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Journal of Clinical Microbiology, February 2009, p. 345-351, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01597-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Longer Intestinal Persistence of Enterococcus faecalis Compared to Enterococcus faecium Clones in Intensive-Care-Unit Patients

Patricia Ruiz-Garbajosa,^1,2 Rosa del Campo,^1,2 Teresa M. Coque,^1,2 Angel Asensio,³ Marc Bonten,⁴ Rob Willems,⁴ Fernando Baquero,^1,2 and Rafael Cantón^1,2*

Servicio de Microbiología, Hospital Universitario Ramón y Cajal, and CIBER en Epidemiología y Salud Pública (CIBERESP), Madrid 28034, Spain,¹ Unidad de Resistencia a Antibióticos y Virulencia Bacteriana, Asociada al Consejo Superior de Investigaciones Científicas (CSIC), Hospital Ramón y Cajal, Madrid 28034, Spain,² Servicio de Medicina Preventiva, Hospital Universitario Puerta de Hierro, Madrid 28035, Spain,³ Department of Medical Microbiology, University Medical Center, Utrecht 3584 CX, The Netherlands⁴

Received 18 August 2008/ Returned for modification 4 November 2008/ Accepted 25 November 2008

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The dynamics of intestinal colonization with enterococcal clones in intensive-care-unit (ICU) patients was evaluated. Eight patients admitted directly to the neurosurgical ICU at the Ramón y Cajal University Hospital (Madrid, Spain) from the community and with no overlapping stay during a 10-month period in 2006 were studied. Rectal swab specimens were collected on admission and daily until the patients were discharged. Clonality was determined by pulsed-field gel electrophoresis and multilocus sequence typing. Clonal colonization dynamics were estimated by using two new parameters: the clonal diversity per patient per day (CDPD) and the clonal persistence ratio (CPR). Enterococcus faecalis isolates (n = 123) and Enterococcus faecium isolates (n = 66) were resolved into 13 and 15 clones, respectively. The CDPD of E. faecalis steadily increased during admission, and E. faecalis showed a higher (P = 0.001) CPR value than E. faecium (0.86 and 0.42, respectively). E. faecium, with the exception of an ampicillin-resistant clone belonging to clonal complex 17, frequently appeared as a short-term colonizer, even though the E. faecalis clones had significantly (P = 0.03) more days under antibiotic exposure than E. faecium (77.5 and 65 days/100 colonization days, respectively). E. faecalis had a longer persistence than E. faecium, except for the CC17 ampicillin-resistant clone, and E. faecalis showed a cumulative increase in CDPD, whereas E. faecium did not. CDPD and CPR were useful for measuring the dynamics of intestinal colonization with enterococcal clones.

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Intensive care units (ICUs) are hospital compartments that present the highest frequencies of nosocomial infection with multiresistant bacteria due to the combination of the presence of seriously ill patients, frequent events of cross transmission, and high levels of antibiotic pressure. Enterococci have emerged as a significant cause of nosocomial infections, causing about 7% of infections among ICU patients (28). Traditionally, enterococcal infections were thought to be of endogenous origin, but modern typing systems have permitted the identification of genotypes that are well adapted to the hospital setting and that are frequently cross transmitted. Population structure studies based on multilocus sequence typing (MLST) data for Enterococcus faecalis (25) and Enterococcus faecium (14), the enterococcal species most often isolated from clinical infections, have revealed the existence of particular clonal complexes (CCs) that are frequently selected in the hospital environment. These CCs have been named high-risk CCs (HrCCs), as they are the cause of most enterococcal infections and hospital outbreaks all over the world (18).

Bacterial persistence in hosts constitutes an important factor driving both bacterial transmission and infection. Different studies that have focused on the longitudinal follow-up of patients colonized with multiresistant enterococci, especially vancomycin-resistant isolates, demonstrated the prolonged persistence of vancomycin-resistant enterococci in the intestinal tract of hospitalized patients (3, 22). Exposure to extended-spectrum cephalosporins and long ICU or hospital stays are commonly associated with prolonged colonization with multidrug-resistant enterococci (2, 4). Studies that have focused on the dynamics of colonization with enterococci have demonstrated a trend for the increased acquisition of resistant genotypes and cross-transmission episodes during patients' hospitalizations (11, 21). However, prospective studies on the persistence of specific enterococcal clones and the colonization dynamics during patients' ICU stays are still lacking. The same is true for the investigation of differences in persistence among E. faecalis and E. faecium clones, both multiresistant and nonmultiresistant ones. To address these points, we designed the present study to evaluate the dynamics of the enterococcal clonal populations in the intestinal compartment of ICU patients and introduced new indexes that can be used to measure bacterial persistence.

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Patients and study design. The Ramón y Cajal University Hospital is a 1,100-bed university hospital in Madrid, Spain. The study was conducted in the neurosurgical ICU (N-ICU) over 10 months in 2006. The N-ICU has eight beds, and during the time of the study, the occupancy rate was 0.81 and the rotation rate was 0.71. The nurse/patient ratio was 0.46. Patients were included in the study if they met all of the following criteria: (i) they were 18 years of age; (ii) they had an expected length of stay (LOS) of >4 days (which is longer than the average for this ICU); (iii) they were admitted to the N-ICU directly from the community setting due to an acute trauma; (iv) they had had no previous admissions to other ICUs and/or hospital wards during the previous 3 months or during the same hospitalization period in another ICU or ward of the hospital; (v) they had no previous stay in day care centers; and (vi) there was no overlap in time with another patient already included in the study, in order to avoid biases in clonal composition due to direct patient-to-patient transmission. Rectal swab specimens were collected from each patient on admission and daily until they were discharged from the N-ICU. Medical records were reviewed; and the following information was collected: sex, age, reason for admission, LOS, previous hospital admissions, and antibiotic therapy during the hospitalization. The study was approved by the local ethics committee.

Sampling. The rectal swab specimens were suspended in 1 ml of saline; and aliquots of 100 µl were seeded on m-Enterococcus agar (Difco, Detroit, MI) plates with and without 125 µg/ml of gentamicin, 256 µg/ml of kanamycin, 512 µg/ml of streptomycin, 4 µg/ml of tetracycline, 6 µg/ml of vancomycin, 2 µg/ml of erythromycin, and 10 µg/ml of ampicillin. Five colonies of each different morphology were subcultured from each agar plate with and without antibiotics. In order to reduce duplicate isolates, all colonies recovered were newly seeded on the antibiotic plates described above to determine the resistance phenotype. Only one colony of each resistance phenotype and morphology was finally selected for further analysis.

Bacterial identification and susceptibility testing. Identification of E. faecalis and E. faecium was performed by amplification of species-specific genes coding for EfaA and and AAC(6')-Ii, respectively (6). Other Enterococcus species were identified by using API Rapid galleries (bioMérieux, la Balme les Grottes, France). Antibiotic susceptibility testing was performed by the standard microdilution technique, and the results were interpreted according to the guidelines of the CLSI (5).

Clonal diversity analysis. Clonal relatedness was established by pulsed-field gel electrophoresis (PFGE) (6). One representative strain of each one of the E. faecalis and E. faecium PFGE types was further studied by MLST (14, 25). Sequence types (STs) were assigned according to the STs in the MLST database (http://www.mlst.net). The PFGE patterns were interpreted by using the criteria suggested by Tenover et al. (30), with isolates with closely related patterns (no more than three bands of difference) being designated as belonging to a single clone. The genetic diversity (GD) of the E. faecalis and E. faecium populations was expressed as the ratio of distinguishable PFGE and MLST genotypes among the total number of recovered isolates. A hospital-acquired clone was defined as a strain that was first isolated more than 48 h after patient admission to the ICU and that was absent in the admission culture samples. An endogenous patient clone was defined as a strain isolated at admission or during the first 48 h of the hospital stay.

Clonal colonization parameters. Two new parameters were designed to evaluate the dynamics of E. faecalis and E. faecium clonal colonization among the N-ICU patients. Both of them are ecological parameters, in which the sequentially enrolled patients serve as successive observation points for clonal persistence in the N-ICU environment. The first parameter was the clonal diversity per patient per day (CDPD), expressed as the number of clones detected per day divided by the number of patients per day, and was computed for every single day of the hospital stay for all patients under observation. Changes in this ratio describe variations in the genetic diversity of the E. faecalis and E. faecium isolates from N-ICU patients across their hospitalizations. The second parameter, the clonal persistence ratio (CPR), expresses the ratio of the number of days in which a specific clone was found to colonize a patient to the number of hospitalization days since the first positive culture with that specific clone.

Clonal selective antimicrobial exposure. The antimicrobial treatment that every patient received might influence the enterococcal clonal diversity and/or persistence. The number of days that antimicrobial agents were administered among the N-ICU patients was calculated, and the results were expressed as the number of days of antimicrobial exposure per 100 colonization days for individual E. faecalis or E. faecium clones and for the total number of clones of both enterococcal species identified. Furthermore, antimicrobial agents were divided into effective and ineffective categories, according to their corresponding intrinsic and in vitro activities against every E. faecium and E. faecalis clone. The numbers of days of exposure to effective and ineffective antimicrobials per 100 colonization days for the E. faecalis and E. faecium clones were also calculated. When a patient received simultaneous therapy with an effective antimicrobial and an ineffective antimicrobial, the combination was considered effective against enterococci.

Statistical analysis. Categorical variables were compared by the ² test, while the mean CDPD indexes were compared by the paired t test. Linear regression and the Spearman coefficient were used to correlate CDPD and the length of hospitalization. All tests of significance were two tailed and were set at 0.05. Mean values are reported with standard deviations.

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Patient characteristics. Eight patients (five females) with a mean age of 50.6 ± 18.6 years (age range, 26 to 88 years) were enrolled during the study period (one patient per month). The mean LOS in the N-ICU was 12.2 ± 4 days (LOS range, 7 to 17 days). None of the patients were hospitalized at the same time, and all patients were admitted directly to the N-ICU because of subarachnoidal hemorrhage and/or head trauma. Two of eight patients (patients 1 and 8) had already been hospitalized 12 and 5 months before their N-ICU admissions, respectively. The previous admissions were in medical or surgical wards.

Bacterial isolates and genetic diversity. Two hundred eight enterococcal isolates (123 E. faecalis isolates, 66 E. faecium isolates, 17 Enterococcus avium isolates, and 2 Enterococcus casseliflavus isolates) were recovered from 100 rectal swab specimens from the eight patients according to the stated criteria. All swabs contained enterococcal isolates. These isolates were grouped into 32 PFGE types (13 for E. faecalis, 15 for E. faecium, 3 for E. avium, and 1 for E. casseliflavus) (Tables 1 and 2). The PFGE patterns for isolates from the same patient were nearly stable during the observation time, as only two clones, E. faecalis 3 (Efc-3) (ST17) and Efc-4 (ST16), presented two different PFGE patterns (differences of one or two bands) and coexisted in the same patient on the same day. E. faecalis isolates belonging to each PFGE type corresponded to 13 different STs (Table 1). E. faecium isolates belonging to each PFGE type corresponded to 14 different STs, since the E. faecium 13 (Efm-13) and Efm-19 PFGE types were grouped in ST32 (Table 2). Among these E. faecium PFGE types, only one ampicillin-resistant clone (Efm-6, ST18) belonged to the previously described clone E. faecium HrCC17 (4) (Table 2). The GDs determined by using the results of PFGE were higher for the E. faecium population (GD = 0.23) than for the E. faecalis (GD = 0.11) and the E. avium (GD = 0.18) populations. The GDs were similar for E. faecium (GD = 0.21) and E. faecalis (GD = 0.11) when MLST data were used.

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TABLE 1. Characteristics of E. faecalis clonesa

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TABLE 2. Characteristics of E. faecium clonesa

Dynamics of enterococcal clonal colonization. E. faecalis and E. faecium clones were detected in all patients with the exception of patient 4, who was colonized only by E. faecium. The numbers of enterococcal populations between the patients varied considerably, such that the number of clones detected ranged from 2 (in patients 4, 6, and 7) to 15 in patient 2. Three E. faecalis clones (clones Efc-1 [ST44], Efc-3 [ST17], and Efc-4 [ST16]) (Table 1) and five E. faecium clones (clones Efm-6 [ST18], Efm-7 [ST22], Efm-14 [ST228], Efm-21 [ST289], and Efm-22 [ST416]) (Table 2) were isolated from different patients, whereas all three E. avium clones were detected only in patient 5 and the E. casseliflavus clone was detected only in patient 8.

Six of the 13 E. faecalis clones (46%) were acquired during the patient's N-ICU admission. Clone Efc-3, which belonged to HrCC9 (18), was acquired by patients 1 and 2. Clone Efc-4, which corresponded to ST16, was acquired by patients 1 and 2 and was found to be endogenous in patients 5 and 8 (Table 1). Among the 15 E. faecium clones, 9 of them were considered acquired (60%). Clone Efm-6, which belonged to HrCC17 (18), was isolated from patient 1 from the beginning of the N-ICU stay, whereas it was acquired by patient 5 (Table 2).

CDPD. On average, we observed a higher index of CDPD for E. faecalis (1.74) than for E. faecium (0.85). Furthermore, throughout the hospitalization period the CDPD showed a statistically significant positive trend for E. faecalis (regression coefficient = 0.089; 95% confidence interval = 0.071 to 0.955; r² = 0.51), while this trend was negative for E. faecium (regression coefficient = –0.031; 95% confidence interval = –0.025 to –0.037; r² = 0.53). Furthermore, the CDPD among the E. faecalis isolates increased, irrespective of whether HrCC9 was included. In contrast, the CDPD among the E. faecium isolates increased only when HrCC17 was included in the analysis (Fig. 1).

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FIG. 1. CDPD values for E. faecalis (A) and E. faecium (B) throughout the hospital stay. The gray areas represent the CDPD values for all E. faecalis (A) and all E. faecium (B) clones, with the exception of E. faecalis HrCC9 and E. faecium HrCC17, respectively. The light gray areas represent the increases in the CDPD values when E. faecalis HrCC9 and E. faecium HrCC17 are included. No statistically significant difference in the E. faecalis CDPD increase when HrCC9 was considered was found, while the differences in the E. faecium CDPD values with and without HrCC17 were statistically significant (P = 0.03).

CPR. The CPR value was significantly higher for the E. faecalis clones (0.86) than for the E. faecium clones (0.42) (P = 0.001) (Fig. 2). During the study period and by considering the entire patient population, the E. faecalis clones accounted for 169 colonization days, whereas the E. faecium clones accounted for 80 colonization days. The mean numbers of colonization days per clone were 13 ± 12.6 and 5.3 ± 5.35 days, respectively. The pooled CPR was significantly (P = 0.001) higher for endogenous E. faecalis clones (0.92) than for endogenous E. faecium clones (0.46) (Fig. 2). Endogenous E. faecalis clones, detected from the beginning of the N-ICU stay, were able to persist for 120 colonization days, which represents an average of 15 ± 9.25 colonization days per clone. This value was 56 days for E. faecium, with an average of 7 ± 3.9 colonization days per clone. No statistically significant differences in pooled CPRs were found among the acquired E. faecalis (0.75) and E. faecium (0.35) clones, with the average numbers of colonization days being 8 ± 8.5 and 2.6 ± 2.6, respectively. Nevertheless, a clear tendency was observed. Nine of 13 E. faecalis clones were recovered almost every day during the study period and thus had CPR values of 1 or nearly 1 (Table 1). On the other hand, only one E. faecium clone, which corresponded to HrCC17, showed a CPR value of 1 (Table 2).

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FIG. 2. CPR values for E. faecalis and E. faecium clones. The CPR value for E. faecalis was significantly (*, P = 0.001) higher than that for E. faecium. There were no significant differences in CPR values among acquired clones, but the CPR values for endogenous E. faecalis clones were significantly (**, P = 0.001) higher than those for endogenous E. faecium clones.

Clonal selective antimicrobial exposure. All patients except patient 4 received systemic antimicrobial therapy; patient 4 did not receive any antimicrobial agent during the N-ICU stay. Monotherapy was administered to seven patients, and three of these seven patients also received antimicrobial combinations. These combinations are shown in Table 1 and 2.

Overall, per 100 days of colonization with E. faecalis clones, patients received 77.5 days of antibiotic treatment. The corresponding value was significantly (P = 0.03) lower (65 days) for E. faecium clones. The number of days with ineffective selective antimicrobial exposure was slightly higher for E. faecium than for E. faecalis (46% of the colonization days for E. faecium and 33% for E. faecalis; P = 0.04). In general, the number of days under effective antimicrobial exposure was higher for E. faecalis (44%) than for E. faecium (19%) (P = 0.0001).

The three E. faecalis clones detected in more than one patient were obviously challenged by the highest antibiotic pressure: Efc-1 (ST44), Efc-3 (ST17), and Efc-4 (ST16) had 13.6, 10.6, and 21.3 days of antibiotic exposure per 100 colonization days, respectively (Table 1). The clones persisted, despite exposure to effective selective antibiotics. This was true not only for multiresistant clones, like Efc-3 (ST17, HrCC9) and Efc-4 (ST16), which were partly exposed to effective antibiotics (5 effective days versus 6 ineffective days/100 colonization days for Efc-3 and 14 effective days versus 7 ineffective exposure days/100 colonization days for Efc-4), but also for susceptible clone Efc-1 (ST44) (13.6 effective antibiotic exposure days/100 colonization days) (Table 1). Among the E. faecium clones, Efm-6 (ST18, HrCC17) encountered the highest antibiotic exposure (18.7 days per 100 colonization days) (Table 2). For this clone, the ineffective antimicrobial exposure was similar to the effective exposure (7.5 ineffective days versus 10 effective days per 100 colonization days). Other E. faecium clones received shorter periods of antimicrobial exposure, which was ineffective in most cases (Table 2). During the observation period, no vancomycin-resistant enterococcal clones were recovered in the hospital.

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This study documents different features of the dynamics of enterococcal intestinal colonization in patients admitted to an N-ICU. However, the aim of this work was not to document the rates of transmission or acquisition as an epidemiological type of study but to report the rates of persistence of the different clones from an ecological perspective. Therefore, we designed a narrow observational protocol that included a limited but sequential number of patients who had no overlapping stays in the ICU and who were admitted over a 10-month period. The enterococcal populations present in the patients on ICU admission were assumed to be part of the healthy gut microbiota before hospitalization and were considered endogenous clones, whereas clonal variation was considered to be a result of hospitalization.

Previous studies of hospital enterococcal colonization focused on the prevalence of multiresistant clones or described only the dynamics of colonization (11, 21). In our ecological study, we evaluated the role of persistence in the dynamics of enterococcal colonization using two new indexes. The first was CDPD and the second was CPR. These parameters allowed us to measure and compare changes in the GD and the persistence among patients' enterococcal populations during their stays in the N-ICU. Similar rates have been used in hospital epidemiology for other purposes, for instance, to document device-associated infection rates in ICU patients (10).

Overall, our results showed a significant increase in the clonal diversity of E. faecalis isolates during patient hospital stays. This was a consequence of the increase in the number of different clones instead of the replacement of preexisting ones after clonal acquisition and might be associated with the higher overall persistence of E. faecalis (as a species). It is well known that E. faecalis is a species that is well adapted for human intestinal colonization (29). This adaptation could be the result of certain physiological properties, including its ability to degrade mucin, its mucosal adherence properties, and a specific host tolerance (15). We cannot discard the possibility that a number of the apparently new E. faecalis clones could respond to minority populations that were already present in the gut. In contrast, the clonal diversity of E. faecium decreased during the patients' ICU stays, and E. faecium frequently appeared to be a transient colonizer. Nevertheless, we cannot rule out the possibility of fluctuations in the population density and the low sensitivity of culture. Remarkably, the ampicillin-resistant E. faecium HrCC17 clone showed significantly higher persistence rates than the other E. faecium clones.

Mathematical modeling suggested that persistent gastrointestinal colonization with vancomycin-resistant enterococci resulted in an increased prevalence of endemicity in particular wards (8). In our institution, an ampicillin-resistant E. faecium clone (Efm-6, ST18) belonging to HrCC17 and a multiresistant E. faecalis clone (Efc-3, ST17) belonging to HrCC9 were endemic and caused several bacteremic episodes (P. Ruiz-Garbajosa et al., unpublished data). Unlike other clones, they persisted for a long time in certain wards and ICUs in our hospital (7, 26). The specific role of the accessory genome involved in clonal persistence is not yet known, but in E. faecalis and E. faecium, hospital-acquired clones have been shown to be enriched in adaptive mechanisms, like the presence of putative virulence genes and novel metabolic pathways (12, 13, 17, 19, 27). Some of these traits might facilitate tenacity in the intestinal gut and could also help the organism to reach a high cell density, which might increase the chance of transmission and persistence (27).

It is well known that antimicrobial agents cause disturbances in the intestinal microbiota, particularly those agents that reach high concentrations in the bile, such as certain expanded-spectrum cephalosporins and fluoroquinolones. Moreover, it has been considered that antimicrobials with activities against anaerobic organisms promoted high-density and persistent enterococcal colonization through the inhibition of intestinal anaerobes (9). More recently, some studies suggested that antimicrobial exposure could induce functional changes in enterococcal populations, resulting in the expression of factors other than resistance that could promote adherence to the intestinal epithelial lining (1, 16, 24). This effect of antibiotics as signaling molecules that modify bacterial adaptive phenotypes has also recently been suggested to exist in chronic bacterial colonizers (20). The results of our study might indicate that the persistence of enterococcal clones during patients' ICU stays might not be greatly affected by systemic antimicrobial treatments, but more detailed studies are required to ascertain whether this point is true. Even though the E. faecalis clones exhibited a higher CPR than the E. faecium clones, the former were under effective antimicrobial treatment for significantly more days than the latter. This was not the case for E. faecium HrCC17, whose behavior was similar to that of E. faecalis. E. faecium HrCC17 and E. faecalis are possibly enriched populations that undergo functional changes after exposure to certain antibiotics, thus facilitating colonization and persistence in the gastrointestinal tract. Nevertheless, in vitro susceptibility phenotypes cannot explain the effects of antimicrobials in the intestinal enterococcal population, since different factors, including the amount of active drug attained in the intestinal tract and the interference of antimicrobials with the bowel content or the intestinal mucosa, should be considered. We are conscious that our conclusions might be limited because of the small number of patients included in this study and also because sampling was performed with rectal swabs and the samples obtained might not represent the contents of the upper intestine. The recovery of fresh feces from these patients was difficult; however, we confirmed visually that the swabs in our study contained feces.

Our approach was more ecological than epidemiological, as it explored persistence on the basis of the recovery and the characterization of a high number of enterococcal clones (32 clones) at a number of sequential observation points (eight noncohospitalized patients) over a relatively long period of time (10 months).

In summary, we used two new indexes, CDPD and CPR, to measure the dynamics of E. faecalis and E. faecium clonal gut colonization and persistence among ICU patients in this pivotal study. These indexes could be applied to other hospital colonization studies. Our results suggest more persistent colonization abilities for the ensemble of E. faecalis clones and also for an ampicillin-resistant E. faecium clone belonging to HrCC17, in contrast to the non-CC17 E. faecium clones. The cumulative increase in the clonal diversity of E. faecalis over time might be a consequence of the long persistence rates in both the individual patient and the ICU. On the contrary, the lower rates of persistence of E. faecium prevent the cumulative increase in clonal diversity. Knowledge of the dynamics of colonization of the different enterococcal clones, including HrCCs, could help provide an understanding of the epidemiology of enterococci in hospitals and could also be useful in the design and implementation of infection control measures.

 
P. Ruiz-Garbajosa was the recipient of a post-MIR contract from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (contract CM04/0013). This work was partially supported by grants from the Ministerio de Sanidad y Consumo of Spain (grant PI061141), the European Union (grant LSHE-CT-2007-037410), and the DeReMicrobiana Project of the Madrid Autonomous Community.

We are grateful to Vicente Pintado for being a permanent advisor and for support of the infection control team.

 
* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Madrid 28034, Spain. Phone: 34913368330. Fax: 34913368809. E-mail: rcanton.hrc{at}salud.madrid.org

Published ahead of print on 3 December 2008.

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Journal of Clinical Microbiology, February 2009, p. 345-351, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01597-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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