International Journal of Antimicrobial Agents 32 (2008) 374377

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International Journal of Antimicrobial Agents

journal homepage: http://www.elsevier.com/locate/ijantimicag

Virulence and antimicrobial resistance in clinical Enterococcus faecium

Hanna Billstrm a , Bodil Lund a , sa Sullivan a,b , Carl Erik Nord a,
a b

Division of Clinical Microbiology, F68, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden Division of Oral and Maxillofacial Surgery, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

a r t i c l e

i n f o

a b s t r a c t
The aim of the present study was to determine the occurrence of seven virulence determinants in Enterococcus faecium clinical blood culture isolates over a 6-year period and to investigate possible correlations between virulence and antibiotic resistance. Two hundred and sixty-three isolates were screened for the presence of genes coding for aggregation substance (asa1), cytolysin (cylA), collagen-binding protein (ace), Enterococcus faecalis endocarditis antigen (efaAfs ), enterococcal surface protein (espfm ), gelatinase (gelE) and hyaluronidase (hylfm ) by polymerase chain reaction. The minimum inhibitory concentrations (MICs) of ampicillin, ciprooxacin, gentamicin, imipenem, linezolid and vancomycin were determined by the agar dilution method and the MIC of daptomycin was determined by Etest. The espfm gene was found in 56% of the isolates, hylfm in 4%, whilst the other virulence genes were detected only sporadically (1%). The level of antibiotic resistance was 77% to ampicillin, 90% to ciprooxacin and 83% to imipenem; 5% of the isolates were resistant to vancomycin and 2% were resistant to gentamicin (high-level resistance, MIC 500 mg/L). A signicant correlation was found between the presence of espfm and resistance to ampicillin, ciprooxacin and imipenem (P < 0.01). Twelve isolates were espfm -positive and ampicillin-susceptible. 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Article history: Received 28 April 2008 Accepted 28 April 2008 Keywords: Enterococcus faecium Clinical isolates Virulence espfm Antimicrobial resistance

1. Introduction Enterococci have emerged as one of the leading pathogens in nosocomial infections. The most commonly encountered species are Enterococcus faecalis and Enterococcus faecium and the proportion of E. faecium has increased over time [1,2]. Enterococci are generally considered to be of low virulence and the virulence determinants of E. faecium are largely unknown. However, several characteristics have been suggested to contribute to the virulence of E. faecium, i.e. aggregation substance, cytolysin, collagen-binding protein, E. faecalis endocarditis antigen, enterococcal surface protein, gelatinase and hyaluronidase [3,4]. Aggregation substance, cytolysin and E. faecalis endocarditis antigen have so far been identied in E. faecalis isolates. Collagen-binding protein has been involved in the adhesion of E. faecium [5]. Enterococcal surface protein is important for the initial adherence and biolm formation of E. faecium [6,7]. It has also been shown that E. faecium strains harbouring the gene espfm (coding enterococcal surface

protein) have signicantly higher conjugation rates than strains without espfm [8]. The extracellular zinc metalloprotease gelatinase that hydrolyses gelatin and collagen has been identied in dairy strains of E. faecium [9]. The exact role of hyaluronidase in E. faecium is not known, but both the genes hylfm (coding hyaluronidase) and espfm have been shown in E. faecium isolates [10,11]. Enterococci have intrinsically reduced susceptibility to several -lactam antibiotics and aminoglycosides [3]. Enterococci may acquire resistance to several antimicrobial agents such as aminoglycosides, -lactams and glycopeptides. The aim of the present study was to determine the occurrence of seven virulence determinants in E. faecium blood culture isolates and to investigate whether there was any correlation between the presence of these genes and antibiotic resistance. 2. Material and methods 2.1. Bacterial isolates and species identication All consecutive non-duplicate E. faecium isolates from patients with clinical signs of septicaemia at the Karolinska University Hospital, Huddinge, Sweden, isolated between January 2000 and December 2005 were included in the study. All isolates were identied to species level by Gram-staining, colony morphology and biochemical tests [12]. Species identity of strains that were

These results were presented in part at the 16th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 14 April 2006, Nice, France, and at the 17th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 31 March3 April 2007, Munich, Germany. Corresponding author. Tel.: +46 8 585 878 38; fax: +46 8 585 879 33. E-mail address: carl.erik.nord@ki.se (C.E. Nord).

0924-8579/$ see front matter 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.04.026

H. Billstrm et al. / International Journal of Antimicrobial Agents 32 (2008) 374377 Table 1 Description of primers used in the polymerase chain reaction (PCR) and sequencing reactions and the corresponding references Target gene Aggregation substance Collagen-binding protein Cytolysin Enterococcus faecalis endocarditis antigen Enterococcal surface protein Gelatinase Hyaluronidase Primers Asa 11; Asa 12 Ace 1; Ace 2 Cyt I; Cyt IIb EfaA 1; EfaA 2 Esp 14F; Esp 12R Gel 11; Gel 12 Hyl n1; Hyl n2 Sequence 5 -CACGCTATTACGAACTATGA-3 ; 5 -TAAGAAAGAACATCACCACGA-3 5 -GGAATGACCGAGAACGATGGC-3 ; 5 -GCTTGATGTTGGCCTGCTTCCG-3 5 -ACTCGGGGATTGATAGGC-3 ; 5 -GCTGCTAAAGCTGCGCTT-3 5 -CGTGAGAAAGAAATGGAGGA-3 ; 5 -CTACTAACACGTCACGAATG-3 5 -AGATTTCATCTTTGATTCTTG G-3 ; 5 -AATTGATTCTTTAGCATCTGG-3 5 -TATGACAATGCTTTTTGGGAT-3; 5 -AGATGCACCCGAAATAATATA-3 5 -ACAGAAGAGCTGCAGGAAATG-3 ; 5 -GACTGACGTCCAAGTTTCCAA-3 Product size 375 bp 616 bp 688 bp 499 bp 510 bp 213 bp 276 bp

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Reference [11] [14] [11] [4] [11] [11] [11]

espfm -positive and ampicillin-sensitive was conrmed by polymerase chain reaction (PCR) [13]. 2.2. PCR and sequencing DNA was puried by re-suspension of an overnight culture of bacteria in 100 L of Milli-Q water, heated at 95 C for 15 min and centrifuged at 15,000 rpm for 5 min. Primer sequences for the seven virulence genes (aggregation substance (asa1), cytolysin (cylA), collagen-binding protein (ace), E. faecalis endocarditis antigen (efaAfs ), enterococcal surface protein (espfm ), gelatinase (gelE) and hyaluronidase (hylfm )) are shown in Table 1. The genes were analysed in separate PCR reactions using 2 L of template in a total of 50 L of Mastermix containing 1 PCR Gold Buffer without MgCl2 , 2.5 mM MgCl2 , 200 M dNTP 4 (Sigma-Aldrich, St Louis, MO), 1 U Taq Polymerase Gold (AmpliTaq Gold ; Applied Biosystems, Solna, Sweden) and 0.4 M each of one of the following primer pairs for ace, asa1, gelE, cylA, efaAfs and hylfm or 0.8 M of primer for espfm (Thermo Electron GmbH, Ulm, Germany). The presence of espfm and hylfm was analysed in a multiplex PCR reaction. Initial denaturation at 95 C for 10 min was followed by 30 cycles of denaturation for 30 s (94 C), amplication for 30 s (56 C for asa1, cylA, espfm , gelE and hylfm and 58 C for ace and efaAfs ) followed by 30 s elongation (72 C). Amplication was terminated with a nal elongation step for 10 min (72 C). Identity between the amplicons and the genes of interest was conrmed by sequencing the PCR product yielded from the reference strains and one positive representative clinical isolate for each target gene. Briey, amplication was achieved by PCR using the same primer pairs previously utilised in the PCR analysis. The PCR products were puried using a QIAquick PCR Purication Kit (QIAGEN, Solna, Sweden). Sequence analysis was performed using an ABI 310 Genetic Analyser according to the manufacturers instructions (Perkin-Elmer, Waltham, MA). The sequences were aligned using PC software FinchTV (http://www.geospiza.com/nchtv/) and ClustaW Multiple sequence alignment (http://www. ebi.ac.uk/Tools/clustalw/index.html) at the European Bioinformatics Institute, Cambridge, UK, and positively identied using the BLAST program (http://www.ncbi.nlm.nih.gov/). Positive controls in the PCR reactions were E. faecalis MMH 594 (asa1, cylA and esp), E. faecalis ATCC 29212 (gelE) and two Swedish clinical isolates (E. faecalis HS1 for ace and efaAfs and E. faecium HS2 for hylfm ). 2.3. Antibiotic susceptibility testing Minimum inhibitory concentrations (MICs) were determined for ampicillin (AstraZeneca, Sdertlje, Sweden), ciprooxacin (Bayer, Elberfeld, Germany), gentamicin (Sigma-Aldrich), imipenem (Merck Sharp & Dohme B.V, Haarlem, The Netherlands), linezolid (Pharmacia & Upjohn, Sollentuna, Sweden) and vancomycin (Abbott Scandinavia AB, Solna, Sweden) using the

agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) [15]. Etest strips (AB BIODISK, Solna, Sweden) were used for detection of daptomycin resistance. The susceptibility breakpoints used were those recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org) when available, and otherwise those from the CLSI [16]: ampicillin, MIC 16 mg/L; ciprooxacin, MIC 4 mg/L; gentamicin, MIC 500 mg/L (highlevel resistance); imipenem, MIC 8 mg/L; linezolid MIC 4 mg/L; vancomycin MIC 8 mg/L; and for daptomycin susceptible at 4 mg/L. Enterococcus faecalis ATCC 29212 was used as a reference strain. 2.4. Statistical analysis Correlation between the occurrence of virulence genes and antibiotic resistance was calculated using the 2 test. A P-value of <0.05 was considered statistically signicant. Correction for multiple signicance tests was calculated according to the Bonferroni method [17]. 3. Results 3.1. Bacterial isolates A total of 263 E. faecium isolates was collected between 2000 and 2006; the numbers ranged between 22 and 59 per year. 3.2. PCR and sequencing The espfm gene was the most widespread virulence determinant and was found in 146 isolates (56%). The presence of espfm was equally distributed over the study period and ranged from 50% to 61% (Table 2). The second most frequent virulence gene, hylfm , was detected in 4% of the isolates (n = 11), of which two isolates also carried espfm . The more rare virulence determinants asa1, ace, efaAfs and gelE were mainly detected during years 2004 and 2005 (Fig. 1). Five isolates harboured two or more virulence genes and none of the isolates carried cylA.
Table 2 Isolates harbouring espfm in relation to the total number of Enterococcus faecium collected each year Year 2000 2001 2002 2003 2004 2005 Total No. of isolates 36 22 41 54 59 51 263 espfm -positive (n (%)) 19 (53) 11 (50) 21 (51) 33 (61) 33 (56) 29 (57) 146 (56) espfm -negative (n (%)) 17 (47) 11 (50) 20 (49) 21 (39) 26 (44) 22 (43) 117 (44)

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H. Billstrm et al. / International Journal of Antimicrobial Agents 32 (2008) 374377 Table 3 Fourteen isolates found with any of the putative virulence determinants ace, asa1, efaAfs , gelE and hylfm and their corresponding antibiotic resistance prole Virulence determinant Antibiotic resistancea AMP hylfm hylfm hylfm hylfm hylfm hylfm hylfm hylfm hylfm hylfm , espfm hylfm , espfm espfm , gelE, efaAfs , ace espfm , gelE, efaAfs , ace espfm , gelE, efaAfs , ace, asa1 R R S R R R R R R S R R S S CIP R R S R R R R R R R R R S R IMI R R S R R R S R R S R R R R GEN R S S S S S S S S S S S S S VAN S S R S S S S S S S S S S S

Fig. 1. Occurrence of virulence determinants ace, asa1, efaAfs , gelE and hylfm during a 6-year period in blood culture isolates of Enterococcus faecium.

3.3. Antibiotic susceptibility testing During the 6-year period, resistance to ampicillin was found in 202 strains (77%), resistance to ciprooxacin in 237 (90%) and resistance to imipenem in 218 (83%). The resistance levels remained at the same level over the study period. The majority of imipenemresistant strains were also resistant to ampicillin. However, 16 imipenem-resistant strains were identied that were ampicillinsusceptible. Fourteen strains (5%) were resistant to vancomycin and ve (2%) were highly resistant to gentamicin. These isolates were collected between the years 2000 and 2003. None of the isolates were resistant to linezolid or daptomycin. Fifteen isolates (6%) were multidrug-resistant (resistant to four or more antimicrobial agents) (Fig. 2). 3.4. Correlation between virulence genes and antibiotic resistance The majority of the espfm -positive isolates (n = 146) were resistant to ampicillin (94%; n = 137), ciprooxacin (96%; n = 140) and imipenem (96%; n = 140). The corresponding resistance rates in espfm -negative isolates (n = 117) were 57% (n = 67), 83% (n = 97) and 67% (n = 78), respectively. Correlation between the presence of the espfm gene and antibiotic resistance was statistically signicant (P < 0.001 for ampicillin and imipenem; P < 0.01 for ciprooxacin). No statistical analyses were performed on the correlation between more rare virulence genes and antibiotic resistance as the numbers were too low. Twelve isolates, all positive for the espfm gene and susceptible to ampicillin, were found. The ve isolates positive for two or more virulence determinants all displayed antibiotic resis-

AMP, ampicillin; CIP, ciprooxacin; IMI, imipenem; GEN, gentamicin; VAN, vancomycin. a Susceptibility breakpoints were as follows: AMP, MIC 16 mg/L; CIP, MIC 4 mg/L; IMI, MIC 8 mg/L; GEN, MIC 500 mg/L; VAN, MIC 8 mg/L.

tance but with different resistance proles. Details of all detected virulence determinants, except espfm , and their corresponding antibiotic resistance patterns are illustrated in Table 3. 4. Discussion In this study, more than one-half of the clinical E. faecium isolates collected carried espfm . The vast majority of the espfm -positive isolates were resistant to ampicillin, ciprooxacin and imipenem. However, 12 espfm -positive isolates that were susceptible to ampicillin were found, which to our knowledge has not previously been reported in clinical E. faecium isolates. A possible explanation for the strong correlations between espfm carriage and antimicrobial resistance might be the higher conjugation frequencies observed in isolates that are espfm -positive compared with those that are espfm -negative [8]. A relatively low number of isolates were found to carry hylfm , which is in contrast to previous ndings [10,11,18]. No apparent correlation between espfm and hylfm could be identied because of the low number of hylfm -positive isolates. Other virulence traits were found in sporadic isolates collected during the last 2 years of the study. Noteworthy is the fact that only ve of the isolates harboured multiple virulence genes, whilst the remaining isolates were positive for single genes only. Although the numbers are small, there is a tendency of increasing levels of virulence genes other than espfm in the E. faecium isolates. Since the role of these genes in the development of an infection is unclear, further investigations are needed to elucidate the importance of these ndings. However, the presence of a specic gene does not automatically mean phenotypic expression of the same, as reported in a recent study where none of the seven gelE-positive E. faecium human isolates were found to produce gelatinase [19]. The cylA gene does not appear to be a characteristic in clinical E. faecium strains since it was not detected in any of the isolates. This assumption is also supported by results from other groups [11,20]. Levels of resistance to ampicillin, ciprooxacin and imipenem were steadily high during the study period. In a previous report, all E. faecium isolates from the normal intestinal microora of healthy subjects were susceptible to ampicillin [21]. Together these data indicate a non-endogenous origin of the currently investigated infection-causing isolates. This suggestion is also in agreement

Fig. 2. Antimicrobial resistance in clinical Enterococcus faecium isolates. n = total number of isolates each year. Breakpoints for antimicrobial resistance were as follows: ampicillin, minimum inhibitory concentration (MIC) 16 mg/L; ciprooxacin, MIC 4 mg/L; imipenem, MIC 8 mg/L; gentamicin, MIC 500 mg/L; and vancomycin, MIC 8 mg/L.

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with the nding of a hospital-adapted E. faecium clonal complex, CC-17, causing infections worldwide [22]. Ampicillin-resistant E. faecium are usually also simultaneously resistant to imipenem. In the present study, 16 of the imipenem-resistant isolates were susceptible to ampicillin, a rare but previously described resistance prole [23]. The number of isolates with resistance to vancomycin and gentamicin (high-level) was low, in accordance with previous reports from Sweden (http://www.STRAMA.org). As expected, no resistance against linezolid or daptomycin was found, probably reecting the novel approval and limited usage of these antimicrobial agents. In conclusion, occurrence of espfm among infection-causing E. faecium isolates was common whilst the other virulence traits were rare. A high proportion of the isolates were resistant to ampicillin, ciprooxacin and imipenem, whilst only a few isolates were resistant to vancomycin and gentamicin. Twelve of the isolates carrying the espfm gene were susceptible to ampicillin. A strong correlation between the presence of espfm and resistance against ampicillin, ciprooxacin and imipenem was found. Funding: This work was supported by grants from the Scandinavian Society for Antimicrobial Chemotherapy. Competing interests: None declared. Ethical approval: Not required. References
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