Diversity among high-level aminoglycoside-resistant enterococci

J. Papaparaskevasa,c,*, A. Vatopoulosb, P. T. Tassiosc, A. Avlamia, N. J. Legakisc and V. Kalapothakib

a Microbiology Department, ‘Laikon’ General Hospital, Athens; b Department of Hygiene and Epidemiology and c Department of Microbiology, University of Athens Medical School, Athens, Greece


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 55 Enterococcus faecalis and 21 Enterococcus faecium non-replicate isolates were obtained from routine clinical specimens, during a 1 year period, in a tertiary care hospital in Athens, Greece. The most common isolation site was the urinary tract (44% of E. faecalis and 33% of E. faecium isolates). No vancomycin resistance was detected. Ampicillin-resistant isolates did not produce ß-lactamase. High-level gentamicin resistance was detected in 22% and 0% of E. faecalis and E. faecium isolates, respectively. The corresponding figures for high-level streptomycin resistance were 40% and 33%. The aminoglycoside-modifying enzyme gene aac(6')+aph(2'') was detected by PCR in 10 of 12 high-level gentamicin-resistant E. faecalis isolates, and the ant(6)-I gene in all high-level streptomycin-resistant isolates of both species. DNA fingerprinting by PFGE grouped 31 of 55 E. faecalis isolates into 10 clusters, and 10 of 21 E. faecium isolates into two clusters, containing two to seven isolates each. Two E. faecalis PFGE types, comprising isolates expressing high-level aminoglycoside resistance, and not observed among non-high-level aminoglycoside-resistant strains, were disseminated in building A of the hospital. In contrast, high-level aminoglycoside resistance seemed to have been acquired nosocomially by a number of genotypically different E. faecium types. Molecular typing was therefore instrumental in understanding the differences in the mode of spread and acquisition of high-level aminoglycoside resistance among these two different enterococcal species.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Enterococci have become important causes of nosocomial infection in many parts of the world.1,2 The same situation is apparent in Greece where the genus represents 7.1% of all bacteria and 23.9% of all Gram-positive bacteria isolated in hospitals, as reported by the Greek Electronic Network for the Surveillance of Antimicrobial Resistance in Bacterial Nosocomial Isolates (GENSAR)3 (see also www.mednet.gr/whonet).

High-level aminoglycoside resistance among enterococci is increasingly being reported world-wide.4–6 In Greece, high-level gentamicin resistance is encountered in 15.4% of nosocomial Enterococcus faecalis and in 11% of Enterococcus faecium, whereas the rates of high-level streptomycin resistance are 42.3 and 52.9%, respectively.3 The presence of high-level aminoglycoside resistance is predictive of the loss of synergy between a cell-wall-active agent (e.g. penicillin, ampicillin or vancomycin) and an aminoglycoside, which makes the treatment of serious enterococcal infections such as endocarditis difficult. This renders understanding of the epidemiology of these organisms and, more specifically, the possible significance of clonal spread, important for infection control.

In the present study, we analysed all clinical isolates of E. faecalis and E. faecium from a large teaching hospital, over a 1 year period, with respect to the prevalence of high-level aminoglycoside resistance, and the possible spread of resistant clones.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study location

Laikon General Hospital, a university-affiliated tertiary care general hospital of 500 beds, is located in central Athens and consists of two closely situated four-floor buildings connected by corridors. Care is provided to patients by house staff in all subspecialties. The hospital antibiotic policy is based on a drug formulary, written local guidelines for empirical and documented therapy and daily consultancy with infectious disease specialists and clinical microbiologists for management of serious infections and use of reserved drugs.

Clinical isolates

All E. faecalis and E. faecium non-replicate isolates (the first isolate from each patient) obtained from routine clinical specimens submitted to the Clinical Microbiology Laboratory between March 1995 and March 1996 were included in the study. All isolates were identified to the genus level by conventional tests based on colony morphology on McConkey agar No. 2 and on 5% blood agar, Gram's stain, catalase test, growth in the presence of 6.5% NaCl and bile, and hydrolysis of aesculin. Identification to the species level was done with the PASCO MIC/ID semi-automated system (Difco, Detroit, MI, USA) for Gram-positive organisms. Identification of 10 randomly selected isolates of each species was further confirmed by a PCR assay.7

Susceptibility testing

Susceptibility to antibiotics was determined by a microdilution method using the PASCO MIC/ID semi-automated system (Difco) for Gram-positive organisms. Interpretation of the results was according to NCCLS guidelines.8 The following antibiotics were tested: ampicillin (Am), erythromycin (Ery), ciprofloxacin (Cip), vancomycin (Van), gentamicin (Gm) and streptomycin (Str). High-level resistance to streptomycin (MIC > 2000 mg/L) and gentamicin (MIC > 500 mg/L) was additionally confirmed by an agar screening method described previously.4 Production of ß-lactamases was tested by the nitrocefin disc method (BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD, USA).

Haemolysis

Haemolysis was sought after 24 and 48 h inoculation, on plates of brain–heart infusion agar base containing 5% sheep blood (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA).

DNA preparation for PCR assays

Pelleted growth from 5 mL overnight broth cultures was resuspended in 500 µL of 50 mM Tris–HCl pH 8, 50 mM EDTA, supplemented with RNase (10 µL of 10 mg/mL) and lysozyme (2 µL of 50 mg/mL). After incubation for 30 min at 37°C, 25 µL of 10% SDS was added, the mixture was incubated again for 30 min at 37°C, proteinase K was added (5 µL of 20 mg/mL) and the mixture was incubated for 30 min at 50°C. The cell lysate was extracted with phenol/chloroform/isoamyl alcohol (24:24:1) and chloroform. DNA was precipitated with cold ethanol, pelleted by centrifugation, dried and resuspended in distilled water.

Confirmation of species identification and detection of aminoglycoside resistance genes by PCR

A PCR assay described previously7 was used to confirm the identity of 10 randomly selected isolates of E. faecalis and E. faecium. Presence of the genes that encode the ANT(6)-I enzyme (responsible for high-level streptomycin resistance) and the AAC(6')+APH(2'') enzyme (responsible for high-level gentamicin resistance) was also confirmed by PCR, as described previously.4

Pulsed-field gel electrophoresis (PFGE) of macrorestricted genomic DNA

Plug preparation was according to a modification of a published protocol.9 Briefly, cell lysis by lysozyme at 37°C was followed by a proteinase K treatment at 55°C, and DNA digestion with SmaI (New England Biolabs, Beverly, MA, USA) at 37°C. Electrophoresis through a 1% agarose gel in 0.5 x TBE was performed with a CHEF DRIII apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Conditions were 14°C, 6 V/cm, 120° switch angle and a run time of 21 h using a linear switch time ramp of 0.5–40 s. The gel was stained in 0.5 mg/L ethidium bromide and documented under UV illumination. Lambda phage DNA concatamers (New England Biolabs) were used as DNA size markers. PFGE band patterns were compared with the GelCompar software (Applied Maths, Kortrijk, Belgium) using the Dice co-efficient10 and clustering by the unweighted pair group method, using arithmetic averages. For dendrogram construction, a 2% difference in band position was tolerated. PFGE patterns assigned to the same cluster did not differ by more than six bands, suggesting that the corresponding isolates were related, according to published guidelines.11


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 55 E. faecalis and 21 E. faecium clinical isolates were obtained during the 1 year study period, with the majority from urine (44 and 33%, respectively). Other isolation sites included surgical wounds (35 and 19%) and blood or other normally sterile body fluids (16 and 28%). The distribution of E. faecalis and E. faecium isolates among hospital wards indicated that the majority were from medical (49 and 19%, respectively) and surgical wards (33 and 33%).

Resistance to ampicillin among the E. faecalis and the E. faecium isolates was 7 and 76%, respectively. None of the ampicillin-resistant isolates was found to produce ß-lactamase. Resistance rates to erythromycin were 47 and 95%, and to ciprofloxacin 24 and 62%, respectively. In contrast, high-level gentamicin resistance was detected among 22 and 0%, and high-level streptomycin resistance among 40 and 33% of E. faecalis and E. faecium isolates, respectively. No vancomycin resistance was detected.

The aminoglycoside-modifying enzyme gene aac(6')+ aph(2'') was detected in 10 of 12 high-level gentamicin-resistant (HLGmR) E. faecalis isolates, and the ant(6)-I gene was detected in all 22 high-level streptomycinresistant (HLStrR) E. faecalis isolates and all seven HLStrR E. faecium isolates.

Based on their high-level aminoglycoside resistance phenotypes, the E. faecalis isolates were divided into three subgroups (Table IGo). Group I consisted of isolates with high-level gentamicin and streptomycin resistance and included four phenotypes: CipEryHLGmStrR (nine isolates), AmCipEryHLGmStrR (one isolate), EryHLGmStrR (one isolate) and CipHLGmStrR (one isolate). Group II comprised 10 isolates with high-level streptomycin resistance, nine of which were EryHLStrR and one CipEryHLStrR. Group III included all non-high-level aminoglycoside-resistant (non-HLAR) isolates, with five distinct phenotypes: fully susceptible (23 isolates), EryR (four isolates), AmCipR (one isolate), AmEryR (one isolate) and AmR (one isolate). Haemolytic activity was detected in eight of the 12 E. faecalis HLGmStrR isolates (67%), but only in two of 10 HLStrR isolates (20%) and three of 33 non-HLAR isolates (9%).


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Table I. Distribution of E. faecalis isolates by HLAR phenotype and PFGE pattern
 
The E. faecium isolates were divided into two groups according to their resistance phenotypes (Table IIGo). Group 1 consisted of seven isolates, five of the AmCipEryHLStrR phenotype and two AmEryHLStrR, whereas group 2 included 14 non-HLAR strains. Of the 14 non-HLAR strains of group 2, one was susceptible to all antibiotic agents and the rest were divided into three phenotypes, AmCipEryR (four isolates), AmEryR (five isolates) and CipEryR (four isolates). In contrast to the situation in E. faecalis, haemolytic activity was detected in only two E. faecium isolates, both HLStrR.


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Table II. Distribution of E. faecium isolates by HLAR phenotype and PFGE pattern
 
DNA fingerprinting by PFGE grouped 31 of 55 E. faecalis isolates into 10 clusters (Figure 1Go and Table IGo), containing two to seven related isolates each, with similarities ranging from 84% to 100%. The remaining 24 strains had unique PFGE patterns. Group I was the most genotypically homogeneous, since seven of 12 HLGmStrR isolates (all of them belonging to the CipEryHLGmStrR phenotype) formed a single cluster, J. One was similar to a susceptible strain (cluster B), and four had unique patterns (Table IGo).



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Figure 1. Dendrogram showing relationships between all E. faecalis isolates, based on their PFGE patterns.

 
The single CipEryHLStrR strain of group II, together with four EryHLStrR strains formed a cluster (E), whilst two other EryHLStrR strains formed cluster C, together with two susceptible strains. Of the remaining three EryHLStrR strains, one had a unique pattern, one belonged to a cluster, D, together with two susceptible strains, and one was similar to an Ery strain (cluster G) (Table IGo).

In contrast, 14 (42%) of the 33 non-HLAR isolates in group III were distributed among eight clusters, whereas the majority (19 isolates, 58%) had unique PFGE types (Table IGo).

When the E. faecalis strain distribution among hospital wards was examined, cluster J (CipEryHLGmStrR) was found only in building A, mainly in second and third floor wards, between June 1995 and January 1996 (Table IIIGo). Cluster E (CipEryHLStrR and EryHLStrR) was found also in building A with a single exception of a strain in building B, mainly in first and second floor wards, between May 1995 and October 1995, and one strain found in March 1996.


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Table III. Distribution of E. faecalis PFGE clusters by ward and month of isolation
 
With respect to E. faecium, PFGE grouped 10 of the 21 isolates into two clusters of related strains, whereas the remaining 11 had unique PFGE patterns. Cluster A consisted of six strains, one AmCipEryHLStrR, one AmEryHLStrR, two AmCipEryR and two AmEryR, whilst cluster B consisted of four strains, two AmCipEryHLStrR, one AmCipEryR and one AmEryR (Figure 2Go and Table IIGo).



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Figure 2. Dendrogram showing relationships between all E. faecium isolates, based on their PFGE patterns.

 
A study of the distribution of E. faecium isolates among hospital wards revealed that four out of the six strains of cluster a were found in the Nephrology ward and the Transplantation Unit (two wards which share common patients), between September and November 1995. The E. faecium strains belonging to PFGE clusters were more evenly distributed in both buildings of the hospital (six in building A and four in building B) than the E. faecalis ones, which were found mainly in building A (Table IVGo).


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Table IV. Distribution of E. faecium PFGE clusters by ward and month of isolation
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study focused mainly on HLAR enterococcal isolates. No vancomycin resistance was detected among these isolates, as resistance rates to vancomycin are very low in Greece, and vancomycin-resistant enterococci (VRE) are sporadic and infrequent.3

Resistance rates to other antibiotics differed between the two enterococcal species. As in other Greek studies,3,12 resistance rates of E. faecium were higher than those of E. faecalis with respect to ampicillin, erythromycin and ciprofloxacin. Also, resistance rates to ciprofloxacin were found to be higher among E. faecalis isolates with high-level gentamicin resistance (11 of 12 isolates in Group I, one of 10 isolates in group II and one of 33 isolates in group III), a fact also evident in a previous study.13

In the present study, high-level gentamicin resistance was confined to E. faecalis, in contast to studies from other countries, including the UK, Singapore, Australia9 and Ireland,14 where high-level gentamicin resistance was more frequent in E. faecium (24%) than E. faecalis (4%), or the USA, where high-level gentamicin resistance rates were equal between the two species.15

In Greece, GENSAR, which represents 18 hospitals, reported an 11% HLGmR prevalence in E. faecium. In a previous study conducted in the northern part of the country,12 HLGmR prevalence in E. faecium was almost twice this figure (26.3%), in contrast to the 0% rate observed in this study. Similarly the rates of high-level streptomycin resistance in E. faecium and high-level gentamicin resistance in E. faecalis observed in this study were lower than those reported by the other two sources.3,12 These variations should be attributed to the distinct epidemiology of antibiotic resistance among different hospitals, underlining the importance of hospital-based research on antibiotic resistance.

For E. faecalis, ward-specific differences in antibiotic resistance rates were noted. In the three main medical wards (first, second and third), with approximately the same number of beds and patient admissions per year, the high-level gentamicin resistance rates were 22, 41 and 25%, respectively, whilst the high-level streptomycin resistance rates were 56, 75 and 0%, respectively. On the other hand, no HLAR isolates were found in the Nephrology and Transplantation Unit. Nine of the 22 HLAR E. faecalis isolates (41%) were obtained from the second internal medicine ward, while only three of 33 non-HLAR E. faecalis strains (9%) were from the same ward. The three internal medicine wards together, contributed 15 of the 22 HLAR isolates (68%), but only 10 of the 33 non-HLAR isolates (30%).

Building-specific distribution of E. faecalis was also evident, since all the HLGmStrR isolates of group I were found in medical and surgical departments in building A of the hospital. Similarly, HLStrR E. faecalis isolates of group II were found mainly in building A (eight of 10 isolates). In contrast, resistance rates for E. faecium were more evenly distributed among wards and buildings, with the exception of the Nephrology and Transplantation Unit, where five of the nine AmEryR isolates were isolated.

The presence of the bifunctional enzyme AAC(6')-I + APH(2'')-I in 10 of 12 HLGmR E. faecalis isolates, together with the simultaneous presence of the ANT(6)-I enzyme, rendered these strains resistant to all clinically useful aminoglycosides. The molecular mechanism of HLGmR in the remaining two strains is under investigation. Although the world-wide dissemination of HLGmR in enterococci, as well as in staphylococci, is believed to have resulted from the dissemination of the bifunctional enzyme gene,16 the novel gentamicin resistance genes (aph(2'')-Ic and aph(2'')-Id) have recently been described in isolates of Enterococcus gallinarum,17 Enterococcus casseliflavus and various clinical E. faecium isolates.18 The two isolates (one of EryHLGmStrR phenotype, the other of CipHLGmStrR) had unique PFGE patterns. The MIC of gentamicin for both isolates was >512 mg/L by both the microdilution and agar screening test methods. The MIC of amikacin for both of these isolates, as well as all E. faecalis isolates of group I, was >256 mg/L by Etest (data not shown). APH(2'')-Ic usually gives a MIC of gentamicin of around 128–256 mg/L and of amikacin of 32 mg/L. Enzyme APH(2'')-Id was reported to produce an amikacin MIC of 32 mg/L in the original E. casseliflavus isolate and has also been detected in vancomycin-resistant E. faecium. Although we have not formally excluded the presence of these more recently described enzymes, it is probably unlikely, based on the MICs observed here. The ANT(6)-I enzyme was responsible for HLStrR among all group II E. faecalis isolates and all group I E. faecium isolates.

The higher frequency of E. faecalis infections, as opposed to those by E. faecium,1,2,6 has been proposed as indirect evidence for the greater intrinsic virulence of E. faecalis. Haemolytic activity, considered to be a virulence factor,19 was detected more frequently (70%) among EryHLGmStrR, than among HLStrR (20%) or susceptible E. faecalis (9%), in agreement with previous studies.20 On the contrary, haemolytic activity was noted in only two of the seven (29%) AmEryHLStrR E. faecium isolates.

DNA fingerprinting by PFGE provided an explanation for the differences in antibiotic resistance, haemolytic activity and ward distribution between the two species, as it revealed the genotypic identity of each isolate, and its relatedness to others. Two main genotypic clusters of E. faecalis, consisting exclusively of HLAR isolates, were present in building A. Clusters J and E contained the highest number of HLGmStrR and HLStrR isolates, respectively. In addition, six of the seven isolates of the epidemic cluster J expressed haemolytic activity. Furthermore, the only confirmed outbreaks were due to strains belonging to these two genotypic groups. In contrast, non-HLAR isolates were both genotypically more varied and more widely scattered in the hospital.

The distribution of E. faecium between hospital wards was different from that of E. faecalis. Isolates belonging to the two main clusters (A and B) were found in both buildings of the hospital, approximately evenly distributed. There was no consistent correlation between phenotypic groups and genotypic clusters, nor was there any confirmed outbreak.

In contrast to E. faecalis, where there was putative evidence of clonal spread of HLAR strains, genotypically distinct E. faecium isolates seem to have acquired the ant(6)-I gene. This acquisition may have been nosocomial, as suggested by the fact that cluster B contained two pairs of non-distinguishable strains from the same wards, but differing in their resistance phenotype. In both cases, the HLStrR strain was isolated 2 or 3 months after the non-HLStrR one. The same phenomenon was seen in cluster A, with an AmCipEryR strain and an AmEryHLStrR strain (in the same ward). This was not observed with E. faecalis, where, in the clusters containing both HLAR and non-HLAR strains, isolation of the susceptible strains did not always precede that of the resistant ones in the same ward.

In conclusion, molecular typing was shown to be necessary for understanding the differences in the mode of spread and acquisition of antibiotic resistance among different species of enterococci. Such information is indispensable for the implementation of effective infection control measures.


    Acknowledgments
 
We thank V. Kontoyanni and J. Pournou for their excellent technical assistance. This study was supported in part by a grant from the Greek Ministry of Health and Social Services.


    Notes
 
* Correspondence address. Department of Microbiology, Medical School, University of Athens, M. Asias 75, 115 27 Athens, Greece. Tel: +30-1-7785638; Fax: +30-1-7709180; E-mail: iosifp{at}otenet.gr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 21 June 1999; returned 20 September 1999; revised 27 October 1999; accepted 12 November 1999