ORIGINAL ARTICLE 10.1111/j.1469-0691.2006.01533.x Genetic and phenotypic differences among Enterococcus faecalis clones from intestinal colonisation and invasive disease P. Ruiz-Garbajosa 1, R. Cantón 1, V. Pintado 2, T. M. Coque 1, R. Willems 3,4, F. Baquero 1 and R. del Campo 1 Departments of 1 Microbiology and 2 Infectious Diseases, University Hospital Ramón y Cajal, Madrid, Spain, 3 Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation and 4 Division of Acute Internal Medicine and Infectious Diseases, University Medical Center Utrecht, Utrecht, The Netherlands ABSTRACT This study investigated the differences among Enterococcus faecalis isolates from the intestinal compartment of healthy volunteers (n = 36), intensive care unit (ICU) patients (n = 29) and blood isolates (n = 31) from the same institution, in comparison with seven epidemic clones from other institutions. In general, isolates from colonised ICU patients and from bacteraemic patients showed higher rates of antimicrobial resistance than isolates from colonised healthy volunteers, particularly for erythromycin and aminoglycosides. The proportion of isolates clone was 1.05 in the community, 2.63 in the ICU, and 1.47 among bacteraemic cases, suggesting low clonal variation in ICUs. Two clones, RENC1 and RENC2, were frequently found as intestinal colonisers of ICU patients, and RENC1 was also found to colonise healthy volunteers. These two clones were a cause of bacteraemia in the institution studied, and RENC2 was also detected in various other Spanish hospitals. Both RENC1 and RENC2 were esp+, bacteriocin producers, and were resistant to all antibiotics tested except vancomycin and ampicillin. RENC1 produced haemolysin whereas RENC2 produced protease. The ace, agg, cyla, esp and gele genes were more common among colonising strains from ICU patients than among isolates from individuals in the community. In both colonised groups (ICUs and the community), 40 50% of isolates harbouring the gele and cyla genes did not express the corresponding phenotypes. Thus, the study indicated that particular E. faecalis clones might be well-adapted to hospital environments, and that surveillance should be directed specifically towards rapid detection of these disseminating clones in order to prevent infections and clonal spread. Keywords Antimicrobial resistance, colonisation, Enterococcus faecalis, invasive disease, population structure, virulence genes Original Submission: 27 September 2005; Revised Submission: 31 March 2006; Accepted: 27 April 2006 Clin Microbiol Infect 2006; 12: 1193 1198 INTRODUCTION Enterococcus spp. have traditionally been considered to be normal members of the human gut microbiota that are involved occasionally in human infections. In the nosocomial setting, the origin of enterococcal infections has been conventionally attributed to the resident strains in each patient s gut flora. However, epidemiological Corresponding author and reprint requests: R. del Campo, Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Ctra. Colmenar, Km 9.1, Madrid 28 034, Spain E-mail: rosacampo@yahoo.com studies designed to control the spread of multiresistant strains, particularly those that are vancomycin-resistant, have demonstrated the importance of cross-transmission of enterococci among inpatients, carried by hands, or fomites [1 4], and also among humans and animals [5]. The current concept of enterococcal epidemiology requires an efficient enterococcal circulation among different ecological niches, including the environment, the animal gut, the human gut in the community, and hospital patients. Nevertheless, genetic differences can be found among strains recovered from these ecological compartments, and molecular epidemiological studies of Ó 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases
1194 Clinical Microbiology and Infection, Volume 12 Number 12, December 2006 Enterococcus faecium have revealed the existence of a hospital-adapted genetic sub-population that is clearly distinct from the community and animal reservoirs [6]. The presence of specific genes associated with virulence or invasiveness has been described recently in enterococci recovered from infection sites, and these have been considered presumptively to be pathogenic traits [7]. In general, these traits should be absent (or very rare) in strains colonising the gut of healthy volunteers, or in food isolates [8]. Nevertheless, recent studies have demonstrated the presence of virulence genes in environmental and food isolates, although in such cases these genes tend to remain phenotypically silent [9 11]. Previous studies, mostly focused on vancomycin-resistant enterococci (VRE), have explored the genetic differences among colonising or pathogenic strains, and have demonstrated that specific clones are involved in outbreaks, particularly in intensive care units (ICUs) or when affecting several different institutions [12 16]. The present study was designed to study prospectively the differences in population structure and pathogenic traits among Enterococcus faecalis isolates colonising two different environments, namely ICU patients and healthy volunteers in the community. In addition, the involvement of different clones in invasive infections was studied in a set of isolates causing bacteraemia or other forms of invasive disease. Three epidemic clones from hospitals located in different regions of Spain were also investigated. MATERIALS AND METHODS Bacterial strains and sampling process Rectal swabs from 21 inpatients in four ICUs located in different areas of the Ramón y Cajal University Hospital in Madrid, Spain (medicine n = 1; general surgery n = 11; cardiovascular surgery n = 1; paediatric cardiology n = 8) were collected prospectively between March and May 2001. In parallel, faecal samples were obtained from 36 healthy volunteers (20 males and 16 females) with no previous exposure to antibiotics or treatment for at least 6 months. Only one isolate per patient was retained. Rectal swabs (or 0.5 g of faeces from the healthy volunteers) were suspended in 1 ml or 5 ml of saline, respectively, and 100-lL aliquots were inoculated on m-enterococcus agar (Difco, Detroit, MI, USA) with and without antibiotics (gentamicin 125 mg L, kanamycin 256 mg L, streptomycin 512 mg L, tetracycline 4 mg L, vancomycin 6 mg L, erythromycin 2mg L or ampicillin 10 mg L). To enhance the recovery of vancomycin-resistant enterococci (VRE), samples were also pre-enriched in brain-heart infusion (BHI) broth (Difco) containing vancomycin 6 mg L. Five colonies of each morphology were selected initially, but only one colony of each resistance phenotype and morphology was finally studied. Thirty-one enterococcal isolates from blood samples recovered in the same hospital during 2001, and seven epidemic E. faecalis clones causing invasive infections in six hospitals in the Spanish provinces of Galicia [17], Mallorca [18], Asturias, Cataluña, La Rioja and Zaragoza [14] were also studied. Bacterial identification The presence of the E. faecalis antigen A gene (efaafs) was investigated by PCR using primers efaafs-1 (5 -GACAGA- CCCTCACGAATA) and efaafs-2 (5 -AGTTCATCATGCTGT- AGTA) and the conditions described previously [9]. The blood isolates were also identified using the WIDER Automatic System (Francisco Soria Melguizo S.A, Madrid, Spain). Susceptibility testing Susceptibility to gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, minocycline, ampicillin, vancomycin, teicoplanin, quinupristin-dalfopristin, chloramphenicol, linezolid and moxifloxacin was determined by the agar dilution method, according to CLSI guidelines [19] and susceptibility breakpoints [20]. E. faecalis ATCC 29212 was used as a control strain. Antibiotics were purchased from Sigma Chemical Co. (St Louis, MO, USA) or were provided by their respective manufacturers. Presence of virulence factors The presence of the ace [8], agg, cyla, esp, gele and efaafs [9] genes, coding for adhesin to collagen, aggregation substance, cytolysin haemolysin, surface protein, gelatinase production and E. faecalis antigen A, respectively, was investigated by PCR using primers and conditions described previously [8,9]. The respective amplicons generated from the colonising 4Er1 E. faecalis strain (RENC2 clone) were sequenced and used as positive controls in each PCR. Production of haemolysin was determined following growth on Tryptic Soy Agar (TSA; Difco) containing horse blood 5% w v, with a clear zone of b-haemolysis considered to be a positive reaction. Protease activity was determined on TSA containing skimmed milk 1.5% w v, with a clear halo around the colonies after incubation for 24 h at 37 C considered to be a positive reaction. Bacteriocin production was tested as described previously [21], with plates being incubated at 37 C for 48 h and then examined for inhibition zones around the strains. Pulsed-field gel electrophoresis (PFGE) Chromosomal DNA was prepared as described previously [22], digested with SmaI, and analysed following electrophoresis (CHEF DR-II apparatus; Bio-Rad, Hercules, CA, USA) in an agarose 1.2% w v gel with 0.5 TBE buffer (89 mm Tris, 89 mm boric acid, 2 mm EDTA, ph 8) for 24 h at 6 V cm 2 and a ramped pulse time of 1 35 s. PFGE results were interpreted according to the criteria proposed by Tenover et al. [23], and by means of Phoretrix v.5.0 software (Bio-Rad) using the Dice coefficient [24]. The genetic relatedness among the isolates of this study was compared with an existing Phoretrix database
Ruiz-Garbajosa et al. Intestinal and invasive E. faecalis clones 1195 that included E. faecalis isolates from food-handlers and animal faeces. Statistical analysis Statistical significance of comparison proportions was calculated by the chi-square test, with quantitative values compared by Student s t-test; p values < 0.05 were considered to be statistically significant. RESULTS Isolates colonising ICU patients Table 1. Antibiotic resistance among enterococcal isolates cultured from healthy volunteers (HV), intensive care unit (ICU) patients or bloodstream infections Antimicrobial agent HV (36 isolates) ICU (29 isolates) Blood (31 isolates) n % n % n % Erythromycin 10 27.7 26 89.6 27 87.1 Ampicillin 0 0 0 0 0 0 Moxifloxacin 0 0 11 37.9 1 3.2 Linezolid 0 0 0 0 0 0 Chloramphenicol 10 27.7 18 62 10 32.2 Tetracycline 27 75 28 96.5 9 29 Minocycline 20 55.5 17 58.6 ND Vancomycin 0 0 0 0 0 0 Teicoplanin 0 0 0 0 0 0 HLR gentamicin 1 2.7 19 65.5 12 38.7 HLR kanamycin 9 25 25 86.2 30 96.7 HLR streptomycin 8 22.2 24 82.7 16 51.6 ND, not determined; HLR, high-level resistance. Table 2. Virulence factors identified in the enterococcal isolates from healthy volunteers (HV), intensive care unit (ICU) patients or bloodstream infections Virulence factor HV (34 clones) ICU (11 clones) Rectal swabs from 21 ICU patients yielded a total of 29 E. faecalis isolates. The antibiotic resistances found in these isolates are shown in Table 1. In general, high percentages of antibiotic resistance were observed, particularly for tetracycline (96.5%), erythromycin (90%), chloramphenicol (62%) and aminoglycosides (high-level resistance (HLR) to gentamicin, 65%; HLR to kanamycin, 86%; and HLR to streptomycin, 83%). Resistance to glycopeptides, ampicillin or linezolid was not detected. In total, 11 different E. faecalis PFGE clones were identified among the ICU isolates. Nine of these clones showed co-resistance to aminoglycosides, erythromycin and tetracycline. The ace, agg, esp and gele virulence genes were detected in 73% of the clones, while cyla was only present in 45% (Table 2). Although gele and cyla were detected in eight (73%) and five (45%) PFGE clones, respectively, protease and haemolysin production were detected under laboratory conditions in only five (45%) and three (27%) of the clones, respectively. Bacteriocin production was detected in eight (73%) of the 11 clones. Two of the 11 E. faecalis PFGE clones detected among the ICU patients proved to be widespread in the institution studied. The RENC1 clone, represented by 11 isolates recovered from all four of the ICUs studied, colonised 52% of ICU patients. The RENC2 clone, represented by five isolates from two ICUs, colonised 23% of ICU patients. Only one patient was co-colonised with both the RENC1 and the RENC2 clones. Isolates colonising healthy volunteers Blood (21 clones) n % n % n % ace 9 26.4 8 72.7 15 71.4 agg 9 26.4 8 72.7 10 47.6 cyla 7 20.5 5 45.4 2 9.5 efaa 34 100 11 100 21 100 esp 12 35.2 8 72.7 11 52.3 gele 13 38.2 8 72.7 18 85.7 Bacteriocin production 22 64.7 8 72.7 18 85.7 Protease production 7 20.5 5 45.4 11 52.3 Haemolysin production 4 11.7 3 27.2 2 9.5 The antimicrobial resistance rates among the 36 E. faecalis isolates from healthy volunteers in the community were low in comparison with the ICU isolates (Table 1), especially for erythromycin (p < 0.00001), chloramphenicol (p 0.005), HLR to gentamicin (p < 0.00001), HLR to kanamycin (p < 0.00001) and HLR to streptomycin (p < 0.00001). Only resistance to tetracycline was high (p 0.01). Resistance to ampicillin, moxifloxacin, linezolid or glycopeptides was not observed. PFGE revealed a high genetic diversity among these isolates, with 34 different clones among the 36 E. faecalis isolates. Only one of these clones was related closely to the widespread hospital RENC1 clonal type, with two band differences, and no clones related to RENC2 were encountered. The community RENC1 strain was susceptible to all antibiotics tested, except tetracycline, and only the ace virulence gene was detected (Table 3). Except for efaafs, the prevalence of the different virulence genes was relatively low (20 38%) in 22 of the 34 different community-derived clones (Table 2). The frequency of bacteriocin, protease
1196 Clinical Microbiology and Infection, Volume 12 Number 12, December 2006 Table 3. Main characteristics of the RENC1 and RENC2 clones of Enterococcus faecalis PFGE group Origin No. isolates Antibiotic susceptibility Virulence factors RENC1 ICU 10 HLR-Gm, HLR-Km, HLR-Sm, Tet, Er Bac, Hem, ace, agg, cyla, esp, gele Blood 1 HLR-Gm, HLR-Km, HLR-Sm, Tet, Er Bac, Hem, ace, agg, cyla, esp, gele RENC1 a ICU 1 HLR-Km, HLR-Sm, Tet, Er Bac, Hem, ace, agg, cyla, esp, gele RENC1 b HV 1 Tet ace RENC2 Blood 1 HLR-Km, HLR-Sm, Tet, Er Bac, ace, agg, esp, gele ICU 5 HLR-Km, HLR-Sm, Tet, Er Bac, ace, agg, esp, gele RENC2 a Blood 7 HLR-Km, HLR-Sm, Tet, Er Bac, ace, agg, esp, gele HLR-Gm, high-level resistance to gentamicin; HLR-Km, high-level resistance to kanamycin; HLR-Sm, high-level resistance to streptomycin; Tet, tetracycline; Er, erythromycin; Bac, bacteriocin production; Hem, haemolysin production. a RENC1 and C2 showed two different pulsed-field gel electrophoresis (PFGE) bands compared with the RENC1 and C2 clones. b RENC1 showed three different PFGE bands compared with RENC1. or haemolysin production was lower than among ICU isolates, although statistical significance was not reached. As with the ICU clones, protease and haemolysin production under laboratory conditions was detected only in a sub-set of isolates carrying the gele and cyla genes (seven of 13, and four of seven isolates, respectively). Bloodstream isolates Twenty-one different clones were distinguished among the 31 E. faecalis isolates from blood. All isolates identified as E. faecalis by the Wider System were positive by PCR for the efaa gene. The ICU-derived epidemic RENC1 and RENC2 clones were also identified among the blood isolates from one (3%) and eight (26%) patients, respectively (Table 3). No differences in the antibiotic resistance markers or the presence of virulence factors were detected among the colonising or invasive isolates that belonged to the same clone. RENC1 carried the haemolysin gene cyla, and the phenotypic expression of this gene was detected, while all isolates belonging to the RENC2 clone were consistently negative for this virulence factor. Four clones colonising healthy volunteers were related genetically to clones associated with cases of bacteraemia. The antibiotic resistance rates, the prevalence of virulence genes, and the production of virulence factors among blood isolates are listed in Tables 1 and 2. In general, lower percentages of antibiotic resistance were observed among the isolates causing bacteraemia than among the faecal isolates colonising ICU inpatients; however, resistance was higher than among the isolates from patients in the community. Statistically significant differences for resistance to tetracycline (p < 0.00001), chloramphenicol (p 0.02), and HLR to gentamicin (p 0.04) were observed between the isolates causing bacteraemia and those colonising ICU patients. The same trend was true for virulence genes, but only the lower prevalence of the cyla gene among the blood isolates was statistically significant (p < 0.00001). Among the E. faecalis epidemic strains causing invasive infections in six Spanish hospitals in different regions, the RENC2 clone was closely related (only one different band) [23] to the epidemic clones from La Coruña [17] and from Palma de Mallorca [18]. However, it is important to note that the epidemic clones from La Coruña and Palma de Mallorca were resistant to vancomycin, whereas the RENC2 clone from Madrid was susceptible. Differences in the other antibiotic susceptibility patterns or the presence of virulence genes were not observed, except that the Palma de Mallorca clone was susceptible to tetracycline. DISCUSSION During the last decade, many studies of VRE epidemiology have shown that particular clones are capable of intra- and inter-hospital dissemination and cause most outbreaks of nosocomial enterococcal infection [25]. Presumably, these clones originated from the community and, once introduced into hospitals, adapted progressively, probably favoured by the acquisition of antibiotic resistance genes and virulence factors. Once a given threshold is reached, these hospital-adapted clones were able to flow back into the community or the environment [26]. Hospital reacquisition of such clones after community or environmental exposure completes the epidemiological loop. However, the epidemiological circulation of particular clones has been poorly studied to date, and particularly so in the case of glycopeptide-susceptible isolates.
Ruiz-Garbajosa et al. Intestinal and invasive E. faecalis clones 1197 The present study investigated vancomycinsusceptible E. faecalis populations in faecal isolates from patients admitted to four independent ICUs in one hospital, and also from healthy volunteers. These colonising isolates were compared with contemporary isolates from blood in order to identify epidemiological trends. Using PFGE analysis, different degrees of genetic diversity were observed among isolates recovered from different origins. The proportion of isolates clone was 2.63 in ICUs, 1.47 among bacteraemic cases, and 1.05 in the community, suggesting a clonal condensation in ICUs. The relatively high clonal diversity in blood isolates suggests that numerous clones are able to cause invasive infection. The results also indicated that some clones, such as RENC1 and RENC2, are widespread as intestinal colonisers of ICU patients and also among invasive isolates. The same E. faecalis clone (RENC2) was found as a cause of bacteraemia in hospitals far from Madrid, i.e., La Coruña in the north-west of Spain [17] and Palma de Mallorca in the east [18]. This clone was not detected among the isolates colonising the healthy volunteers. Virulence genes were detected more frequently among ICU isolates than among invasive isolates, suggesting that the presence of the virulence genes analysed was not necessarily linked to pathogenicity. Comparing only the colonising isolates, virulence genes were more frequent among isolates from ICU patients than among those from healthy volunteers, with the results for ace, agg, cyla, esp and gele being statistically significant. None of the invasive isolates that belonged to the RENC2 clone carried the cyla gene or displayed corresponding haemolysin production, whereas all isolates from the RENC1 clone colonising the intestine carried and expressed this virulence factor. Moreover, while ICU isolates harboured the gele or cyla genes more frequently than isolates from communitybased patients, the proportion of isolates carrying these genes and expressing them phenotypically was similar in both groups. These genes may have a biological cost outside the hospital, perhaps also in association with environmental factors and or the quorum-sensing mechanism for autoinduced cylr1 and cylr2 regulator genes [27]. The active cyla protein consists of two subunits produced by the cylm gene that are related to bacteriocins classified as lantibiotics [28]. If these factors were important for hospital maintenance, both a higher frequency of genes and a higher rate of phenotypically expressed genes might be expected in hospitals. There was a relatively low percentage of haemolysin production among blood isolates in comparison with both groups of faecal isolates. As expected, the frequency of antibiotic resistance was much higher among blood isolates than among faecal isolates from the community, but surprisingly, the faecal ICU isolates had the highest percentages of resistance, with statistically higher levels of resistance to erythromycin, moxifloxacin, chloramphenicol, tetracycline and HLR to gentamicin. The HLR to gentamicin among E. faecalis isolates colonising ICU patients in this study was much higher (65.5%) than the figure of 20% reported in Sweden [29,30]. Antibiotic resistance may be considered to be both the effect and the cause of the adaptation of certain clones to the hospital setting. In practice, the most likely antibiotics involved in the selection of ICU clones are probably gentamicin and fluoroquinolones. Overall, the present study demonstrated that specific E. faecalis clones might be well-adapted to and disseminated in hospital environments, not only causing infection, but also colonising inpatients. Surveillance of resistant E. faecalis should be targeted specifically at the rapid detection of these highly adapted clones in order to prevent infections and further clonal spread of antibioticresistant E. faecalis strains in the hospital. The development of new molecular typing tools such as multilocus sequence typing (44th Interscience Conference on Antimicrobial Agents and Chemotherapy, abstract K-371), will provide the opportunity to evaluate the global population structure of E. faecalis, including all ecological and geographical compartments. ACKNOWLEDGEMENTS We are grateful to A. Oliver (Hospital de Sondureta, Palma de Mallorca) and G. Bou (Hospital Juan Canalejo, La Coruña) for supply of their epidemic clones. P. Ruiz-Garbajosa is the recipient of a post-mir contract from the Health Ministry in Spain. This work was supported in part by research grants from the Spanish Pneumococcal Infection Study Network (G03 103) and the Microbial Sciences Foundation. REFERENCES 1. Bonilla HF, Zervos MJ, Lyons MJ et al. Colonization with vancomycin-resistant Enterococcus faecium: comparison of a
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