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Antimicrobial Agents and Chemotherapy, November 2008, p. 4187-4190, Vol. 52, No. 11
0066-4804/08/$08.00+0     doi:10.1128/AAC.00367-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Prospective Study of Fecal Colonization by Extended-Spectrum-β-Lactamase-Producing Escherichia coli in Neutropenic Patients with Cancer{triangledown}

L. Calatayud,1,2* M. Arnan,3 J. Liñares,1,2 M. A. Dominguez,1 C. Gudiol,4 J. Carratalà,4 M. Batlle,5 J. M. Ribera,5 and F. Gudiol4

Microbiology Department, Hospital Universitari de Bellvitge, University of Barcelona, IDIBELL, Barcelona,1 Ciber de Enfermedades Respiratorias, ISCIII, Madrid,2 Hematology Department, Hospital Duran y Reynals, Barcelona,3 Infectious Diseases Department, Hospital Universitari de Bellvitge, University of Barcelona, IDIBELL, Barcelona,4 Hematology Department, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain5

Received 17 March 2008/ Returned for modification 9 April 2008/ Accepted 10 September 2008


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ABSTRACT
 
Fecal colonization by extended-spectrum-β-lactamase-producing Escherichia coli in 912 stool samples collected from 154 neutropenic patients with cancer, hospitalized at two teaching institutions, was prospectively studied. Forty-nine (31.8%) patients were colonized, 22 of them at hospital admission. Most strains were clonally unrelated and carried a CTX-M-9 group enzyme.


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TEXT
 
Infections due to extended-spectrum-β-lactamase-producing Escherichia coli (ESBL E. coli) are increasing in many countries (15). Neutropenic patients with cancer may constitute a special risk group for these infections and present additional therapeutic difficulties (8, 14). In these patients, the gastrointestinal tract is a significant source of infecting organisms, with intestinal colonization being the prelude to bacterial translocation across the gut and systemic dissemination (2). Although ESBL E. coli has been recognized sporadically as a cause of bloodstream infection in neutropenic patients (9, 21), no studies to date have prospectively analyzed fecal colonization by ESBL E. coli among this particular population. We sought to determine the prevalence of ESBL E. coli among neutropenic patients with cancer and to characterize the ESBLs and genotype of isolates.

This prospective cohort study was conducted between 1 May 2006 and 30 November 2007 in Barcelona, Spain, at two teaching hospitals: Hospital Duran i Reynals (hospital A) and Hospital Germans Trias i Pujol (hospital B). The ethical committees at both institutions approved the study. One hundred fifty-four consecutive patients (75 from hospital A and 79 from hospital B) with acute leukemia or undergoing hematopoietic stem cell transplantation, who received cytotoxic chemotherapy and developed neutropenia (<500 neutrophils/µl), were included. No antibacterial prophylaxis was administered during the study period.

Fecal samples were obtained at hospital admission and weekly thereafter during neutropenia until recovery or death. Rectal swabs were cultured on three plates: MacConkey agar without antibiotics and MacConkey agar supplemented with cefotaxime (2 µg/ml) and ceftazidime (4 µg/ml). Identification of isolates and antibiotic susceptibility testing were performed using commercial panels (MicroScan). CLSI criteria were used to define susceptibility or resistance to antimicrobial agents (3). ESBL screening was performed by use of a double-disc synergy test (7). Molecular typing was done by pulsed-field gel electrophoresis (PFGE) (XbaI) with the first isolate from each of the colonized patients; strains that differed in three or fewer bands were considered as belonging to the same cluster (22). ESBLs were characterized by a multiplex PCR (4) and isoelec-tric focusing. Sequencing of PCR products was performed with 22 selected strains on the basis of pI and PCR results.

Nine hundred twelve stool samples collected from 154 patients were studied. At admission, 22 (14.3%) patients were colonized by ESBL E. coli: 11 (14.7%) of 75 patients at hospital A and 11 (13.9%) of 79 patients at hospital B. Seven (31.8%) of the 22 patients colonized at admission had been hospitalized in the previous 6 months, and 4 (18.2%) patients had received antimicrobial therapy in the previous 30 days. Overall, ESBL E. coli strains were isolated from feces of 49 (31.8%) of the 154 patients studied: 17 (22.7%) of 75 patients in hospital A and 32 (40.5%) of 79 patients in hospital B (Fig. 1). Forty-six of the 49 patients presented one or more episodes of febrile neutropenia during the study period and received broad-spectrum antibiotics according to the institution protocols based on current international guidelines (5).


Figure 1
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  		FIG. 1. Distribution of the first ESBL E. coli isolate from each of 49 fecal carriers. (a) Hospital A. (b) Hospital B. Admission*, three patients were colonized by E. coli simultaneously harboring CTX-M and OXA ESBLs.

Rates of resistance to non-β-lactam antibiotics were as follows (in hospital A versus hospital B, respectively): gentamicin, 11.8% versus 40.6%; tobramycin, 11.8% versus 46.9%; ciprofloxacin, 76.5% versus 90.6%; and cotrimoxazole, 64.7% versus 68.8%. All of the strains were susceptible to imipenem. Nine resistance patterns were identified (Table 1).


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  		TABLE 1. Characteristics of ESBL E. coli strains isolated from colonized patients

Results of β-lactamase characterization are summarized in Table 1. The ESBLs were identified as 32 from the CTX-M-9 group, 8 from the CTX-M-1 group, 6 from the SHV family, 5 from the OXA family, and 1 from the TEM family (TEM-52). Fecal detection of ESBL E. coli strains harboring different ESBL genes over time is shown in Fig. 1.

By use of PFGE, 44 different genotypes were found. Among patients of hospital B, three epidemic genotypes (EC1, EC2, and EC3) carrying blaCTX-M-14 ESBL were detected. Three of four strains of genotype EC1 were isolated in November 2006 and shared the same resistance pattern. The two strains of cluster EC2 were isolated in May 2006, and the two strains of cluster EC3 were isolated in June 2006 (Table 1).

Simultaneous colonization by ESBL E. coli and ESBL Klebsiella pneumoniae was observed for six patients (12.3%): three patients hospitalized at hospital A and three patients at hospital B. This finding was not related to clonal spread because all strains (six E. coli strains and six K. pneumoniae strains) showed different PFGE patterns. However, a possible dissemination of ESBLs in the gut of each patient could have occurred, because E. coli and K. pneumoniae isolated in each patient carried the same ESBL (Table 1).

Our study documented that ESBL E. coli fecal colonization among hospitalized neutropenic patients with cancer was frequent. The rectal carriage of ESBL E. coli at admission was 14.3%. This figure is higher than those found by other Spanish investigators analyzing the carriage of ESBL-producing Enterobacteriaceae among different nonneutropenic populations, including healthy volunteers, outpatients, and hospitalized patients (11, 12, 23). The higher rate of fecal colonization found among neutropenic patients may be related, at least in part, to the frequent use of antibiotics in this population. Nevertheless, studies aimed at identifying specific factors for ESBL E. coli rectal carriage at admission and subsequent acquisition during hospitalization among neutropenic patients are needed.

Molecular typing of isolates demonstrated a high degree of genetic diversity, and most of the isolates harbored a CTX-M-9 enzyme (10, 17, 18, 20). Although plasmid transmission between isolates was not studied, the high clonal diversity found suggests horizontal gene transfer among strains rather than spread of specific E. coli clones. Moreover, most of the isolates carried CTX-M group ESBL enzymes, which are often transmissible by conjugation in vitro and thus disseminate easily (1). However, clonal spread of eight strains was detected at hospital B (genotypes EC1, EC2, and EC3), suggesting the presence of some epidemic clones. Our findings agree with a recent study where unrelated ESBL E. coli strains harboring CTX-M group enzymes and epidemic isolates coexisted as causes of nosocomial infection or colonization (19).

As ESBLs are encoded by plasmids, these enzymes could easily spread to different species of Enterobacteriaceae. These plasmids also carry other genes that encode resistance to other group of antibiotics, with the result that ESBL-producing microorganisms are usually multiresistant (6). We found that resistance of ESBL E. coli strains to quinolones, gentamicin, and cotrimoxazole was high, as was previously reported (13, 16).

Our results may have significant prophylactic and therapeutic implications. The high rate of fecal colonization by ESBL E. coli strains among neutropenic patients and the frequent resistance of these strains to quinolones raise questions about the use of these agents for decontamination of the gastrointestinal tract. On the other hand, the administration of oxyimino β-lactams, such as ceftazidime and cefepime, which are frequently used for treatment of febrile neutropenia, should be avoided in rectal carriers of ESBL E. coli.

In summary, we found that ESBL E. coli fecal colonization among neutropenic patients with cancer was common and that it was frequently documented at admission. Most ESBL E. coli strains were unrelated and carried CTX-M-9 group enzymes. Our findings may have significant therapeutic implications for this population of severely immunocompromised patients.


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ACKNOWLEDGMENTS
 
This work was supported by research grant 051610 from Fundació la Marató de TV3, Generalitat de Catalunya, Barcelona, Spain, and by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III-FEDER, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008).

We thank Carmen Ardanuy for expert advice and critical review of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Hospital Universitari de Bellvitge, 08907 L'Hospitalet de Llobregat, Barcelona, Spain. Phone: 34-932607930. Fax: 34-932607547. E-mail: lcalatayud{at}csub.scs.es Back

{triangledown} Published ahead of print on 22 September 2008. Back


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Antimicrobial Agents and Chemotherapy, November 2008, p. 4187-4190, Vol. 52, No. 11
0066-4804/08/$08.00+0     doi:10.1128/AAC.00367-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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