Risk factors associated with ampicillin resistance in patients with bacteraemia caused by Enterococcus faecium

Jesús Fortún1,*, Teresa M. Coque2, Pilar Martín-Dávila1, Leonor Moreno1, Rafael Cantón2, Elena Loza2, Fernando Baquero2 and Santiago Moreno1

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
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Epidemiological characteristics of ampicillin-resistant, vancomycin-susceptible Enterococcus faecium are not well known. Recently, these strains have been proposed as the substratum for the later appearance of vancomycin-resistant E. faecium. To analyse this problem, the medical charts of patients with bacteraemia caused by E. faecium diagnosed in our institution during a 6 year period (1994–1999) were reviewed. Demographic data, clinical characteristics, antibiotic exposure and outcome were compared among patients with ampicillin-resistant (MIC > 16 mg/L, NCCLS criteria) and ampicillin-susceptible strains. Clonality between different strains was analysed by pulsed-field gel electrophoresis (PFGE). We evaluated 49 cases of E. faecium bacteraemia; 29 patients with ampicillin-resistant strains and 20 patients with -susceptible strains were identified. By logistic regression analysis, only previous administration of ß-lactams (OR: 6.3; 95% CI: 1.12–20.0) and urinary catheterization (OR:4.2; 95% CI: 1.3–30.0) were identified as predictors of ampicillin resistance in enterococcal bacteraemic patients. An elevated APACHE II score was the only independent factor associated with mortality in enterococcal bacteraemia (OR:13.5; 95% CI: 1.04–175.4). PFGE analysis revealed a strong association between specific ampicillin-resistant clones and the location of patients during hospitalization, suggesting nosocomial transmission. Bacteraemia caused by ampicillin-resistant enterococci was not associated with increased mortality when compared with bacteraemias caused by ampicillin-susceptible strains.


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Enterococci have become the fourth most common cause of bloodstream infections in Spain, with Enterococcus faecium and Enterococcus faecalis the predominant species.1 In recent years, increasing resistance to first line antibiotics has been observed among enterococcal species worldwide, especially in E. faecium, thus limiting the already reduced therapeutic options to treat severe enterococcal infections.2,3

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
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Setting

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 physician’s 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 manufacturer’s 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 Tris–borate–EDTA 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 Student’s t-test for continuous data and {chi}2 analysis for categorical data. Yates’ correction and two-tailed Fisher’s 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
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Demographic data

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).



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Figure 1. Number of blood culture isolates of E. faecalis (black bars), E. faecium (hatched bars) and other enterococci (white bars) during the period 1994–1999 in Ramón y Cajal Hospital.

 
The evolution of resistance to ampicillin was clearly different for E. faecalis and E. faecium isolates in our centre in the last 10 years. No cases of ampicillin resistance were observed among E. faecalis isolates, whereas a high proportion of ampicillin-resistant strains was detected in E. faecium. A significant increase was observed from 1991 (18% of total E. faecium isolates) to 2000 (71%), with a mean of 52% during this period and reaching 80% in 1998 (Figure 2).



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Figure 2. Evolution of ampicillin resistance in total numbers of E. faecium isolated at Ramón y Cajal Hospital, 1991–2000.

 
We had access to the clinical records in 49 of the 60 patients with E. faecium bacteraemia. Of the 49 patients analysed, bacteraemia was caused by ampicillin-resistant strains in 29 (59%) and by ampicillin-susceptible strains in 20 (41%). Data for analysis were not available for the other 11 patients (seven with resistant isolates). In these patients the data included in their clinical records were too scarce to be analysed. However, patients who were and were not analysed did not differ with respect to clinical data and outcome.

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 1995–1997 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 1997–2000 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.


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Table 1.  Demographic, clinical data and antibiotic exposure in patients with bacteraemia caused by ampicillin-resistant (AREF) and ampicillin-susceptible (ASEF) E. faecium
 
By logistic regression analysis, previous administration of ß-lactams (OR: 6.3; 95% CI: 1.12–20.0) and urinary catheterization (OR: 4.2; 95% CI: 1.3–30.0) were confirmed as predictors of ampicillin resistance in enterococcal bacteraemic patients.

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: 0.47–9.95). Mortality attributed to bacteraemia was 21% and 15%, respectively (OR: 1.5; 95% CI: 0.27–8.85).

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: 1.04–175.4) was independently associated with mortality.


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Table 2.  Risk factors associated with mortality in patients with enterococcal bacteraemia
 

    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Enterococci were the third most common cause of bloodstream infections in our hospital during the study period. The prevalence of bacteraemia caused by Enterococcus spp. has remained stable for the last 5 years, and most enterococcal blood isolates were E. faecalis or E. faecium, which is comparable with the distribution of species in previous studies.1,17,19,21 Ampicillin resistance has developed during the last decade in E. faecium, being present in 70% of the isolates during 2000. The prevalence of vancomycin resistance among blood isolates of E. faecalis and E. faecium in our hospital during the study period was <3%; all were classified as vanA-type.

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
 
Presented in part at the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Toronto, Canada, September 2000.


    Footnotes
 
* Corresponding author. Tel: +34-91-336-8709; Fax: +34-91-336-8792; E-mail: fortun{at}mi.madritel.es Back


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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
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