Departments of 1 Infectious Diseases and 2 Microbiology, Ramón y Cajal Hospital, University of Alcalá, Crtra Colmenar Km 9.1, Madrid 28034, Spain
Received 5 April 2002; returned 3 July 2002; revised 9 August 2002; accepted 22 August 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Epidemiology of enterococci resistant to different antibiotics (vancomycin, aminoglycosides and ampicillin) is complex and varies among hospitals and countries. Susceptibility of enterococci to ampicillin remained stable until 1982, when the first nosocomial outbreak of ampicillin-resistant E. faecium was reported.4 This resistance is related to an alteration of penicillin-binding proteins, mainly PBP5.5,6 PBP5-mediated ampicillin resistance is thought to be intrinsic to the enterococci and is usually non-transferable. Recently, cotransfer of ampicillin and vancomycin resistance has been described, and ampicillin resistance has been proposed as a risk factor for endemic spread of vancomycin-resistant strains.69
Several studies have been published on risk factors for vancomycin-resistant enterococcal infection or colonization;1015 however, information about risk factors for acquisition of ampicillin-resistant enterococci has focused on colonization,1619 although there are a few studies in relation to cases with bacteraemia.2022 We recently noted an increased number of ampicillin-resistant E. faecium being isolated by the clinical microbiology laboratory at our 1200 bed tertiary care hospital.23 This prompted an investigation to determine the risk factors for the acquisition of ampicillin-resistant enterococci in bacteraemic patients as well as its related mortality.
![]() |
Material and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hospital Ramón y Cajal is a university teaching hospital providing gynaecological, paediatric and adult medical and surgical care, including liver, lung, kidney and bone marrow transplantation units. The institution provides health care to a population of 600 000.
Patients and chart review
All patients who had E. faecium isolated from blood cultures submitted to the clinical microbiology department at the Ramón y Cajal Hospital from January 1994 to December 1999 were identified. Patients with bacteraemia caused by ampicillin-resistant strains of E. faecium were considered as cases and were compared with patients with bacteraemia caused by ampicillin-susceptible strains that were considered as controls.
Patients medical charts were reviewed, and demographic, clinical and microbiological data were collected. Exposure to antimicrobials was evaluated from the last month to the date of the initial enterococcal blood culture. Outcome of patients was analysed until hospital discharge or death occurred.
Microbial identification and susceptibility testing
Blood cultures were obtained at the physicians discretion and processed by the BACTEC 9240 blood culture system (Becton Dickinson Diagnostic Instruments, USA). Only one isolate per patient was further analysed. Preliminary identification and susceptibility tests were performed by using the automated microdilution PASCO (Difco, Detroit, MI, USA) or WIDER Systems (Fco. Soria Melguizo, Madrid, Spain). For identification of enterococci to the species level, the biochemical scheme of Facklam & Collins,24 and the criteria for motility and pigment production were used. Identification at species level was confirmed by amplification of the aac-6'-Ii gene, which is specific for E. faecium. Antimicrobial susceptibility to different antibiotics was determined by an agar dilution method according to the NCCLS guidelines.25 Ampicillin resistance was defined by MICs > 16 mg/L. ß-Lactamase production was tested by placing a heavy suspension of organisms into a microtitre well containing nitrocefin (100 µmol/mL) (BBL Microbiology, Cockeysville, MD, USA).
Detection of genes coding for glycopeptide resistance
E. faecium isolates resistant to glycopeptides were examined for the presence of the vanA and vanB genes using oligonucleotides and PCR conditions previously described.26 Total DNA was extracted from E. faecium isolates by InstaGene (BioRad, La Jolla, CA, USA) following manufacturers instructions.
Pulsed-field gel electrophoresis (PFGE)
Genomic DNA was prepared and digested with SmaI (Amersham Pharmacia) as previously described.27 After digestion, DNA fragments were separated by electrophoresis in 1.2% agarose gels (Pulsed-Field Agarose Certified; BioRad) and 0.5 x TrisborateEDTA buffer using a contour-clamped homogeneous electric field apparatus (CHEF-DRIII system; BioRad). Electrophoresis conditions were 12°C at 6 V/cm for 27 h with pulse times ranging from 1 to 27 s. The DNA banding patterns were analysed by visual examination by two independent investigators. According to the standard criteria given by Tenover et al.27 to establish clonal relationships, isolates were considered to be related if they exhibited differences of up to six bands, if there was good epidemiological evidence to suggest relatedness among isolates, or if they had been isolated over extended periods of time.
Statistical analysis
Characteristics of cases and controls were compared with the Students t-test for continuous data and 2 analysis for categorical data. Yates correction and two-tailed Fishers exact test were performed if necessary. All variables having a P value of <0.1 were included in logistic regression modelling. Multivariate analysis was carried out with the use of logistic regression, with significant variables selected by a backward stepwise procedure. Statistical analysis of the data was performed with Epi-Info version 6 (CDC, Atlanta) and SPSS 7.5 for Windows (SPSS Inc., Chicago, IL, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A total of 691 enterococcal blood isolates were identified through the period of study, with E. faecalis being isolated in most episodes (522, 75%). E. faecium was isolated from 104 blood cultures, corresponding to 60 patients, and represented 15% of all enterococcal isolates (Figure 1).
|
|
The source of bacteraemia was mainly related to a urinary or venous catheter origin in most cases, but this information was not available for several patients.
Antimicrobial susceptibility
Most of the ampicillin-resistant isolates (n = 29) were also resistant to erythromycin (93%), clindamycin (87%), co-trimoxazole (70%) and ciprofloxacin (70%), and were highly resistant to streptomycin (74%) and kanamycin (81%). Three isolates (10%) showed high-level resistance to gentamicin (HLRGm). Glycopeptide resistance was found in two isolates (vancomycin MIC >128 mg/L, teicoplanin MIC >64 mg/L), both of which contained vanA. Production of ß-lactamase was not detected in any ampicillin-resistant isolate.
The occurrence of resistance to other antibiotics was lower for ampicillin-susceptible isolates (n = 20): erythromycin (42%), clindamycin (37%), co-trimoxazole (4%), ciprofloxacin (4%), high level of resistance to streptomycin (4%) and kanamycin (25%). All were vancomycin susceptible and did not show HLRGm.
Pulsed-field gel electrophoresis (PFGE)
Analysis of SmaI-digested genomic DNA banding patterns from 29 ampicillin-resistant isolates revealed 13 clonal types. Isolates from six different patients obtained during 19951997 showed identical DNA banding patterns and were classified as Type A. Four of them were isolated in patients hospitalized in Gastroenterology or General Surgery Departments (placed on the 10th and 11th floor, respectively). Isolates from 12 different patients obtained during 19972000 showed a DNA banding pattern that differed from each other by one to six bands and from the common pattern by one to six bands. We considered these as a clonal group (Type B). Type B was found in 12 patients, nine of whom were included in our analysis. The other isolates corresponded to 11 clones that were largely unrelated: six patients were included in our analysis and all of them were hospitalized in other wards not on the 10th and 11th floor. The association between clones and place of hospitalization was statistically significant (P = 0.01).
Risk factors for ampicillin resistance
A comparison of demographic data, clinical characteristics and microbiological features among patients with ampicillin-resistant and -susceptible strains is shown in Table 1. In univariate analysis, urinary catheterization was significantly more frequent in patients with resistant isolates. In addition, a trend towards a higher number of central venous catheters was observed in patients with resistant isolates. The analysis of drug exposure showed a significant association between exposure to ß-lactams in the last month and the development of bacteraemia by resistant isolates. No association was found, however, with any specific ß-lactam (penicillins, cephalosporins and carbapenems). Patients with resistant isolates were more likely to have received quinolones.
|
Outcome and mortality
There were no significant differences in the outcome of patients with ampicillin-resistant and -susceptible strains.
We did not find significant differences in mortality between the two groups. Overall mortality in patients with bacteraemia caused by ampicillin-resistant and -susceptible E. faecium was 34% and 21%, respectively (OR: 2.1; 95% CI: 0.479.95). Mortality attributed to bacteraemia was 21% and 15%, respectively (OR: 1.5; 95% CI: 0.278.85).
An elevated APACHE II score and urinary catheterization were significantly associated with a higher mortality in univariate analysis (Table 2). A trend was observed among patients receiving parenteral nutrition and renal failure (expressed as a creatinine serum level higher than 2 mg/L). Although mortality was higher in patients with ampicillin-resistant E. faecium bacteraemia, the difference was not statistically significant. By logistic regression analysis, only a high APACHE score (OR: 13.5; 95% CI: 1.04175.4) was independently associated with mortality.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We found that previous exposure to ß-lactams and urinary catheterization were two major risk factors for ampicillin resistance in patients with bacteraemia caused by E. faecium. Since the early studies of Boyce et al.,18 which related ampicillin-resistant enterococci to the use of imipenem, different authors have demonstrated the association of ampicillin-resistant enterococci with ß-lactams,16,18,20 quinolones,17,19 aminoglycosides21 and co-trimoxazole.21 Antibiotics may facilitate colonization and infection by depleting the gastrointestinal tract of its normal anaerobic flora and by selecting enterococci due to limited bactericidal activity against these organisms. Exposure to multiple antimicrobial agents is favoured by prolonged hospitalization, a circumstance that has been associated with the selection of ampicillin-resistant enterococci,20,21,28 as well as of enterococci with high-level resistance to aminoglycosides.2830
The second factor identified as independently associated with the development of ampicillin-resistant E. faecium bacteraemia in our study was urinary catheterization. Gross et al.31 demonstrated that nosocomial enterococcal urinary pathogens come from the gastrointestinal tract. Bladder catheterization has been shown to increase urinary enterococcal colonization in patients with ampicillin-resistant enterococcal bacteraemia, and a gastrointestinal origin of urinary colonization has been indicated by plasmid analysis.32
Positive clinical specimens in the absence of rectal carriage provide evidence for exogenous acquisition. E. faecium, including multiply resistant strains, has been isolated from the hands of hospital staff during some outbreaks.33,34 Patterns of environmental contamination compatible with staff hand carriage35 and the ability of E. faecium to survive on fingertips and gloves indicate that nosocomial transmission via staff hands is feasible.36 Nosocomial transmission was suggested for most isolates in our study. Two-thirds of the strains analysed belonged to two of 13 clonal types. The strains were grouped by time and hospital location: clone A was observed during 1995 and 1997 and accounted for 66% of strains from the Gastroenterology and General Surgery Departments; and clone B, observed from 1997 to 1999, accounted for 50% of the strains obtained in these departments. These departments account for <10% of hospital beds.
Based on similar data, some authors have recommended infection control precautions. By containing the nosocomial spread of ampicillin-resistant enterococci, the need for vancomycin use is reduced and may therefore delay the occurrence of vancomycin-resistant enterococci in those institutions where such organisms are not yet a problem. Recently, four cases of infection caused by a vancomycin-resistant (vanB-type) strain occurred during a clonal outbreak caused by genomically related ampicillin-resistant E. faecium, and in association with an increase in vancomycin use.37
The spread of ampicillin-resistant enterococci in our hospital was not associated with a higher mortality in bacteraemic patients. Other authors have found an increased intrahospital death rate for patients infected by ampicillin-resistant enterococci. However, mortality has been ultimately related to the underlying disease of the patients or to inappropiate antimicrobial therapy in these studies.21 Our data support these findings; although mortality was higher in patients with ampicillin-resistant enterococcal bacteraemia, the only factor independently associated with mortality was a high APACHE score.
In conclusion, we have observed a rise in ampicillin-resistant enterococci among bacteraemic patients. This increase has been favoured by different factors. Prolonged antibiotic use during hospitalization, and urinary catheterization in patients with perianal colonization, may have contributed to the selection of these strains and the later development of bacteraemia. Nosocomial transmission may account for the increase of ampicillin-resistant enterococci observed, but fortunately, bacteraemia caused by these bacteria was not associated with increased mortality.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Murray, B. E. (2000). Vancomycin resistant enterococcal infections. New England Journal of Medicine 342, 7109.
3 . Murray, B. E. (1998). Diversity among multidrug-resistant enterococci. Emerging Infectious Diseases 4, 3747[ISI][Medline]
4 . Coudron, P. E., Mayhall, C. G., Facklam, R. R., Spadora, A. C., Lamb, V. A., Lybrand, M. R. et al. (1984). Streptococcus faecium outbreak in a neonatal intensive care unit. Journal of Clinical Microbiology 20, 10448.[ISI][Medline]
5 . Ligozzi, M., Pittaluga, F. & Fontana, R. (1996). Modification of penicillin-binding protein 5 associated with high-level ampicillin resistance in Enterococcus faecium. Antimicrobial Agents and Chemotherapy 40, 3547.[Abstract]
6 . Rice, L. B. (2001). Emergence of vancomycin-resistant enterococci. Emerging Infectious Diseases 7, 1837.[ISI][Medline]
7
.
Carias, L. L., Rudin, S. D., Donskey, C. J. & Rice, L. B. (1998). Genetic linkage and cotransfer of a novel, vanB-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate. Journal of Bacteriology 180, 442634.
8
.
Hanrahan, J., Hoyen, C. & Rice, L. B. (1999). Geographic distribution of a large mobile element that transfers ampicillin and vancomycin resistance between Enterococcus faecium strains. Antimicrobial Agents and Chemotherapy 44, 134951.
9
.
Suppola, J. H., Jolho, E., Salmenlinna, S., Tarkka, E., Vuopio-Varkila, J. & Vaara, M. (1999). VanA and vanB incorporate into endemic ampicillin-resistant vancomycin-sensitive Enterococcus faecium strain: effect on interpretation of clonality. Journal of Clinical Microbiology 37, 39349.
10 . Beltrami, E. M., Singer, D. A., Fish, L., Manning, K., Young, S., Banerjee, S. N. et al. (2000). Risk factors for acquisition of vancomycin-resistant enterococci among patients on a renal ward during a community hospital outbreak. American Journal of Infection Control 28, 2825.[ISI][Medline]
11 . Loeb, M., Salama, S., Armstrong-Evans, M., Capretta, G. & Olde, J. (1999). A case-control study to detect modifiable risk factors for colonization with vancomycin-resistant enterococci. Infection Control and Hospital Epidemiology 20, 7603.[ISI][Medline]
12 . Fuller, R. E., Harrell, L. J., Meredith, F. T., Sexton, D. J. & Colvin, L. G. (1998). Vancomycin-resistant enterococci: risk related to the use of intravenous vancomycin in a university hospital. Infection Control and Hospital Epidemiology 19, 8213.[ISI][Medline]
13 . Lucas, G. M., Lechtzin, N., Puryear, D. W., Yau, L. L., Flexner, C. W. & Moore, R. D. (1998). Vancomycin-resistant and vancomycin-susceptible enterococcal bacteremia: comparison of clinical features and outcomes. Clinical Infectious Diseases 26, 112733.[ISI][Medline]
14 . Roghmann, M. C., McCarter, R. J., Jr, Brewrink, J., Cross, A. S. & Morris, J. G., Jr (1997). Clostridium difficile infection is a risk factor for bacteremia due to vancomycin-resistant enterococci (VRE) in VRE-colonized patients with acute leukemia. Clinical Infectious Diseases 25, 10569.[ISI][Medline]
15 . Linden, P. K., Pasculle, A. W., Manez, R., Kramer, D. J., Fung, J. J., Pinna, A. D. et al. (1996). Differences in outcomes for patients with bacteremia due to vancomycin-resistant Enterococcus faecium or vancomycin-susceptible Enterococcus faecium. Clinical Infectious Diseases 22, 66370.[ISI][Medline]
16 . Suppola, J. P., Volin, L., Valtonen, V. V. & Vaara, M. (1996). Overgrowth of Enterococcus faecium in the feces of patients with hematologic malignancies. Clinical Infectious Diseases 23, 6947.[ISI][Medline]
17 . Mohn, S. C., Harthug, S. & Langeland, N. (2000). Outbreak of ampicillin-resistant Enterococcus faeciumrisk factors for faecal colonisation. Acta Pathologica, Microbiologica, et Immunologica Scandinavica 108, 296302.
18 . Boyce, J. M., Opal, S. M., Potter-Bynoe, G., LaForge, R. G., Zervos, M. J., Furtado, G. et al. (1992). Emergence and nosocomial transmission of ampicillin-resistant enterococci. Antimicrobial Agents and Chemotherapy 36, 10329.[Abstract]
19
.
Torell, E., Cars, O., Olsson-Liljequist, B., Hoffman, B. M., Lindback, J. & Burman, L. G. (1999). Near absence of vancomycin-resistant enterococci but high carriage rates of quinolone-resistant ampicillin-resistant enterococci among hospitalized patients and nonhospitalized individuals in Sweden. Journal of Clinical Microbiology 37, 350913.
20 . Harthug, S., Eide, G. E. & Langeland, N. (2000). Nosocomial outbreak of ampicillin resistant Enterococcus faecium: risk factors for infection and fatal outcome. Journal of Hospital Infection 45, 13544.[ISI][Medline]
21 . McCarthy, A. E., Victor, G., Ramotar, K. & Toye, B. (1994). Risk factors for acquiring ampicillin-resistant enterococci and clinical outcomes at a Canadian tertiary-care hospital. Journal of Clinical Microbiology 32, 26716.[Abstract]
22 . Chirurgi, V. A., Oster, S. E., Goldberg, A. A., Zervos, M. J. & McCabe, R. E. (1991). Ampicillin-resistant Enterococcus raffinosus in an acute-care hospital: case-control study and antimicrobial susceptibilities. Journal of Clinical Microbiology 29, 26635[ISI][Medline]
23 . Coque, M. T., Loza, E., Cantón, R., Moreno, L., Martín-Dávila, P. & Baquero, F. (2000). Antimicrobial susceptibility and molecular typing of Enterococcus faecium responsible for bacteraemia in Madrid, Spain. In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada. Abstract 165, p. 71. American Society for Microbiology, Washington, DC, USA.
24 . Facklam, R. R. & Sahm, D. A. (1995). Enterococcus. In Manual of Clinical Microbiology, 6th edn (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. & Yolken, R. H., Eds), pp. 308314. American Society for Microbiology, Washington, DC, USA.
25 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition: Approved Standard M7-A. NCCLS, Villanova, PA, USA.
26 . Dutka-Malen, S., Evers, S. & Courvalin, P. (1995). Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. Journal of Clinical Microbiology 33, 247.[Abstract]
27
.
Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 22339.
28
.
Silverman, J., Thal, L. A., Perri, M. B., Bostic, G. & Zervos, M. J. (1998). Epidemiologic evaluation of antimicrobial resistance in community-acquired enterococci. Journal of Clinical Microbiology 36, 8302.
29 . Axelrod, P. & Talbot, G. H. (1989). Risk factors for acquisition of gentamicin-resistant enterococci. Archives of Internal Medicine 149, 1397401.[Abstract]
30 . Patterson, J. E. & Zervos, M. J. (1990). High-level gentamicin resistance in Enterococcus: microbiology, genetic basis, and epidemiology. Review of Infectious Diseases 12, 64452.[ISI][Medline]
31 . Gross, P. A., Harkavy, L. M., Barden, G. E. & Flower, M. F. (1976). The epidemiology of nosocomial enterococcal urinary tract infection. American Journal of the Medical Sciences 272, 7581.[ISI][Medline]
32 . Chirurgi, V. A., Oster, S. E., Goldberg, A. A. & McCabe, R. E. (1992). Nosocomial acquisition of beta-lactamase-negative, ampicillin-resistant Enterococcus. Archives of Internal Medicine 152, 145761.[Abstract]
33 . Coudron, P. E., Mayhall, C. G., Facklam, R. R., Spadora, A. C., Lamb, V. A., Lybrand, M. R. et al. (1984). Streptococcus faecium outbreak in a neonatal intensive care unit. Journal of Clinical Microbiology 20, 10448.[ISI][Medline]
34 . Wells, C. L., Juni, B. A., Cameron, S. B., Mason, K. R., Dunn, D. L., Ferrieri, P. et al. (1995). Stool carriage, clinical isolation, and mortality during an outbreak of vancomycin-resistant enterococci in hospitalized medical and/or surgical patients. Clinical Infectious Diseases 21, 4550.[ISI][Medline]
35 . Wade, J. J. (1997). Enterococcus faecium in hospitals. European Journal of Clinical Microbiology and Infectious Diseases 16, 1139.[ISI][Medline]
36 . Noskin, G. A., Stosor, V., Cooper, I. & Peterson, L. R. (1995). Recovery of vancomycin-resistant enterococci on fingertips and environmental surfaces. Infection Control and Hospital Epidemiology 16, 57781.[ISI][Medline]
37 . Harthug, S., Digranes, A., Hope, O., Kristiansen, B. E., Allum, A. G. & Langeland, N. (2000). Vancomycin resistance emerging in a clonal outbreak caused by ampicillin-resistant Enterococcus faecium. Clinical Microbiology and Infection 6, 1928.[ISI][Medline]