Risk factors associated with extended-spectrum ß-lactamase-producing organisms at a tertiary care hospital

Eileen M. Graffunder1,*, Karen E. Preston2, Ann M. Evans2 and Richard A. Venezia3

1 Department of Epidemiology MC-45, Albany Medical Center, 43 New Scotland Avenue, Albany, NY 12208, USA; 2 Department of Laboratory Medicine, Albany Medical Center, Albany, NY, USA; 3 Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA


* Corresponding author. E-mail: Graffue{at}mail.amc.edu

Received 14 June 2004; returned 22 February 2005; revised 27 February 2005; accepted 28 April 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background: In 1995, ß-lactam inhibitor combinations replaced third-generation cephalosporins as empirical therapy in an effort to manage extended-spectrum ß-lactamase (ESBL) resistance. This study investigated the relationship between antibiotic usage and ESBL organisms from 1994 through 2002 using epidemiological and molecular analysis.

Methods: A case–control study of 119 patients with ESBL organisms and 132 patients with non-ESBL organisms was conducted. Demographics, co-morbidities, device utilization and antibiotic use were analysed for all patients and infected patients only (cases = 75, controls = 83). Both exposure and degree of exposure (in grams) to antibiotics were included. A dot blot hybridization technique was used to identify genes in plasmid extracts from the ESBL organisms.

Results: Ventilator days OR 1.1 (1.06, 1.15) P < 0.001, adult respiratory distress syndrome (ARDS) OR 3.1 (1.0, 9.7) P = 0.05, prior aminoglycoside use OR 2.7 (1.2, 6.1) P = 0.02, prior third-generation cephalosporin use OR 7.2 (2.6, 20) P < 0.001, and prior trimethoprim/sulfamethoxazole use OR 8.8 (3.1, 26) P < 0.001 were significantly associated with ESBL organisms by multivariate analysis. All models were concordant with a significant association of ventilator days, third-generation cephalosporins and trimethoprim/sulfamethoxazole with ESBL organisms. ß-Lactamase inhibitor combinations were not associated with ESBL organisms. Hybridization of plasmid extracts demonstrated that 95% of the ESBL organisms carried intI1, a mobile DNA element with a sulphonamide-resistance (R) gene and a frequent carrier of other R factors. Genes for specific types of trimethoprim-R and aminoglycoside-R were present in 26% and 40% of the extracts, respectively.

Conclusions: These data indicate that, besides patient risk factors and third-generation cephalosporins, other antibiotics may provide selective pressures in maintaining ESBL organisms due to multiple resistance genes on plasmids. ß-Lactamase inhibitor combinations appear to be an acceptable substitute to third-generation cephalosporins in strategies to control ESBL organisms.

Keywords: cephalosporins , epidemiology , Gram-negative organisms , ESBLs


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial resistance is an increasing threat afflicting hospitals worldwide. Resistant bacteria can result from resistance genes in their chromosome or by acquisition of genes from other bacteria. This resistance may be expressed through a number of mechanisms such as enzymic modification or degradation of antimicrobial compounds. Once established, bacterial resistance spreads rapidly in response to antimicrobial challenges and through lapses in infection control practices. The introduction of extended-spectrum oxyimino ß-lactam antibiotics (third-generation cephalosporins) in the 1980s was rapidly followed by the emergence of resistance.1,2 Extended-spectrum ß-lactamases (ESBLs), are enzymes that hydrolyse extended-spectrum oxyimino ß-lactam antibiotics such as ceftazidime, ceftriaxone, cefotaxime and aztreonam.1,3 Bacteria that express plasmid-mediated ESBLs have continued to remain susceptible to ß-lactamase inhibitor combinations and carbapenems.4,5

The first ESBL organisms were identified in Germany in 1983.1,6,7 Since that time, an increase in this resistance has occurred throughout Europe and the United States.68 Among the Enterobacteriaceae, members of the TEM and SHV enzyme families are the most commonly encountered plasmid-mediated ESBLs.2,9 More than 150 different ESBL enzymes have been described.1 ESBL organisms pose therapeutic challenges for physicians as resistance genes for other antimicrobials such as aminoglycosides, tetracyclines and trimethoprim/sulfamethoxazole are often present on the same plasmid.1015

Risk factors identified as predictors of ESBL organisms are previous hospitalization, prolonged length of stay, increased severity of illness, ICU admission, mechanical ventilation, central venous and urinary catheters and prior exposure to antimicrobial agents, especially the oxyimino ß-lactam antibiotics.1,7,8,1620 Many outbreaks of ESBL organisms have been successfully managed with either enhanced infection control practices or antimicrobial restrictions.6,2126 In the studies that have addressed antimicrobial restrictions, oxyimino ß-lactam antibiotics have been replaced with either ß-lactamase inhibitor combinations or carbapenems with varying success in controlling ESBL organisms.2126

In 1995, ß-lactamase inhibitor combinations were substituted for third-generation cephalosporins as empirical therapy in an effort to manage ESBL resistance. Despite a subsequent reduction in third-generation cephalosporins, an increase in ESBL organisms occurred in 2001–2002. A previous report indicated that multiple strains and plasmids, with both temporal and geographic clustering, were responsible for the ESBL organisms in our population.27 Although some of the ESBL isolates from that analysis were included in this study these two studies are mutually exclusive. This study investigated the risk factors and relationship between antibiotic usage and the acquisition of ESBL organisms from 1994 through 2002 using epidemiological and molecular analyses.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study was conducted at Albany Medical Center, a 600 bed tertiary care facility located in upstate New York. Cases were defined as patients diagnosed with a nosocomial ESBL Citrobacter, Enterobacter, Klebsiella, Serratia or E. coli from 1994 through 2002. Controls were randomly selected from a database of all patients diagnosed with a nosocomial non-ESBL Citrobacter, Enterobacter, Klebsiella, Serratia or E. coli during the same time period. Both cases and controls could not possess another type of resistant organism such as MRSA, VRE, multidrug-resistant Pseudomonas or Acinetobacter prior to the Enterobacteriaceae under study. Patients with incomplete data in the medical record were excluded in both populations. Patient isolates were considered nosocomial when the first culture-positive specimen was obtained >48 h after admission and the patient had not presented with symptoms of infection on admission. A retrospective review of the patient's medical record was conducted and data were collected from admission to the time of the first positive culture using a standardized data collection form. Infections were defined using the Centers for Disease Control National Nosocomial Infection Surveillance definitions.28 Antibiotic data were grouped into antimicrobial classes for analysis and were analysed by exposure and degree of exposure in grams. All organisms were identified by standard laboratory techniques.

Antimicrobial susceptibility testing was performed by disc diffusion according to the National Committee for Clinical Laboratory Standards (NCCLS).29 ESBL production was confirmed by the NCCLS document M100-S12 recommended method.30 Screening of plasmid extracts for resistance factors and associated mobile elements was done according to the dot blot protocol in the Genius Systems user's guide for filter hybridization version 2.0 (Boehringer-Mannheim, Indianapolis, IN, USA) with the reagents of the DIG DNA labelling and detection kit (Roche).27 This study was approved by the institutional review board as an exempt study and did not require written informed consent.

Statistical analysis

Statistical analysis was conducted using SPSS, 8.0 (Chicago, IL, USA) and NCSS (Keysville, UT, USA). Continuous variables were compared by the two-sample t-test and dichotomous variables were compared by Fisher's exact test for two by two comparisons or Pearson {chi}2 for greater than two responses. Antimicrobial therapy was grouped into classes of antimicrobials to assure adequate cell counts. Logistic regression analysis was conducted to obtain unadjusted odds ratios. To identify risk factors that were independently associated with an ESBL organism, multivariate analysis was conducted using a logistic regression model, likelihood ratio test. Automated forward selection functions were used in preliminary analysis to assess the contribution of risk factors that did not meet statistical significance but had a P value ≤0.25. Since there were no differences in the log likelihood function for the full models compared to the reduced models, only the risk factors that reached statistical significance were included in the final models presented. Risk factors that reached statistical significance (P < 0.05) using a forward selection process remained in the model. Non-significant variables were assessed as confounding variables and had to have a meaningful change in the regression coefficients of other variables to remain in the model. Multivariate logistic regression models were assessed by the Hosmer and Lemeshow Goodness of Fit Statistic and the C-statistic representing the area under the ROC curve. Four separate logistic regression models were conducted. Separate analyses were conducted for all patients and for infected patients only. For each population, two models were conducted one for exposure to antimicrobial agents and one for the degree of exposure to antimicrobial agents (in grams) to examine potential dose effect. Additional analysis was conducted using SPSS Answer Tree, Chi-Squared Automatic Interaction Detector (CHIAD) to examine relationships between risk factors and the development of an ESBL organism. CHIAD is a statistical technique for segmentation. Using the criterion of statistical tests, CHIAD evaluates the values of potential predictor variables and merges values that are judged to be statistically homogeneous with respect to the target variable. Significance was defined as a P value ≤0.05. All tests were two-tailed.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two hundred and fifty-one patients were evaluated. When comparing the 119 cases with the 132 controls, there was no difference in the percentage of patients defined as ‘infected’ [63% (75/119) of the ESBL and 62.9% (83/132) of the non-ESBL population, respectively, P = 0.98]. The organisms and sites of infection and sources of colonization were similar in both groups (Table 1 and 2). The risk factors significant by univariate analysis were also similar in both groups (Tables 3 and 4) excluding ICU stay, total parenteral nutrition (TPN), ß-lactamase inhibitor combinations, and carbapenems, which were significantly higher (P < 0.05) in the analysis that included all patients with ESBL organisms. Patients with ESBL organisms had more comorbidities, more device days and were exposed to more antimicrobial agents than the patients with non-ESBL organisms.


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Table 1. Types of Enterobacteriaceae included in this study

 

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Table 2. Sites of infection and sources of colonization

 

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Table 3. Characteristics of all patients

 

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Table 4. Characteristics of infected patients

 
The results of the four different multivariate analyses were concordant with significant risks of ESBL organisms associated with the number of ventilator days and treatment with third-generation cephalosporins or trimethoprim/sulfamethoxazole. In the multivariate analyses of all patients, ARDS and prior treatment with an aminoglycoside were also independently associated with ESBL organisms in both models (Table 5a and b). When analysing the subset of infected patients only, the two models were concordant with number of ventilator days, presence of chronic obstructive pulmonary disease (COPD), and treatment with third-generation cephalosporins or trimethoprim/sulfamethoxazole. Central line and urinary catheter days were significant in the patients with ESBL organisms in the model examining exposure to antimicrobial agents. When analysing the degree of exposure to antimicrobial agents, the only additional significant variable was treatment with an aminoglycoside (Table 5c and d).


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Table 5. Results of logistic regression analysis

 
CHIAD was also used to further examine the relationship between risk factors and the development of ESBL organisms. All variables that were significant by univariate analyses were entered into the CHAID model. The model identified ventilator days as the most significant risk factor for the acquisition of an ESBL organism. Durations of ventilator days were divided into three groups with rates of ESBL organisms 29.4% (n = 153) in patients with ≤7 days of ventilation, 67.1% (n = 73) in patients with >7 to 27 days of ventilation and 100% (n = 25) in patients with ventilator days that exceeded 27 days. As the rate of ESBL organisms increased with ventilator days so did the rate of exposure to at least one of the antimicrobial agents identified as an independent risk factor for ESBL organisms. The rate of associated antimicrobial exposure was 32.4% for <7 days of ventilation, 42.5% for >7 to 27 days of ventilation and 88% for >27 days of ventilation. In the group of patients with ventilator days >7 to 27 days, prior exposure to ß-lactamase inhibitor combinations appeared to have a protective effect. The rate of ESBL organisms was 56.9% (n = 51) for those with prior exposure to ß-lactamase inhibitor combinations and 90.9% (n = 22) in patients with no exposure to ß-lactamase inhibitor combinations (P = 0.005).

A majority, 93.3% of the 119 ESBL organisms screened with the gene probes carried a gene encoding a member of the SHV enzyme family. Ninety-five percent (113) of the ESBL organisms were positive for intI1, evidence of a type I integron, a mobile DNA element noted for carrying a fixed sulphonamide resistance gene cassette. The dfrA1 gene for trimethoprim resistance and the aac(3)-Ia gene for gentamicin resistance were present in 26% and 40% of the extracts, respectively (Table 6). These genes are among those found in mobile integron cassettes associated with intI1.


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Table 6. Gene probes used on plasmid extract and the percentage identified in ESBL organisms (n = 119)

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
These data indicate that in addition to patient risk factors, device utilization and third-generation cephalosporin use, other classes of antimicrobial agents may provide selective pressures in maintaining ESBL organisms. Multivariate analysis identified that third-generation cephalosporin use, aminoglycosides and trimethoprim/sulfamethoxazole were the antimicrobial classes independently associated with ESBL organisms in our study. The purpose of including the results of the hybridization studies is to illustrate that there is a correlation with the antimicrobial agents identified as risk factors for ESBL organisms and the presence of related resistant genes residing on the plasmids. Similar to our study, Wiener et al. determined that prior exposure to trimethoprim/sulfamethoxazole was an independent risk factor for ESBL organisms and that the plasmid responsible for their outbreak also conferred resistance to trimethoprim/sulfamethoxazole.15

Patients with prolonged ventilation were at the greatest risk of having an ESBL organism and the selective pressure of antimicrobial agents appears to be the greatest in this population. In total, 88% of patients with >27 days of ventilation had exposure to at least one of the antimicrobial agents associated with ESBL organisms. Prolonged ventilation is consistent with the acquisition of any resistant organism as these patients tend to be more debilitated, have greater exposure to acid suppressors, antimicrobial agents and more opportunities for aspiration and nosocomial acquisition. This risk factor has also been confirmed by other studies.31,32 Patients with COPD and ARDS, two of the comorbidities associated with ESBL organisms, are more likely to require longer durations of ventilator support due to the clinical nature of their underlying disease. In both populations, the mean duration of the ventilator days was significantly longer than in patients who lacked either risk factor. Because of the increased risk of developing an infection with an ESBL organism, this population could be targeted to receive alternative therapy to oxyimino ß-lactamase antibiotics depending on their present condition and clinical presentation.

Since the objective of this study was to identify risk factors for the acquisition of ESBL organisms, we did not differentiate between infection and colonization in the main analysis. Colonization with a resistant organism is often the precursor to infections and the duration of medical devices increases the risk of a patient developing an infection.33 Because the risk factors may be different for infected patients, we conducted a subset analysis, which included infected patients only. The risk factors for ESBL organisms in the infected group were concordant with the total study population in regard to duration of ventilation, third-generation cephalosporins, trimethoprim/sulfamethoxazole and aminoglycosides. The only risk factor specific to an increased risk in the infected population was central line days.

The control of resistant organisms usually requires enforcement of antimicrobial usage or infection control practices.6,2126 It is difficult to define the relationship between these two practices in antimicrobial resistance studies. Although we did substitute ß-lactamase inhibitor combinations as empirical therapy in 1995, this study was not designed to measure its effect. This study was designed to identify the risk factors associated with ESBL organisms. ß-Lactamase inhibitor combinations were not independently associated with a reduction in ESBL organisms, but they did demonstrate a protective effect in a subset of our population. The protective effect of ß-lactamase inhibitor combinations for ESBL organisms in ventilated patients has previously been reported by Piroth et al.32

There are several limitations to this study. In order to have sufficient data for analysis, we had to collect data over a long period of time. It is difficult to ascertain the relative influence and the effects that other changes over time may be influencing our results. Obviously, advances in medical technology, changes in patient populations, formulary restrictions and changes in standards of practice or infection control measures may have affected our results. Secondly, the increase in ESBL organisms in 2001 followed a clonal outbreak of multiresistant Acinetobacter (non-ESBL) in one of our intensive care units. None of the patients in this study were involved in that outbreak. Although residing in an ICU did not retain statistical significance in the model, we cannot conclude that a lapse in infection control practices may not be a contributing factor.

In conclusion, the risk factors identified in this study, duration of ventilation, ARDS, prior exposure to third-generation cephalosporins, aminoglycosides and trimethoprim/sulfamethoxazole are consistent with risk factors identified by other investigators. Patients on prolonged ventilation should be targeted and assessed for the risk of ESBL organisms when prescribing antimicrobial therapy.


    Acknowledgements
 
We thank Anthony Harris (University of Maryland School of Medicine) for his helpful comments. This study was supported in part through a grant from Wyeth-Ayerst Pharmaceutical, Inc.


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