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Clinical and Molecular Epidemiology of Extended-Spectrum β -Lactamase—Producing Escherichia coli as a Cause of Nosocomial Infection or Colonization: Implications for Control

  1. Jesús Rodríguez-Baño1,2,
  2. Maria D. Navarro1,
  3. Luisa Romero2,
  4. Miguel A. Muniain1,3,
  5. Evelio J. Perea2,4,
  6. Ramón Pérez-Cano1,3,
  7. Jose R. Hernández4, and
  8. Alvaro Pascual2,4
  1. 1Sección de Enfermedades Infecciosas, Servicio de Medicina Interna, Seville, Spain
  2. 2Servicio de Microbiología, Hospital Universitario Virgen Macarena, Seville, Spain
  3. 3Departamento de Medicina, Seville, Spain
  4. 4Departamento de Microbiologia, Universidad de Sevilla, Seville, Spain
  1. Reprints or correspondence: Dr. Jesús Rodríguez-Baño, Sección de Enfermedades Infecciosas, Hospital Universitario Virgen Macarena, Avda Dr Fedriani 3, 41071 Seville, Spain(jrb{at}nacom.es).
 
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Abstract

Background. Extended-spectrum β -lactamase (ESBL)—producing members of the Enterobacteriaceae family are important nosocomial pathogens. Escherichia coli producing a specific family of ESBL (the CTX-M enzymes) are emerging worldwide. The epidemiology of these organisms as causes of nosocomial infection is poorly understood. The aims of this study were to investigate the clinical and molecular epidemiology of nosocomial infection or colonization due to ESBL-producing E. coli in hospitalized patients, consider the specific types of ESBLs produced, and identify the risk factors for infection and colonization with these organisms.

Methods. All patients with nosocomial colonization and/or infection due to ESBL-producing E. coli in 2 centers (a tertiary care hospital and a geriatric care center) identified between January 2001 and May 2002 were included. A double case-control study was performed. The clonal relatedness of the isolates was studied by repetitive extragenic palindromic—polymerase chain reaction and pulsed-field gel electrophoresis. ESBLs were characterized by isoelectric focusing, polymerase chain reaction, and sequencing.

Results. Forty-seven case patients were included. CTX-M—producing E. coli were clonally unrelated and more frequently susceptible to nonoxyimino— β -lactams. Alternately, isolates producing SHV- and TEM-type ESBL were epidemic and multidrug resistant. Urinary catheterization was a risk factor for both CTX-M—producing and SHV-TEM—producing isolates. Previous oxyimino—β -lactam use, diabetes, and ultimately fatal or nonfatal underlying diseases were independent risk factors for infection or colonization with CTX-M—producing isolates, whereas previous fluoroquinolone use was associated with infection or colonization with SHV-TEM—producing isolates.

Conclusions. The epidemiology of ESBL-producing E. coli as a cause of nosocomial infection is complex. Sporadic CTX-M—producing isolates coexisted with epidemic multidrug-resistant SHV-TEM—producing isolates. These data should be taken into account for the design of control measures.

Extended-spectrum β -lactamases (ESBLs) are plasmid-encoded β -lactamases that confer significant resistance to penicillins, narrow- and extended-spectrum cephalosporins, and aztreonam. Organisms harboring ESBLs are also frequently resistant to aminoglycosides, trimethoprim-sulfamethoxazole, and quinolones. ESBL-producing members of the Enterobacteriaceae family have played a leading role among nosocomially acquired multidrug-resistant organisms during the past decade [1, 2]. Many outbreaks of infection caused by ESBL-producing Klebsiella pneumoniae have been reported all over the world [1, 2]. The risk factors for infection caused by this organism are similar to those described for other nosocomial multidrug-resistant pathogens [3].

ESBLs have been classified into different types. Initially, the 2 most frequent types of ESBL were TEM types and SHV types. More recently, enzymes belonging to a different type of ESBL, such as the CTX-M type (so called because they are mainly cefotaximases), are being detected with increasing frequency, particularly in ESBL-producing Escherichia coli, which now represents an emerging cause of infection in many areas of the world [4–10]. Some data suggest that the epidemiology of these emerging ESBL-producing E. coli is substantially different from that of ESBL-producing K. pneumoniae [4, 5, 9, 11]. The aims of this study were to investigate the clinical and molecular epidemiology of nosocomial infection due to ESBL-producing E. coli in hospitalized patients, consider the specific types of ESBLs produced, and identify the risk factors for infection and colonization with these organisms.

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Methods

Study Site, Subjects, and Design

The study was performed in the 2 hospitals of the administrative Health Area of North Seville, Spain (the Hospital Universitario Virgen Macarena, a 950-bed teaching hospital, and the Hospital San Lázaro, a 150-bed hospital that mainly provides care for elderly patients). Intensive care is only provided in the acute care hospital. Transfer of patients between the 2 hospitals is frequent.

During the study period (January 2001–May 2002, inclusive), all patients from whom ESBL-producing E. coli had been isolated from any clinical sample were identified through daily review of microbiologic reports and were prospectively investigated. Patients were included as cases if their samples were obtained after 48 h of hospitalization and provided that, at the time of admission to the hospital, there had been no signs or symptoms of infection potentially attributable to ESBL-producing E. coli. Patients colonized by ESBL-producing E. coli detected only by means of surveillance samples were excluded. Case patients were included only once.

Two control groups were studied. Patients in control group 1 were randomly selected among patients who were admitted to the same hospital unit during the same quarter and had a hospital stay previous to the isolation of ESBL-producing E. coli that was at least as long as that of the corresponding case patient. Patients in control group 2 were randomly selected among patients admitted to the same type of hospital service unit (medical or surgical unit or intensive care unit) during the same quarter and from whom a non—ESBL-producing isolate of E. coli had been recovered from any clinical sample. Control group 1 included 2 controls per case patient and control group 2 included 2 controls per case patient, except for 9 case patients for whom only 1 control was found.

Definitions and Variables

The type of infection was defined in accordance with Centers for Disease Control and Prevention (CDC) criteria [12]. Patients who did not meet criteria for infection were considered to be colonized. The following data were collected: age; sex; underlying diseases; severity of the underlying diseases, according to the McCabe classification [13]; previous hospitalizations; residence in a nursing home; receipt of long-term hemodialysis; receipt of antimicrobial agents; invasive procedures; and hospitalization in an intensive care unit. The time at risk was defined as the time from admission to the hospital until the date on which E. coli was isolated for case patients and control group 2, and as the time from admission until the date of discharge or death for patients in control group 1.

Antimicrobial therapy was considered to be appropriate if at least 1 antimicrobial with in vitro activity against the isolated microorganism had been administered at the usual dose for at least 24 h. All case patients were observed until hospital discharge or death. The study protocol was approved by the local ethics committee.

Microbiological Studies

Bacterial isolates and susceptibility assays. The first isolate from each patient was studied. Identification was determined using the Vitek 2 system (bioMérieux) and API 20E strips (bioMérieux). ESBL production was screened and confirmed in accordance with guidelines of the NCCLS [14, 15]. The in vitro activity of antimicrobial agents was determined using a microdilution assay, in accordance with NCCLS guidelines [15].

Molecular typing. Clonal relationships between the isolates were determined by repetitive extragenic palindromic (REP)—PCR, as described elsewhere [16]. Isolates were considered to be clonally related when band patterns differed by <3 bands. Isolates determined by REP—PCR to be clonally related were also studied by PFGE, as described elsewhere [17], using XbaI endonuclease (Roche Applied Sciences). Interpretation of the clonal relatedness of PFGE patterns was performed as described by Tenover et al. [18].

β -Lactamase characterization. Isoelectric focusing [19, 20] and PCR were used for the preliminary characterization of β -lactamases and β -lactamase genes, respectively. The isoelectric focusing ranges were correlated to TEM-, SHV-, or CTX-M—type β -lactamases [21]. The presence of blaTEM, blaSHV, and blaCTX-M in each organism was studied by PCR, as described elsewhere [22, 23]. Oligonucleotide primers designed to amplify the genes encoding the most common subgroups within the ESBL families were used [23–25]. E. coli J53 Rif-R was used as a negative control.

Amplicons were sequenced at an external center (DNA Automatic Sequencing Service, Consejo Superior de Investigaciones Cientifícas, Madrid, Spain) equipped with an ABI Prism 377 sequencer (Applied Biosystems). Sequences were analyzed using the Chromas application, the Basic Local Alignment Search Tool [26], and the Traduction Multiple program [27].

Statistical Analysis

Continuous variables were compared using the Mann-Whitney U test. Qualitative variables were compared using the χ 2 test or the Fisher' exact test, as appropriate; ORs and 95% CIs were calculated. Multivariate logistic regression analysis was performed to determine variables that were independently associated with the risk of colonization or infection with ESBL-producing E. coli. Potential interactions between variables were considered. Variables were selected in a backward stepwise process. Data were analyzed using the SPSS statistical software package (SPSS).

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Results

During the study period, ESBL-producing E. coli was isolated from 96 patients; 47 patients met the inclusion criteria and were included in this study. The other 49 patients were nonhospitalized and have been analyzed elsewhere [5]. During the study period, 4.1% of all E. coli isolated from hospitalized patients were ESBL producers, and only 15 ESBL-producing Enterobacteriaceae isolates were isolated from hospitalized patients.

Twenty-nine case patients (62%) were in the acute care hospital, and 18 (38%) were in the geriatric care hospital. The rate of colonization or infection was significantly higher in the geriatric care hospital (0.6 cases per 1000 patient-days vs. 0.06 cases per 1000 patient-days; P <.001).

The characteristics of the case patients are shown in table 1; 32% of cases occurred during the first week of hospitalization. Thirty-six patients (77%) were considered to be infected. The types of infection included the following: 15 urinary tract infections (42%), 13 soft-tissue infections (36%; 4 pressure ulcers and 5 surgical site infections), 4 respiratory tract infections (11%), 3 cases of primary bacteremia (8%), and 1 intra-abdominal infection (3%). Three patients had secondary bacteremia. Overall, 6 patients (16%) were bacteremic. Only 13 patients (36%) received appropriate empirical antimicrobial therapy. Fourteen patients died (crude mortality rate, 30%). Five patients died of infection (infection-related mortality rate, 14%).

With respect to the type of ESBL, 29 isolates (62%) produced an SHV type (28 were identified by sequencing as SHV-12, and 1 was identified as SHV-2a), 27 (57%) produced a CTX-M type (26 were CTX-M-14, and 1 was CTX-M-9), and 18 (38%) produced an as yet unidentified ESBL with an isoelectric point of 6.5, which is probably a TEM type, according to the results of other experiments (data not shown), here referred to as “TEM-Q” for easier understanding of data. Twenty-four isolates (51%) produced >1 ESBL, the most frequent combination being SHV-12 plus TEM-Q (14 isolates). of the 23 isolates that produced only 1 ESBL, 16 produced CTX-M-14. Isolates producing TEM-Q clustered from January through July 2001 (14 isolates) and from December 2001 through March 2002 (4 isolates).

Twenty-five different genotypes were identified by REP-PCR, including those of 4 clonally related groups (“epidemic isolates”; 55%) and those of 21 clonally unrelated isolates (“sporadic isolates”; 45%). Isolates considered to be clonally related by REP-PCR were also studied by PFGE, with identical results. The distribution of cases throughout the study period according to genotypes is shown in figure 1. Clonal group A comprised 11 isolates; all of them produced SHV-12, 9 isolates also produced TEM-Q, and 5 isolates also produced CTX-M-14. Four of these patients were in the geriatric care hospital, and 7 were in the acute care hospital (3 patients were in the intensive care unit, 3 were in an internal medicine ward, and 1 was in a surgical ward). Clonal group B also consisted of 11 isolates: 9 produced SHV-12, 8 produced TEM-Q, 1 produced CTX-M-14, and 1 produced CTX-M-9. Eight patients were in the geriatric care hospital, and 3 were in the acute care hospital (2 patients were in the intensive care unit, and 1 patient was in a general surgery ward). Among the 6 patients in the intensive care unit included in the study, 5 had an epidemic isolate. The duration of hospitalization of patients colonized or infected with these 2 clonal groups is represented in figure 2. The other 2 clonal groups were constituted by 2 isolates each. Among the 21 sporadic isolates, 17 produced a CTX-M enzyme. Compared with nonepidemic isolates, epidemic isolates were more frequently producers of TEM- and SHV-type ESBLs (62% vs. 10% [P <.001] and 85% vs. 33% [P <.001], respectively) and less frequently producers of CTX-M types (39% vs. 81%; P =.003). Susceptibility data for the isolates are shown in table 2.

Figure 1
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Figure 1

Distribution of the 47 infecting or colonizing extended-spectrum β -lactamase–producing Escherichia coli isolates in hospitalized patients during the study period, according to genotype.

Figure 2
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Figure 2

Duration of hospital stay for 22 patients colonized or infected with extended-spectrum β -lactamase–producing Escherichia coli isolates belonging to clonally-related groups A (grey bars) and B (white bars). The black point represents the date when the microorganism was isolated, and the asterisk indicates stay in the geriatric care hospital.

Table 1
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Table 1

Characteristics of hospitalized patients colonized or infected with extended-spectrum β -lactamase–producing Escherichia coli.

Table 2
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Table 2

Susceptibility data of extended-spectrum β -lactamase–producing Escherichia coli isolated from hospitalized patients.

To investigate the epidemiology of the different types of ESBLs, 2 homogeneous groups were compared: 16 patients with isolates producing only CTX-M-14 (the “CTX-M group”) and 14 patients with isolates producing both SHV-12 and TEM-Q but not CTX-M-14 (the “SHV-TEM group”). All isolates but 1 from the CTX-M group were sporadic, whereas all isolates from the SHV-TEM group belonged to epidemic clones. Patients with isolates from the CTX-M group were admitted more frequently to surgical units (37.5% vs. 0%, P =.01), less frequently to the geriatric care hospital (25% vs. 78.5%; P =.009), and had diabetes more often (56.3% vs. 21.4%; P =.05), than did patients with isolates from the SHV-TEM group; previous use of fluoroquinolones was less frequent among patients in the CTX-M group (25% vs. 64.3%; P =.03), whereas previous use of cefotaxime was somehow more frequent, a difference that was not statistically significant (37.5% vs. 21.4%; P =.4). The median duration of previous hospital stay was shorter among patients in the CTX-M group (14 days vs. 21 days), but the difference was not statistically significant, because of the wide ranges (3 to >99 days in both groups). Isolates in the CTX-M group were more frequently susceptible to amoxicillin-clavulanic acid (81.3% vs. 50%; P =.07), ciprofloxacin (25% vs. 0; P =.04), gentamicin (87.5% vs. 42.9%; P =.01), and tobramycin (87.5% vs. 7.1%; P <.001) than were isolates in the SHV-TEM group.

Results of univariate analysis of risk factors for colonization or infection due to ESBL-producing E. coli is shown in table 3. Multivariate analysis was performed for all case patients, for those with isolates in the CTX-M group, and for those with isolates in the SHV-TEM group (table 4).

Table 3
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Table 3

Univariate analysis of risk factors for colonization or infection with extended-spectrum β -lactamase–producing Escherichia coli in hospitalized patients.

Table 4
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Table 4

Multivariate model of risk factors for colonizationor infection due to extended-spectrum β -lactamase–producing Escherichia coli in hospitalized patients.

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Discussion

ESBL-producing members of the Enterobacteriaceae family have been important causes of nosocomial infections for the past 2 decades [1, 2]. Most attention has been focused on K. pneumoniae, because traditionally it has been the most frequently encountered nosocomial organism that produces ESBL. Specific information about the clinical epidemiology and relevance of nosocomial infection with ESBL-producing E. coli is scarce, because these organisms have usually been investigated together with other ESBL-producing Enterobacteriaceae isolates [28–32] or in studies limited to investigation of their microbiological aspects [33, 34].

The emergence of infections caused by ESBL-producing E. coli (and, particularly, isolates producing CTX-M–type ESBL) has been recently reported throughout the world [4–10]; in our region, the first CTX-M–producing E. coli were detected in 1999 [35]. Some data suggest that the epidemiologic characteristics of these emerging ESBL-producing E. coli are different from those of ESBL-producing K. pneumoniae: the proportion of patients with community-acquired infections due to E. coli is much higher [5, 9, 11], whereas clonally related outbreaks are far less frequent. Our data confirm that, beyond its importance as a pathogen in nonhospitalized patients [5, 9], ESBL-producing E. coli is also an emerging cause of nosocomial infections. Our findings also show that the epidemiologic characteristics of these organisms are complex and somehow different, depending on the type of ESBLs produced.

In acute care hospitals, most previously reported nosocomial infections caused by ESBL-producing E. coli were caused by clonally unrelated isolates. However, some small clonal outbreaks have been described [6, 29, 31, 33, 34, 36–37,38]. Surprisingly, 45% of the cases in our study were caused by clonally related isolates, which predominantly produced both SHV-12 and an as yet undetermined TEM-type ESBL. An epidemiologic relationship was found for many of these case patients, supporting the horizontal transmission of the organism. The lapses of time without cases may be indicative of an unnoticed reservoir, colonized patients being the most probable. Interinstitutional spread was probably caused by the transfer of patients between the hospitals.

The 2 major clonal outbreaks of nosocomial infection seem to have been self-limited. Contact precautions for patients colonized with ESBL-producing E. coli were not established because transmission of these isolates had not been previously noticed in our hospitals. Our study was performed in the context of a very low incidence of nosocomial infection with other ESBL-producing organisms [35]; a very active nosocomial infection–control program, particularly for multiresistant bacteria [39], has been in place at our hospitals since 1997, and use of alcoholic hand rubs have been available in all rooms since 2000. Thus, these measures might have had some impact on the unfolding of the outbreaks.

Several data suggest that the epidemiologic characteristics of CTX-M–producing isolates are different from those of isolates producing SHV-TEM enzymes, something that had not been previously noticed. All isolates in the SHV-TEM group belonged to the main epidemic clonal groups, whereas CTX-M isolates were clonally unrelated. Five of the 6 case-patients in the intensive care unit were infected with isolates from the SHV-TEM group; most of the nosocomial outbreaks of ESBL-producing K. pneumoniae have been caused by TEM-producing or SHV-producing isolates and predominantly occur in high-risk areas, such as intensive care units [2]. In addition, in our area, the proportion of cases caused by TEM- or SHV-producing isolates among hospitalized patients (38% and 62%, respectively) was much higher than that among nonhospitalized patients during the same period (18% and 18%) [5]. Isolates from the SHV-TEM group were more resistant to antimicrobials (although this should be interpreted with caution because these isolates are clonally related). Finally, nonfatal underlying conditions were associated only with CTX-M-14–producing isolates. All these data are indicative of the fact that, although ESBL-producing E. coli from the SHV-TEM group behaved as typical epidemic nosocomial pathogens [3], those from the CTX-M group did not. We hypothesize that many of the patients with CTX-M-14–producing isolates had acquired the organism in the community. This is supported by the fact that many CTX-M cases occurred early during hospitalization and that, in our area, most of the ESBL-producing E. coli in nonhospitalized patients are clonally unrelated and produce an ESBL from the CTX-M-9 group (a group comprising CTX-M-14, among others ESBLs) [5]; in fact, we have recently used sequencing techniques to characterized them as CTX-M-14 isolates (Velasco C, Romer L, Rodriguez-Bańo J, et al., unpublished data).

The information provided by the double case-control study is complementary; comparison with control group 2 overestimated the risk associated with previous antimicrobial use [40], but avoided common risk factors for infection caused by E. coli found in the analysis performed with control group 1. Urinary catheterization and previous administration of oxyimino–β -lactams or fluoroquinolones were independent risk factors for infection with ESBL-producing E. coli. The results of the multivariate models for specific types of ESBLs provide additional data that suggest differences in the epidemiologic characteristics of epidemic SHV-TEM and sporadic CTX-M isolates. Oxyimino–β -lactams, of which cefotaxime was the most frequently used, were related to CTX-M–producing isolates, traducing the cefotaximase activity of these enzymes, whereas fluoroquinolones use was associated with the ciprofloxacin-resistant SHV-TEM–producing isolates.

Our results provided further evidence of the importance of combining clinical and molecular epidemiologic data in the investigation of resistant pathogens and may have implications for the design of control programs for these organisms [41, 42]. Contact precautions, active surveillance in high-risk areas, and decrease of oxyimino–β -lactam consumption, as recommended by Paterson and Yu [1], should be performed when epidemic TEM-SHV–producing E. coli are detected; reduction in fluoroquinolone use should also be considered. Some phenotypic features of these isolates (e.g., a ceftazidime MIC greater than the cefotaxime MIC and resistance to other antimicrobials) might be used as a preliminary epidemiologic marker, although some types of CTX-M ESBLs have been described to efficiently hydrolyze ceftazidime [43]. Alternately, sporadic CTX-M-producing E. coli represent a new challenge for infection control, because some small clonal nosocomial outbreaks have been described and because the spread of genetic elements carrying the blaCTX-M genes is a threat. If most of these cases are imported from the community, the implementation of contact precautions for the cases detected by analysis of clinical samples might be useless, because many other colonized patients will remain undetected. Active surveillance of colonization involving all patients admitted to the hospital is impractical; targeted surveillance involving high-risk patients could be an option, but risk factors for colonization at admission have not been investigated. Also, more studies focused on the epidemiologic behavior of these organisms and the mobile genetic elements harboring the CTX-M enzymes within hospitals are needed. Thus, the control measures that should be implemented for these organisms remain unclear. Meanwhile, strict application of hand hygiene, adequate use of gloves, and judicious use of oxyimino–β -lactams (particularly cefotaxime) are mandatory, and because molecular epidemiologic analyses or techniques for ESBL characterization are not widely available, continuous surveillance based on clinical epidemiology data and susceptibility patterns of the isolates is necessary to promptly recognize possible outbreaks.

Our study has some limitations. We did not perform active surveillance to detect colonized patients. This approach is desirable, but it proved to be impractical in our situation, because the case patients were from many different wards. Also, transmission of plasmids containing the ESBLs between isolates should also be studied. Characterization of the plasmids containing the ESBLs is in progress.

In conclusion, our data indicate that the epidemiology of nosocomial infection with ESBL-producing E. coli in hospitalized patients is complex; outbreaks of infection caused by isolates producing TEM and SHV types of ESBL similar to those caused by ESBL-producing K. pneumoniae and clonally unrelated isolates producing CTX-M enzymes may coexist. These data should be considered for control purposes. Also, ESBL-producing E. coli should be considered in the etiology of nosocomial infections in patients with risk factors.

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Acknowledgements

Financial support. REIPI (Spanish Network for Research in Infectious Diseases), Instituto de Salud Carlos III, Ministerio de Salud y Consumo (C03/14); and Asociación Sanitaria Virgen Macarena (fellowship to M.D.N.).

Potential conflicts of interest. All authors: no conflicts.

  • Received June 28, 2005.
  • Accepted August 23, 2005.
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