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Antibiotic resistance and genotypic characterization by PFGE of clinical and environmental isolates of enterococci
FEMS Microbiology Letters 201 (2001) 205^211
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Antibiotic resistance and genotypic characterization by PFGE of clinical and environmental isolates of enterococci Giordano Dicuonzo a; *, Giovanni Gherardi a , Giulia Lorino b , Silvia Angeletti a , Fabrizio Battistoni a , Lucia Bertuccini c , Roberta Creti d , Roberta Di Rosa e , Mario Venditti e , Lucilla Baldassarri c a
Dipartimento di Medicina di Laboratorio e Microbiologia, Universita© Campus Biomedico, via Emilio Longoni 83, 00155 Rome, Italy b Dipartimento di Scienze di Sanita© Pubblica G. Sanarelli, Universita© `La Sapienza', Piazza Aldo Moro 5, 00185 Rome, Italy c Laboratorio di Ultrastrutture, Istituto Superiore di Sanita©, Viale Regina Elena 299, 00161 Rome, Italy d Laboratorio di Batteriologia e Micologia Medica, Istituto Superiore di Sanita©, Viale Regina Elena 299, 00161 Rome, Italy e Dipartimento di Medicina Clinica, Universita© `La Sapienza', Piazza Aldo Moro 5, 00185 Rome, Italy Received 12 February 2001; received in revised form 23 April 2001; accepted 23 May 2001 First published online 26 June 2001
Abstract Fifty-four Enterococcus faecalis and 20 Enterococcus faecium isolates from clinical and non-human sources in Rome, Italy, were characterized by antibiotic resistance and pulsed field gel electrophoresis (PFGE). Resistance to vancomycin, teicoplanin, ampicillin, and ciprofloxacin was more frequent in E. faecium than in E. faecalis, whereas high-level resistance to aminoglycoside was found primarily in E. faecalis. Multi-resistance was found primarily among clinical isolates, but was also observed among environmental isolates. Common genotypes shared among clinical and environmental isolates were observed, however, the majority of isolates occurred as unique, sourcespecific clones. Several PFGE types were associated with shared features in their antibiotic resistance patterns; evidences of clonal spread between and within wards were also noted. This is the first report indicating clonal relatedness between human and environmental enterococci isolated in Italy. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Pulsed ¢eld gel electrophoresis ; Antibiotic susceptibility; Enterococcus
1. Introduction Enterococcus faecalis and Enterococcus faecium account for greater than 95% of enterococcal infections in humans [1]. Enterococci are listed as the third/fourth cause of nosocomial infections [2] and there has been a rapid increase of glycopeptide and high-level aminoglycoside-resistant strains [3,4]. Fortunately, vancomycin-resistant enterococci still remain scarce in Italy [5]. Enterococci are also commonly isolated from non-human sources [6]. Elucidating the genetic relationships between human and non-human enterococcal isolates provides valuable information concerning the epidemiology of enterococcal infections. Pulsed ¢eld gel electrophoresis (PFGE) has gained wide
acceptance for establishing clonal relatedness within many bacterial species including E. faecalis and E. faecium [7^9]. In this study we determined the prevalence of di¡erent species of clinical and environmental enterococcal isolates in Rome, Italy. We compared the antibiotic resistance pattern and PFGE pro¢les of these strains and documented numerous clonal types, some of which were found both in human and environmental isolates. Certain clonal types were associated with common features in drug resistance patterns. 2. Materials and methods 2.1. Isolates
* Corresponding author. Tel. : +39 (06) 22541790; Fax: +39 (06) 22541456. E-mail address : [email protected] (G. Dicuonzo).
Sixty-four isolates of enterococci were recovered from 20 di¡erent wards of three hospitals in Rome during a
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Table 1 Identi¢cation of the 90 species of enterococci isolated from human and environmental sources Source (no. of isolates)
Species
No. of isolates
Human isolates (64)
E. faecalis E. faecium Enterococcus gallinarum Enterococcus avium
48 14 1 1
Environmental isolates (26)
E. faecalis E. faecium E. hirae Enterococcus casseli£avus Enterococcus durans/hirae
8 8 7 2 1
3-year period (1997^1999) and 26 isolates were recovered from environmental sources in the same period. The initial isolation from environmental samples was done on bileesculin azide agar incubated at 35^37³C for 24 h in 5% CO2 ; three colonies were randomly picked from each plate and identi¢ed to species level. Five out of the 64 human isolates were from the normal £ora of healthy individuals, three being isolated from the oral cavity of healthy carriers, and two being part of the vaginal £ora. The remaining 59 strains causing disease were from urinary tract infections (UTI) (12 isolates), from biliary stents (21), from the blood or endovascular devices of patients with endocarditis or endovascular infections (19), from surgical wound infections (two), from abscess (1), from ascites (one), from urinary catheter (one), and from a subhepatic drain (two). The 26 environmental isolates were recovered
from seawater (eight), wastewater (12), and wells (six) in the same city. Seawater specimens were collected far from wastewater outlet. Wastewater samples were in the catchment area of the hospitals considered. 2.2. Identi¢cation Isolates were identi¢ed to the species level according to standard biochemical tests. In cases of uncertain identi¢cation by the routine phenotypic tests PCR was performed using E. faecalis- and E. faecium-speci¢c primers as previously described [10]. PCR results were resolved by electrophoresis on a 2% agarose gel containing 0.5 Wg Wl31 of ethidium bromide. 2.3. Antimicrobial susceptibility tests Fifty-four E. faecalis and 20 E. faecium isolates from clinical and environmental sources were tested by using the Kirby^Bauer or broth microdilution system according to Swenson et al. [11,12]. Antibiotics tested were ampicillin, vancomycin, teicoplanin, tetracycline, cipro£oxacin, chloramphenicol ; high-level resistance (HLR) to gentamicin and streptomycin was also assessed. Isolates showing intermediate levels of susceptibility were classi¢ed as resistant. 2.4. PFGE analysis Chromosomal SmaI restriction patterns were determined as previously described [13] using the 74 isolates
Table 2 Antibiotic-resistant pro¢les among clinical and environmental E. faecalis and E. faecium isolates Species (no. of isolates)
Antibiotica
(No. of resistant isolates)
(%)
Clinical isolatesb
Environmental isolatesc
no.
%
no.
E. faecalis (54)
Va Tec Amp Te Cip Chl HLSm HLGm
(2) (0) (8) (33) (32) (27) (19) (19)
(4) (0) (15) (61) (59) (50) (35) (35)
2 0 8 31 26 26 18 18
4 0 17 67 56 65 39 39
0 0 0 2 6 1 1 0
E. faecium (20)
Va Tec Amp Te Cip Chl HLSm HLGm
(2) (1) (12) (12) (18) (7) (4) (2)
(10) (5) (60) (60) (90) (35) (20) (10)
1 0 9 8 11 2 4 2
8 0 69 61 85 15 31 15
1 1 3 4 7 5 0 0
a
% 0 0 0 25 75 12.5 12.5 0 14 14 43 57 100 71 0 0
According to National Committee for Clinical Laboratory Standards (NCCLS) de¢nitions. Va, vancomycin ; Tec, teicoplanin ; Amp, ampicillin ; Te, tetracycline; Cip, cipro£oxacin ; Chl, chloramphenicol; HLSm and HLGm, HLR to streptomycin and gentamicin, respectively. Isolates showing intermediate levels to antibiotics were classi¢ed as resistant. b Forty-six and 13 clinical isolates of E. faecalis and E. faecium, respectively, were tested for antimicrobial resistance. c Eight and seven environmental isolates of E. faecalis and E. faecium, respectively, were tested for antimicrobial resistance.
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Fig. 1. Genetic relatedness of the 54 E. faecalis examined, based on the PFGE banding patterns of the isolates. Strain codes and PFGE subtypes are depicted. Cases I^X indicate multiple-strain genetic clusters encountered among study E. faecalis. Environmental isolates are marked with an asterisk.
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Table 3 Genotypic characterization of 54 E. faecalis (EFS) and 20 E. faecium (EFM) isolates in correlation to the site of infection, ward, and antibiotic resistance pro¢les No. sample
Site of infection
Ward
PFGE patternd
Antibiotypea
2-EFS 59-EFS 3-EFS 7-EFS 8-EFS 9-EFS 40-EFS 17-EFS 56-EFS 33-EFS 4-EFS 21-EFS 25-EFS 10-EFS 27-EFS 11-EFS 14-EFS 30-EFS 31-EFS 29-EFS 13-EFS 44-EFS 47-EFS 12-EFS 22-EFS 52-EFS 16-EFS 35-EFS 34-EFS 37-EFS 38-EFS 20-EFS 42-EFS 45-EFS 49-EFS 15-EFS 19-EFS 26-EFS 64b-EFS 36-EFS 39-EFS 55-EFS 43-EFS 51-EFS 28-EFS 1-EFS 23-EFS 65b-EFS 32-EFS 18-EFS 67-EFS 68-EFS 69-EFS 77-EFS 5-EFM 63-EFM 6-EFM 41-EFM 46-EFM 48-EFM 64a-EFM 65a-EFM
biliary stent biliary stent biliary stent biliary stent biliary stent biliary stent pharingeal swab UTIb UTI wastewater biliary stent UTI blood biliary stent vagina biliary stent wound oral tract oral tract wastewater biliary stent vagina V^P shuntb biliary stent abscess blood UTI catheter wastewater UTI UTI UTI ascitis blood VDIb UTI UTI AVIb biliary stent wound blood blood UTI blood wastewater biliary stent blood subhepatic drain wastewater UTI well well UTI well biliary stent biliary stent biliary stent blood VDI VDI biliary stent subhepatic drain
I I I I I I II (carrier) II XVI environment I II XVIII I I (carrier) I III carrier carrier environment IV V (carrier) VI I VII VIII II IX environment X I II XI XII XIII II II XIII I XIV XIV VIII XV XII environment I XVII XIX environment II environment environment XXI environment I I I XX XXIII VIII I XIX
1 1 1.1 1.2 1.3 1.3 2 3 3 3 4 4.1 4.2 5 6 7 8 9 9 9.1 9.2 9.3 9.3 9.4 10 11 12 12.1 13 14 14.1 14.2 14.3 15 15 15.1 16 16 16 17 17 17.1 17.2 17.3 17.4 18 18 18 19 20 21 22 23 24 A A B C D E1 E2 E3
TeR, CipR, ChlR, GmR, SmR AmpR, TeR, ChlR, GmR, SmR AmpR, TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR sc s TeR, CipR CipR, ChlR, CipR, VaR, ChlR CipR CipR, ChlR, GmR TeR, ChlR, GmR s s s CipR TeR s s TeR, CipR, ChlR, SmR AmpR, TeR, CipR TeR, ChlR TeR, CipR s AmpR TeR, ChlR, VaR GmR, SmR TeR s TeR, ChlR TeR, CipR, ChlR TeR, CipR, ChlR, SmR TeR, CipR, ChlR, GmR, SmR AmpR, CipR AmpR TeR, CipR TeR, ChlR, GmR, SmR AmpR, TeR, CipR, GmR, SmR TeR, CipR, ChlR, GmR, SmR TeR,CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR AmpR, TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR CipR TeR, CipR, ChlR s CipR AmpR, TeR, CipR, ChlR CipR AmpR, TeR, CipR AmpR, TeR, CipR AmpR, TeR, GmR, SmR s AmpR, TeR, CipR AmpR, TeR, CipR, SmR AmpR, CipR AmpR, TeR, CipR, ChlR, SmR
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Table 3 (continued) No. sample
Site of infection
Ward
PFGE patternd
Antibiotypea
78-EFM 60-EFM 61-EFM 62-EFM 66-EFM 70-EFM 71-EFM 72-EFM 73-EFM 74-EFM 75-EFM 76-EFM
blood biliary stent biliary stent biliary stent well wastewater seawater seawater seawater seawater wastewater blood
XIX I I I environment environment environment environment environment environment environment VIII
E4 F F G H I J K L M N O
AmpR, TeR, CipR, SmR CipR, GmR CipR VaR, CipR TeR, CipR CipR, ChlR CipR VaR, TecR, AmpR, TeR, CipR, ChlR AmpR, CipR, ChlR AmpR, TeR, CipR, ChlR TeR, CipR, ChlR AmpR, TeR, CipR, ChlR
a
According to NCCLS de¢nitions. Te, tetracycline; Cip, cipro£oxacin; Chl, chloramphenicol; Va, vancomycin; Amp, ampicillin ; Gm and Sm, high levels to gentamicin and streptomycin, respectively. R, resistance; S, susceptibility. b UTI, urinary tract infection ; AVI, aortic valve infection ; VDI, vascular device infection; V^P shunt, ventricular^peritoneal shunt. c s, susceptible to all antibiotics. d Isolates di¡ering by 96 bands from subtype 1 for each group (type) were assigned a common type.
for which antibiotic susceptibility tests were performed. Interpretative criteria of strain relatedness were those followed by Tenover et al. [14]. 2.5. Cluster analysis PFGE types were analyzed with Bionumerics software for Windows, version 2.5 (Applied Maths). The DNA banding patterns were normalized with lambda concatemer ladder standards. Comparison of the banding patterns was performed by the unweighted pair group method with arithmetic averages and with the Dice similarity coe¤cient. A tolerance of 1.5% in band position was applied during comparison of the DNA patterns. 3. Results and discussion The majority of clinical isolates were recovered from UTI (12 isolates), from biliary stents (21 isolates), and from patients with endocarditis or endovascular infections (19 isolates). Among the 64 human isolates of enterococci the most prevalent species found was represented by E. faecalis (Table 1); of the 26 environmental isolates, E. faecalis, E. faecium, and Enterococcus hirae were the predominant species. PCR allowed species identi¢cation in 10 cases (¢ve E. faecium and ¢ve E. faecalis) where biochemical tests were unclear (data not shown). Table 2 summarizes the antibiotic resistance pro¢les found among the E. faecalis and E. faecium strains analyzed in this study. Only four isolates (two E. faecalis and two E. faecium) were found to be vancomycin-resistant, three of these being human isolates. The environmental E. faecium resistant to vancomycin was also the only teicoplanin-resistant strain found in this survey; it is noteworthy that this strain was also multi-drug-resistant. Our data con¢rm the low incidence of glycopeptide resistance
in Italy among clinical enterococci, and are in agreement with results described in a previous surveillance study [5]. Multi-drug-resistant isolates were found from both human and environmental sources (Tables 2 and 3). Three ampicillin-resistant environmental isolates, all genomically unrelated E. faecium strains, were found. The results of this survey indicate that multi-drug resistance is common among clinical isolates of enterococci, and in agreement with a previous report [15] it is found less frequently among a variety of non-clinical human and environmental aquatic sources. Interesting to note that four out of seven E. faecium aquatic isolates were resistant to at least three di¡erent antibiotics versus only two of eight E. faecalis multi-resistant environmental isolates (Table 3). We did not observe a high prevalence of HLR among environmental isolates, in contrast to a previous report [16]; however, it must be underlined that the number of strains examined in this study is much smaller than that in the referenced study. Fifty-four E. faecalis isolates and 20 E. faecium isolates were genotyped by PFGE to explore clonal relatedness of strains from environment and humans (Figs. 1 and 2). Among molecular methods used for subtyping bacterial species, PFGE is considered one of the most reliable due to its discriminatory power, sensitivity and reproducibility [7^9]. Several reports described molecular typing methods useful for epidemiologic surveillance on vancomycin-resistant enterococci [17,18]; the same methods, other than PFGE, proved to be non-appropriate or less reliable for accurate clonality studies on vancomycin-sensitive enterococci [7^9,17,18]. For these reasons we chose PFGE to genetically compare our clinical and environmental isolates of enterococci which only rarely showed glycopeptide resistance. Wide genotypic variability was found among the clinical and environmental isolates of E. faecalis and E. faecium. Twenty-four di¡erent PFGE types and 42 subtypes among E. faecalis strains, and 15 pulsed types with
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Fig. 2. Genetic relatedness of the 20 E. faecium studied. Three multiple-strain genetic groups, de¢ned as I, II, and III, were found. Strain codes, PFGE types, and dendrogram with percentages of similarity are also reported. Environmental isolates are marked with an asterisk.
18 subtypes for E. faecium isolates were identi¢ed (Figs. 1 and 2, Table 3). The majority of isolates clustered as source-speci¢c clones, although examples of common PFGE types shared among clinical and environmental E. faecalis isolates occurred. Among E. faecalis isolates, 10 of the 24 PFGE types were shared between at least two or more isolates and 14 unique PFGE types were found (Fig. 1). All the 46 E. faecalis clinical isolates grouped into one of 19 PFGE types, whereas eight di¡erent genotypes characterized the eight environmental isolates (Fig. 1, Table 3). Types 3, 9, and 17 were shared between clinical and environmental E. faecalis isolates, with one isolate recovered from wastewater sharing the identical PFGE subtype 3 with two urinary isolates (Fig. 1, Table 3). This suggests the spread of environmental strains into human facilities and vice versa. Twenty-one out of 46 E. faecalis isolates recovered from humans clustered in only four PFGE types (1, 9, 14, 17), representing 44% of all human E. faecalis strains examined (Fig. 1, Table 3). More genomic variability was seen among the 20 E. faecium isolates that were subjected to PFGE (Table 3). This was especially apparent with environmental isolates. Each of the seven environmental E. faecium isolates displayed a unique PFGE pro¢le, while eight PFGE types were shared among the 13 clinical E. faecium isolates (Fig. 2, Table 3). Consistencies of antibiotic resistance patterns were observed within several PFGE types. Among E. faecalis, a majority of isolates within PFGE types 1, 16, 17, and 18
were characterized by HLR, and also were resistant to tetracycline, cipro£oxacin, and chloramphenicol. PFGE types 3, 9, and 15 were uniformly sensitive to high-level aminoglycosides (Table 3). Interestingly, PFGE type 1 was exclusive of biliary stent group, and accounted for six of the 13 isolates (Table 3); as all six isolates were recovered from patients hospitalized in the same ward, undergoing the same invasive procedure, this is suggestive of a ward-associated clonal spread [19]. E. faecalis isolates recovered from UTI were mainly PFGE types 3 and 14 (Table 3). Several examples of isolates within the same PFGE type from di¡erent wards of the same or di¡erent hospitals were found. To our knowledge, this is one of the few studies examining the clonal relatedness between clinical and environmental enterococci. Little data have been reported from Italy regarding antibiotic resistance of enterococci from environmental sources ; genetic relationship of Italian clinical and environmental enterococci has not been previously described. We have found genetically related clusters of strains from di¡erent settings. Establishing environmental origins and direction of spread of such strains will require further investigation. Acknowledgements We gratefully acknowledge Dr. Bernard Beall, CDC, Atlanta, USA, for helpful comments and suggestions. We thank Manola Valente for the technical support in
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the cluster analysis. This work was partially supported by the Italian Ministry of Health, Project 1% `Interactions between opportunistic pathogens and biomaterials in the pathogenesis of prosthesis infections' (ICS 080.1/RS 98.43) to L.B., and by `Finanziamento progetti di Ateneo 60%', University `La Sapienza', Rome, Italy, to G.L. References
[10]
[11]
[12]
[1] Devriese, L.A., Collins, M.D. and Wirth, R. (1992) The genus Enterococcus. In: The Procaryotes. A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identi¢cation, Applications (Balows, A., Truper, H.G., Dworkin, M., Harder, W., Schleifer, K.H., Eds.), pp. 1465^1481. Springer-Verlag, New York. [2] Hunt, C.P. (1998) The emergence of enterococci as a cause of nosocomial infection. Br. J. Biomed. Sci. 55, 149^156. [3] Antalek, M.D., Mylotte, J.M., Lesse, A.J. and Sellick, J.A. (1995) Clinical and molecular epidemiology of Enterococcus faecalis bacteremia, with special reference to strains with high-level resistance to gentamicin. Clin. Infect. Dis. 20, 103^109. [4] O'Connell, N.H. and Humphreys, H. (2000) Intensive care unit design and environmental factors in the acquisition of infection. J. Hosp. Infect. 45, 255^262. [5] Fontana, R., Ligozzi, M., Mazariol, A. and Veneri, G. (1998) Resistance of enterococci to ampicillin and glycopeptide antibiotics in Italy. The Italian surveillance group for antimicrobial resistance. Clin. Infect. Dis. 27, S84^S86. [6] Facklam, R.R., Sahm, D.F. and Teixeira, L.M. (1999) Enterococcus. In: Manual of Clinical Microbiology (Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H., Eds.), pp. 297^305. American Society for Microbiology, Washington, DC. [7] Gordillo, M.E., Sigh, K.V. and Murray, B.E. (1993) Comparison of ribotyping and pulsed-¢eld gel electrophoresis for subspecies di¡erentiation of strains of Enterococcus faecalis. J. Clin. Microbiol. 31, 1570^1574. [8] Murray, B.E., Singh, K.V., Heath, J.D., Sharma, B.R. and Weinstock, G.M. (1990) Comparison of genomic DNAs of di¡erent enterococcal isolates using restriction endonucleases with infrequent recognition sites. J. Clin. Microbiol. 28, 2059^2063. [9] Chiew, Y.F. and Hall, L.M. (1998) Comparison of three methods for
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the molecular typing of Singapore isolates of enterococci with highlevel aminoglycoside resistances. J. Hosp. Infect. 38, 223^230. Dutka-Malen, S., Evers, S. and Courvalin, P. (1995) Detection of glycopeptide resistance genotypes and identi¢cation to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33, 24^27. National Committee for Clinical Laboratory Standards (1997) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A4. National Committee for Laboratory Standards, Villanova, PA. Swenson, J.M., Hindler, J.A. and Peterson, L.R. (1999) Special phenotypic methods for detecting antibacterial resistance. In: Manual of Clinical Microbiology (Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H., Eds.), pp. 1563^1577. American Society for Microbiology, Washington, DC. Gherardi, G., Whitney, C.G., Facklam, R.R. and Beall, B. (2000) Major related sets of antibiotic-resistant pneumococci in the United States as determined by pulsed-¢eld gel electrophoresis and pbplapbp2b-pbp2x-dhf restriction pro¢les. J. Infect. Dis. 181, 216^229. Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H. and Swaminathan, B. (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-¢eld gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233^2239. Pinto, B., Pierotti, R., Canale, G. and Reali, D. (1999) Characterization of `fecal streptococci' as indicators of faecal pollution and distribution in the environment. Lett. Appl. Microbiol. 29, 258^263. Rice, E.W., Messer, J.W., Johnson, C.H. and Reasoner, D.J. (1995) Occurrence of high-level aminoglycoside resistance in environmental isolates of enterococci. Appl. Environ. Microbiol. 61, 374^376. Bopp, L.H., Schoonmaker, D.J., Baltch, A.L., Smith, R.P. and Ritz, W.J. (1999) Molecular epidemiology of vancomycin-resistant enterococci from 6 hospitals in New York State. Am. J. Infect. Control 27, 411^417. Van den Braak, N., Power, E., Anthony, R., Endtz, H.P., Verbrugh, H.A. and van Belkum, A. (2000) Random ampli¢cation of polymorphic DNA versus pulsed ¢eld gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci. FEMS Microbiol. Lett. 192, 45^52. Biavasco, F., Miele, A., Vignaroli, C., Manso, E., Lupidi, R. and Varaldo, P.E. (1996) Genotypic characterization of a nosocomial outbreak of VanA Enterococcus faecalis. Microb. Drug Resist. 2, 231^ 237.
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Antibiotic resistance and genotypic characterization by PFGE of clinical and environmental isolates of enterococci Giordano Dicuonzo a; *, Giovanni Gherardi a , Giulia Lorino b , Silvia Angeletti a , Fabrizio Battistoni a , Lucia Bertuccini c , Roberta Creti d , Roberta Di Rosa e , Mario Venditti e , Lucilla Baldassarri c a
Dipartimento di Medicina di Laboratorio e Microbiologia, Universita© Campus Biomedico, via Emilio Longoni 83, 00155 Rome, Italy b Dipartimento di Scienze di Sanita© Pubblica G. Sanarelli, Universita© `La Sapienza', Piazza Aldo Moro 5, 00185 Rome, Italy c Laboratorio di Ultrastrutture, Istituto Superiore di Sanita©, Viale Regina Elena 299, 00161 Rome, Italy d Laboratorio di Batteriologia e Micologia Medica, Istituto Superiore di Sanita©, Viale Regina Elena 299, 00161 Rome, Italy e Dipartimento di Medicina Clinica, Universita© `La Sapienza', Piazza Aldo Moro 5, 00185 Rome, Italy Received 12 February 2001; received in revised form 23 April 2001; accepted 23 May 2001 First published online 26 June 2001
Abstract Fifty-four Enterococcus faecalis and 20 Enterococcus faecium isolates from clinical and non-human sources in Rome, Italy, were characterized by antibiotic resistance and pulsed field gel electrophoresis (PFGE). Resistance to vancomycin, teicoplanin, ampicillin, and ciprofloxacin was more frequent in E. faecium than in E. faecalis, whereas high-level resistance to aminoglycoside was found primarily in E. faecalis. Multi-resistance was found primarily among clinical isolates, but was also observed among environmental isolates. Common genotypes shared among clinical and environmental isolates were observed, however, the majority of isolates occurred as unique, sourcespecific clones. Several PFGE types were associated with shared features in their antibiotic resistance patterns; evidences of clonal spread between and within wards were also noted. This is the first report indicating clonal relatedness between human and environmental enterococci isolated in Italy. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Pulsed ¢eld gel electrophoresis ; Antibiotic susceptibility; Enterococcus
1. Introduction Enterococcus faecalis and Enterococcus faecium account for greater than 95% of enterococcal infections in humans [1]. Enterococci are listed as the third/fourth cause of nosocomial infections [2] and there has been a rapid increase of glycopeptide and high-level aminoglycoside-resistant strains [3,4]. Fortunately, vancomycin-resistant enterococci still remain scarce in Italy [5]. Enterococci are also commonly isolated from non-human sources [6]. Elucidating the genetic relationships between human and non-human enterococcal isolates provides valuable information concerning the epidemiology of enterococcal infections. Pulsed ¢eld gel electrophoresis (PFGE) has gained wide
acceptance for establishing clonal relatedness within many bacterial species including E. faecalis and E. faecium [7^9]. In this study we determined the prevalence of di¡erent species of clinical and environmental enterococcal isolates in Rome, Italy. We compared the antibiotic resistance pattern and PFGE pro¢les of these strains and documented numerous clonal types, some of which were found both in human and environmental isolates. Certain clonal types were associated with common features in drug resistance patterns. 2. Materials and methods 2.1. Isolates
* Corresponding author. Tel. : +39 (06) 22541790; Fax: +39 (06) 22541456. E-mail address : [email protected] (G. Dicuonzo).
Sixty-four isolates of enterococci were recovered from 20 di¡erent wards of three hospitals in Rome during a
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Table 1 Identi¢cation of the 90 species of enterococci isolated from human and environmental sources Source (no. of isolates)
Species
No. of isolates
Human isolates (64)
E. faecalis E. faecium Enterococcus gallinarum Enterococcus avium
48 14 1 1
Environmental isolates (26)
E. faecalis E. faecium E. hirae Enterococcus casseli£avus Enterococcus durans/hirae
8 8 7 2 1
3-year period (1997^1999) and 26 isolates were recovered from environmental sources in the same period. The initial isolation from environmental samples was done on bileesculin azide agar incubated at 35^37³C for 24 h in 5% CO2 ; three colonies were randomly picked from each plate and identi¢ed to species level. Five out of the 64 human isolates were from the normal £ora of healthy individuals, three being isolated from the oral cavity of healthy carriers, and two being part of the vaginal £ora. The remaining 59 strains causing disease were from urinary tract infections (UTI) (12 isolates), from biliary stents (21), from the blood or endovascular devices of patients with endocarditis or endovascular infections (19), from surgical wound infections (two), from abscess (1), from ascites (one), from urinary catheter (one), and from a subhepatic drain (two). The 26 environmental isolates were recovered
from seawater (eight), wastewater (12), and wells (six) in the same city. Seawater specimens were collected far from wastewater outlet. Wastewater samples were in the catchment area of the hospitals considered. 2.2. Identi¢cation Isolates were identi¢ed to the species level according to standard biochemical tests. In cases of uncertain identi¢cation by the routine phenotypic tests PCR was performed using E. faecalis- and E. faecium-speci¢c primers as previously described [10]. PCR results were resolved by electrophoresis on a 2% agarose gel containing 0.5 Wg Wl31 of ethidium bromide. 2.3. Antimicrobial susceptibility tests Fifty-four E. faecalis and 20 E. faecium isolates from clinical and environmental sources were tested by using the Kirby^Bauer or broth microdilution system according to Swenson et al. [11,12]. Antibiotics tested were ampicillin, vancomycin, teicoplanin, tetracycline, cipro£oxacin, chloramphenicol ; high-level resistance (HLR) to gentamicin and streptomycin was also assessed. Isolates showing intermediate levels of susceptibility were classi¢ed as resistant. 2.4. PFGE analysis Chromosomal SmaI restriction patterns were determined as previously described [13] using the 74 isolates
Table 2 Antibiotic-resistant pro¢les among clinical and environmental E. faecalis and E. faecium isolates Species (no. of isolates)
Antibiotica
(No. of resistant isolates)
(%)
Clinical isolatesb
Environmental isolatesc
no.
%
no.
E. faecalis (54)
Va Tec Amp Te Cip Chl HLSm HLGm
(2) (0) (8) (33) (32) (27) (19) (19)
(4) (0) (15) (61) (59) (50) (35) (35)
2 0 8 31 26 26 18 18
4 0 17 67 56 65 39 39
0 0 0 2 6 1 1 0
E. faecium (20)
Va Tec Amp Te Cip Chl HLSm HLGm
(2) (1) (12) (12) (18) (7) (4) (2)
(10) (5) (60) (60) (90) (35) (20) (10)
1 0 9 8 11 2 4 2
8 0 69 61 85 15 31 15
1 1 3 4 7 5 0 0
a
% 0 0 0 25 75 12.5 12.5 0 14 14 43 57 100 71 0 0
According to National Committee for Clinical Laboratory Standards (NCCLS) de¢nitions. Va, vancomycin ; Tec, teicoplanin ; Amp, ampicillin ; Te, tetracycline; Cip, cipro£oxacin ; Chl, chloramphenicol; HLSm and HLGm, HLR to streptomycin and gentamicin, respectively. Isolates showing intermediate levels to antibiotics were classi¢ed as resistant. b Forty-six and 13 clinical isolates of E. faecalis and E. faecium, respectively, were tested for antimicrobial resistance. c Eight and seven environmental isolates of E. faecalis and E. faecium, respectively, were tested for antimicrobial resistance.
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207
Fig. 1. Genetic relatedness of the 54 E. faecalis examined, based on the PFGE banding patterns of the isolates. Strain codes and PFGE subtypes are depicted. Cases I^X indicate multiple-strain genetic clusters encountered among study E. faecalis. Environmental isolates are marked with an asterisk.
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Table 3 Genotypic characterization of 54 E. faecalis (EFS) and 20 E. faecium (EFM) isolates in correlation to the site of infection, ward, and antibiotic resistance pro¢les No. sample
Site of infection
Ward
PFGE patternd
Antibiotypea
2-EFS 59-EFS 3-EFS 7-EFS 8-EFS 9-EFS 40-EFS 17-EFS 56-EFS 33-EFS 4-EFS 21-EFS 25-EFS 10-EFS 27-EFS 11-EFS 14-EFS 30-EFS 31-EFS 29-EFS 13-EFS 44-EFS 47-EFS 12-EFS 22-EFS 52-EFS 16-EFS 35-EFS 34-EFS 37-EFS 38-EFS 20-EFS 42-EFS 45-EFS 49-EFS 15-EFS 19-EFS 26-EFS 64b-EFS 36-EFS 39-EFS 55-EFS 43-EFS 51-EFS 28-EFS 1-EFS 23-EFS 65b-EFS 32-EFS 18-EFS 67-EFS 68-EFS 69-EFS 77-EFS 5-EFM 63-EFM 6-EFM 41-EFM 46-EFM 48-EFM 64a-EFM 65a-EFM
biliary stent biliary stent biliary stent biliary stent biliary stent biliary stent pharingeal swab UTIb UTI wastewater biliary stent UTI blood biliary stent vagina biliary stent wound oral tract oral tract wastewater biliary stent vagina V^P shuntb biliary stent abscess blood UTI catheter wastewater UTI UTI UTI ascitis blood VDIb UTI UTI AVIb biliary stent wound blood blood UTI blood wastewater biliary stent blood subhepatic drain wastewater UTI well well UTI well biliary stent biliary stent biliary stent blood VDI VDI biliary stent subhepatic drain
I I I I I I II (carrier) II XVI environment I II XVIII I I (carrier) I III carrier carrier environment IV V (carrier) VI I VII VIII II IX environment X I II XI XII XIII II II XIII I XIV XIV VIII XV XII environment I XVII XIX environment II environment environment XXI environment I I I XX XXIII VIII I XIX
1 1 1.1 1.2 1.3 1.3 2 3 3 3 4 4.1 4.2 5 6 7 8 9 9 9.1 9.2 9.3 9.3 9.4 10 11 12 12.1 13 14 14.1 14.2 14.3 15 15 15.1 16 16 16 17 17 17.1 17.2 17.3 17.4 18 18 18 19 20 21 22 23 24 A A B C D E1 E2 E3
TeR, CipR, ChlR, GmR, SmR AmpR, TeR, ChlR, GmR, SmR AmpR, TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR sc s TeR, CipR CipR, ChlR, CipR, VaR, ChlR CipR CipR, ChlR, GmR TeR, ChlR, GmR s s s CipR TeR s s TeR, CipR, ChlR, SmR AmpR, TeR, CipR TeR, ChlR TeR, CipR s AmpR TeR, ChlR, VaR GmR, SmR TeR s TeR, ChlR TeR, CipR, ChlR TeR, CipR, ChlR, SmR TeR, CipR, ChlR, GmR, SmR AmpR, CipR AmpR TeR, CipR TeR, ChlR, GmR, SmR AmpR, TeR, CipR, GmR, SmR TeR, CipR, ChlR, GmR, SmR TeR,CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR AmpR, TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR TeR, CipR, ChlR, GmR, SmR CipR TeR, CipR, ChlR s CipR AmpR, TeR, CipR, ChlR CipR AmpR, TeR, CipR AmpR, TeR, CipR AmpR, TeR, GmR, SmR s AmpR, TeR, CipR AmpR, TeR, CipR, SmR AmpR, CipR AmpR, TeR, CipR, ChlR, SmR
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Table 3 (continued) No. sample
Site of infection
Ward
PFGE patternd
Antibiotypea
78-EFM 60-EFM 61-EFM 62-EFM 66-EFM 70-EFM 71-EFM 72-EFM 73-EFM 74-EFM 75-EFM 76-EFM
blood biliary stent biliary stent biliary stent well wastewater seawater seawater seawater seawater wastewater blood
XIX I I I environment environment environment environment environment environment environment VIII
E4 F F G H I J K L M N O
AmpR, TeR, CipR, SmR CipR, GmR CipR VaR, CipR TeR, CipR CipR, ChlR CipR VaR, TecR, AmpR, TeR, CipR, ChlR AmpR, CipR, ChlR AmpR, TeR, CipR, ChlR TeR, CipR, ChlR AmpR, TeR, CipR, ChlR
a
According to NCCLS de¢nitions. Te, tetracycline; Cip, cipro£oxacin; Chl, chloramphenicol; Va, vancomycin; Amp, ampicillin ; Gm and Sm, high levels to gentamicin and streptomycin, respectively. R, resistance; S, susceptibility. b UTI, urinary tract infection ; AVI, aortic valve infection ; VDI, vascular device infection; V^P shunt, ventricular^peritoneal shunt. c s, susceptible to all antibiotics. d Isolates di¡ering by 96 bands from subtype 1 for each group (type) were assigned a common type.
for which antibiotic susceptibility tests were performed. Interpretative criteria of strain relatedness were those followed by Tenover et al. [14]. 2.5. Cluster analysis PFGE types were analyzed with Bionumerics software for Windows, version 2.5 (Applied Maths). The DNA banding patterns were normalized with lambda concatemer ladder standards. Comparison of the banding patterns was performed by the unweighted pair group method with arithmetic averages and with the Dice similarity coe¤cient. A tolerance of 1.5% in band position was applied during comparison of the DNA patterns. 3. Results and discussion The majority of clinical isolates were recovered from UTI (12 isolates), from biliary stents (21 isolates), and from patients with endocarditis or endovascular infections (19 isolates). Among the 64 human isolates of enterococci the most prevalent species found was represented by E. faecalis (Table 1); of the 26 environmental isolates, E. faecalis, E. faecium, and Enterococcus hirae were the predominant species. PCR allowed species identi¢cation in 10 cases (¢ve E. faecium and ¢ve E. faecalis) where biochemical tests were unclear (data not shown). Table 2 summarizes the antibiotic resistance pro¢les found among the E. faecalis and E. faecium strains analyzed in this study. Only four isolates (two E. faecalis and two E. faecium) were found to be vancomycin-resistant, three of these being human isolates. The environmental E. faecium resistant to vancomycin was also the only teicoplanin-resistant strain found in this survey; it is noteworthy that this strain was also multi-drug-resistant. Our data con¢rm the low incidence of glycopeptide resistance
in Italy among clinical enterococci, and are in agreement with results described in a previous surveillance study [5]. Multi-drug-resistant isolates were found from both human and environmental sources (Tables 2 and 3). Three ampicillin-resistant environmental isolates, all genomically unrelated E. faecium strains, were found. The results of this survey indicate that multi-drug resistance is common among clinical isolates of enterococci, and in agreement with a previous report [15] it is found less frequently among a variety of non-clinical human and environmental aquatic sources. Interesting to note that four out of seven E. faecium aquatic isolates were resistant to at least three di¡erent antibiotics versus only two of eight E. faecalis multi-resistant environmental isolates (Table 3). We did not observe a high prevalence of HLR among environmental isolates, in contrast to a previous report [16]; however, it must be underlined that the number of strains examined in this study is much smaller than that in the referenced study. Fifty-four E. faecalis isolates and 20 E. faecium isolates were genotyped by PFGE to explore clonal relatedness of strains from environment and humans (Figs. 1 and 2). Among molecular methods used for subtyping bacterial species, PFGE is considered one of the most reliable due to its discriminatory power, sensitivity and reproducibility [7^9]. Several reports described molecular typing methods useful for epidemiologic surveillance on vancomycin-resistant enterococci [17,18]; the same methods, other than PFGE, proved to be non-appropriate or less reliable for accurate clonality studies on vancomycin-sensitive enterococci [7^9,17,18]. For these reasons we chose PFGE to genetically compare our clinical and environmental isolates of enterococci which only rarely showed glycopeptide resistance. Wide genotypic variability was found among the clinical and environmental isolates of E. faecalis and E. faecium. Twenty-four di¡erent PFGE types and 42 subtypes among E. faecalis strains, and 15 pulsed types with
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Fig. 2. Genetic relatedness of the 20 E. faecium studied. Three multiple-strain genetic groups, de¢ned as I, II, and III, were found. Strain codes, PFGE types, and dendrogram with percentages of similarity are also reported. Environmental isolates are marked with an asterisk.
18 subtypes for E. faecium isolates were identi¢ed (Figs. 1 and 2, Table 3). The majority of isolates clustered as source-speci¢c clones, although examples of common PFGE types shared among clinical and environmental E. faecalis isolates occurred. Among E. faecalis isolates, 10 of the 24 PFGE types were shared between at least two or more isolates and 14 unique PFGE types were found (Fig. 1). All the 46 E. faecalis clinical isolates grouped into one of 19 PFGE types, whereas eight di¡erent genotypes characterized the eight environmental isolates (Fig. 1, Table 3). Types 3, 9, and 17 were shared between clinical and environmental E. faecalis isolates, with one isolate recovered from wastewater sharing the identical PFGE subtype 3 with two urinary isolates (Fig. 1, Table 3). This suggests the spread of environmental strains into human facilities and vice versa. Twenty-one out of 46 E. faecalis isolates recovered from humans clustered in only four PFGE types (1, 9, 14, 17), representing 44% of all human E. faecalis strains examined (Fig. 1, Table 3). More genomic variability was seen among the 20 E. faecium isolates that were subjected to PFGE (Table 3). This was especially apparent with environmental isolates. Each of the seven environmental E. faecium isolates displayed a unique PFGE pro¢le, while eight PFGE types were shared among the 13 clinical E. faecium isolates (Fig. 2, Table 3). Consistencies of antibiotic resistance patterns were observed within several PFGE types. Among E. faecalis, a majority of isolates within PFGE types 1, 16, 17, and 18
were characterized by HLR, and also were resistant to tetracycline, cipro£oxacin, and chloramphenicol. PFGE types 3, 9, and 15 were uniformly sensitive to high-level aminoglycosides (Table 3). Interestingly, PFGE type 1 was exclusive of biliary stent group, and accounted for six of the 13 isolates (Table 3); as all six isolates were recovered from patients hospitalized in the same ward, undergoing the same invasive procedure, this is suggestive of a ward-associated clonal spread [19]. E. faecalis isolates recovered from UTI were mainly PFGE types 3 and 14 (Table 3). Several examples of isolates within the same PFGE type from di¡erent wards of the same or di¡erent hospitals were found. To our knowledge, this is one of the few studies examining the clonal relatedness between clinical and environmental enterococci. Little data have been reported from Italy regarding antibiotic resistance of enterococci from environmental sources ; genetic relationship of Italian clinical and environmental enterococci has not been previously described. We have found genetically related clusters of strains from di¡erent settings. Establishing environmental origins and direction of spread of such strains will require further investigation. Acknowledgements We gratefully acknowledge Dr. Bernard Beall, CDC, Atlanta, USA, for helpful comments and suggestions. We thank Manola Valente for the technical support in
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the cluster analysis. This work was partially supported by the Italian Ministry of Health, Project 1% `Interactions between opportunistic pathogens and biomaterials in the pathogenesis of prosthesis infections' (ICS 080.1/RS 98.43) to L.B., and by `Finanziamento progetti di Ateneo 60%', University `La Sapienza', Rome, Italy, to G.L. References
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