Greece; 2Department of Basic Sciences, School of Health Sciences, University of Athens, 123 Papadiamantopoulou Street, 11527 Athens, Greece; 3Department of Microbiology, Medical School, University of Thessalia, Larissa, Greece