ß-Lactamases Producing Organisms in Household Contacts of Infected ACCEPTED. Community Patients Madrid. Spain

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1 JCM Accepts, published online ahead of print on 18 June 2008 J. Clin. Microbiol. doi: /jcm Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved High rate of Intestinal Colonization with Extended Spectrum ß-Lactamases Producing Organisms in Household Contacts of Infected Community Patients Aránzazu Valverde, 1,2 Fabio Grill, 3 Teresa M. Coque, 1,2 Vicente Pintado 3, Fernando Baquero, 1,2 Rafael Cantón, 1,2* and Javier Cobo 3 Servicio de Microbiología, 1 CIBER en Epidemiología y Salud Pública (CIBERESP), 2 and Servicio de Enfermedades Infecciosas 3. Hospital Universitario Ramón y Cajal Madrid. Spain Running title. ESBL fecal colonization in family groups Key words: ESBL, Escherichia coli, household contacts, fecal carriage *Corresponding author: R. Cantón. Servicio de Microbiología. Hospital Universitario Ramón y Cajal Madrid. Spain. Phone: ; FAX: ; rcanton.hrc@salud.madrid.org 1

2 Fecal carriage with extended-spectrum ß-lactamase (ESBL)-organisms was detected in 70% of index cases (n=40) with community-acquired infections due to ESBL-producers and reached 16.7% in household contacts (n=54). Sixty-six percent of ESBL-organisms from index cases were indistinguishable (PFGE) when compared with household isolates. Community-patients and their households represent a reservoir for ESBL-producers, increasing dispersal of resistance in healthy people. 2

3 Extended spectrum ß-lactamase (ESBL) producing organisms have dramatically increased worldwide (2,22). This has been associated with efficient dispersion of specific clones and plasmids harbouring bla ESBL genes (5,6,20). In addition, co- resistance to non-ß-lactam antibiotics may have fuelled persistence of ESBL-producing isolates, a fact that has been demonstrated in intensive care units (17,19,23). A threatening epidemiological problem is the dispersal of ESBL-organisms to healthy people in the community, which might depend on the frequency of ESBL-fecal carriers as well as their presence in the food chain (15,18,30). In a previous study, we demonstrated that nearly 12% of hospitalized patients might carry ESBL-producing isolates in the intestinal compartment (32). This colonization has been associated with a high risk for developing an infection due to ESBL-producers (23,25). This has been scarcely studied in community patients and to a lesser extent in healthy subjects (24,32). Moreover, there is little information about the pathways of the intestinal colonization. This has been investigated in hospitalized patients and recently in outpatients involved in food-borne outbreaks (9,15). In the present study, we analysed the fecal carriage with ESBL-producing isolates in a group of patients with community-acquired infections (CAIs) due to these organisms and the corresponding status of the people living with them (household contacts). From April 2004 to June 2005 a total of 299 patients presented an infection or colonization process due to an ESBL-producing organism in our institution. Fifty-six percent were outpatients and 95% of them had a urinary tract infection. Forty clinical samples from 40 patients (index cases, ICs) with CAIs (37 urinary tract infections, 2 bacteremia, and one soft tissue infection) due to an ESBL-producing E. coli (n=39) or Klebsiella pneumoniae (n=1) isolates and fecal samples (one per IC) were studied. Only ICs that positively agree to participate in the study and submitted fecal samples were 3

4 enrolled. Thirty-four of the 40 ICs (mean age 63.6 years, range 2-96) were females. In addition, 54 fecal samples from 54 household contacts of 29 IC recovered within twoweeks of IC enrolment were also studied. The number of household contacts for each IC ranged from only one to 7 (mean 2 household per patient). The ethical committee approved the study and signed informed consents were obtained. Fecal samples were screened for ESBL-producers as previously described (32). Bacterial identification was performed by standard methods and CLSI microdilution (4) for susceptibility pattern. ESBL characterization was performed by PCR and sequencing (6,32,33). Population structure was established by PFGE (32). In addition, phylogenetic groups among E. coli isolates were identified by a multiplex PCR (3). Twenty-eight out of 40 ICs (70%, CI95%: ) and 9 out of 54 household contacts (16.7%; CI95%: ) presented fecal carriage with ESBL-producing E. coli isolates (Figure 1). Moreover, 9 ICs out of 29 had at least one colonized household contact (31.0%; CI 95%: ). This figure increased (42.1%; CI 95%: ) in the subset of household contacts of ICs with fecal carriage (8/19). In contrast, only one household contact (1/10, 10%; CI95%:0-28.6) within the subset of those from ICs with negative fecal carriage, presented an intestinal colonization of ESBL-producing E. coli (Figure 1). Characteristics of ESBL-producing isolates from ICs and their corresponding household contacts are shown in tables 1 and 2. Seventy-two percent (21/29) of ICs presented E. coli clinical isolates with the same PFGE type as those from their fecal samples. Moreover, PFGE analysis revealed an indistinguishable pattern among ESBLproducing E. coli from ICs (clinical sample or fecal sample) and their corresponding household contacts in 66% (6/9) of isolates. 4

5 Overall, ESBL characterization revealed a predominance of CTX-M-14 (57%) and SHV-12 (18.6%), the emergence of ESBLs belonging to CTX-M-1 group (7.1%), and a small representation of TEM (5.7%) enzymes. This distribution is in agreement with the epidemiological situation of ESBL-producing isolates from the community at the time of enrollment in our study (2,27,31). It is of note that 2 ICs and 2 household contacts were colonized with two different ESBL-producing isolates. The present study demonstrates a high rate (70%) of intestinal colonization in patients with CAI due to ESBL-producing organisms. The presence of these pathogens in the bowel is considered as a risk factor for suffering infections with these bacteria (28). Moreover, it can explain the high rates of isolation of ESBL-producing organisms in certain anatomic locations (i.e. abdominal and urinary tract infections), than in others where there is not a clear implication of endogenous microbiota (28). It is of note that our 70% figure is similar to that found in hospitalized patients under high antimicrobial selective pressure (17,23). Around 50% of our patients received antimicrobial treatment (18 patients) or medical extrahospitalary assistance (22 patients) at least two months before ESBL infections which have been defined as risk factors for colonization with these pathogens (28). An important finding was the high rate (16.7%) of intestinal colonization in households of patients suffering CAI with ESBL-producing organisms. This figure is higher than that previously found by our group in the same geographic area (3.7%, p=0.004, Chi square test) and in other countries (range, 1.7% to 13.1%) in healthy individuals (13,21,26,32). These values were even higher (42.1%) in the subset of household contacts of ICs with fecal carriage. Despite of potential limitation of our study due to the small sample size, these results revealed the importance of the intestinal colonization as a reservoir for transmission of resistant bacteria and its potential role as 5

6 traffickers in antibiotic resistance genes (14,29). This has been studied in the nosocomial setting where it is generally assumed that for every patient with a clinically significant infection with an ESBL-producing organism at least one other patient exists in the same unit with intestinal colonization with an ESBL-producer (9,17,22). Data from the community remain scarce. It has been shown that methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci may be acquired from different family members (1,10,11) and there is no information contradicting this suspicion with ESBL pathogens. Patient-to-patient transmission has been demonstrated with ESBL-producing K. pneumoniae isolates in the nosocomial setting (7,17,23). In the community, this landscape might have also been produced with E. coli (6,8,9,16). In our study, up to 66% of isolates from ICs and their corresponding households had an indistinguishable PFGE pattern. Epidemic plasmids might be responsible for this situation. This possibility was studied in different CTX-M-14 producing clones (Valverde et al, unpublished data). We cannot rule out a common source as we do not investigate ESBL-producing organisms in food or domestic pets. Both have been involved in the spread of these pathogens and their corresponding bla ESBL genes (12,15,30). In summary, high rates of intestinal colonization with ESBL-producing organisms in community patients infected with these isolates was observed. Household contacts of these patients had high rates of intestinal colonization with ESBL-producing organisms, higher than that in outpatients and the general population (32). These results highlight that community patients and their households represent a clear reservoir for these organisms and the corresponding bla ESBL genes. This fact increases the risk of dissemination of such organisms to normal healthy people and facilitates the acquisition of resistance mechanisms by susceptible bacteria. 6

7 A. Valverde is supported by CIBER-ESP (Network Center for Biomedical Research in Epidemiology and Public Health) from Instituto Carlos III, Ministerio de Sanidad y Consumo of Spain. F. Grill was supported by Red Española de Investigación en Patología Infecciosa (REIPI-ISCIII-C03/14) from Instituto Carlos III, Ministerio de Sanidad y Consumo of Spain. This study was partially supported by research grants from the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (PI040162), DeReMicrobiana Project of the Madrid Autonomous Community, and the European Commission (LSHM-CT ). We thank Azucena Rollán for her excellent technical work. 7

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12 TABLE 1. Characteristics of ESBL-producing isolates from clinical and fecal samples of index cases with no household contacts Index case IC1 IC2 IC4 IC11 IC12 IC13 IC15 IC16 IC18 IC19 E. coli Sample Organism PFGE phylogroup ESBL Urine E.coli CV3C A CTX-M-14 Fecal E.coli CV3HC A CTX-M-14 Urine E.coli CV12C D CTX-M-14 Fecal E.coli CV12C D CTX-M-14 Urine E.coli CV16C A CTX-M-14 Fecal E.coli CV16C A CTX-M-14 Urine E.coli CV37C D CTX-M-14 Fecal E.coli CV37HC D CTX-M-14 Urine E.coli CV39C D CTX-M-14 Fecal E.coli CV39C D CTX-M-14 Urine E.coli CV40C A CTX-M-14 Fecal E.coli CV40C A CTX-M-14 Urine E.coli CV52C A CTX-M-14 Fecal E.coli CV52C A CTX-M-14 Urine E.coli CV59C B1 CTX-M-14 Fecal E.coli CV59C B1 CTX-M-14 Urine E.coli CV77C B1 CTX-M-14 Fecal E.coli CV77C B1 CTX-M-14 Urine E.coli CV80C B1 CTX-M-14 Fecal E.coli CV80HC B1 CTX-M-14 12

13 262 TABLE 1 (cont.) E. coli Index case Sample Organism PFGE ESBL phylogroup IC7 Urine E.coli CV20C D CTX-M-14 Fecal E.coli CV20S D SHV-2 IC10 Urine E.coli CV29C B1 CTX-M-9 Fecal E.coli CV29C B1 CTX-M-9 IC17 Urine E.coli CV70C D CTX-M-9 Fecal E.coli CV70C D CTX-M-9 IC20 Urine E.coli CV86C A CTX-M-9 Fecal E.coli CV86C D CTX-M-9 IC3 Urine E.coli CV14O B1 SHV-12 Fecal E.coli CV14H-D a B1 SHV-12 IC5 Urine E.coli CV17S B1 SHV-12 Fecal E.coli CV17S B1 SHV-12 IC8 Urine E.coli CV24S A SHV-12 Fecal E.coli CV24S A SHV-12 IC6 Urine K. pneumoniae CV18O - TEM-4 Fecal E.coli CV18H-D a B1 SHV-12 IC14 Urine E.coli CV41T A TEM-39 Fecal E.coli CV41T A TEM-39 IC9 Urine E.coli CV28C D CTX-M-32 Fecal E.coli CV28C D CTX-M a. PFGE pattern degradated. 13

14 TABLE 2. Characteristics of ESBL-producing E. coli isolates from all samples of index cases and fecal samples from positive ESBL-household contacts Family Index case Household contact a group (no. of household contacts) Sample PFGE Phylogroup ESBL PFGE Phylogroup ESBL FG1 (1 ) Urine CV5C D CTX-M-14 Fecal CV5C D CTX-M-14 CV5C D CTX-M-14 FG2 (1) Urine CV34C A CTX-M-14 Fecal - b - b - b CV36C A CTX-M-14 FG4 (1) Urine CV61C B1 CTX-M-14 Fecal CV61C B1 CTX-M-14 CV61C B1 CTX-M-14 FG5 (1) Urine CV64C A CTX-M-14 Fecal CV64C B1 CTX-M-14 CV64C B1 CTX-M-14 FG7 (1) Urine CV81C A CTX-M-14 Fecal CV81C A CTX-M-14 CV82C D CTX-M-14 FG8 (1) Urine CV92C B1 CTX-M-14 Fecal CV92HC A CTX-M-14 CV93.1 A TEM-52 CV93.2 A TEM-52 FG9 (1) Urine CV94S D SHV-12 Fecal CV94S D SHV-12 CV94S D SHV-12 FG3 (1) Urine CV44S D SHV-12 Fecal CV44S D SHV-12 CV44C A CTX-M-14 CV44C A CTX-M-14 FG6 (1) Urine CV68C D CTX-M-1 Fecal CV68C D CTX-M-1 CV68C D CTX-M a. Isolates of household contacts were obtained from fecal samples; b. No positive ESBL fecal sample was obtained 14 CV69C D CTX-M-14

15 FIG 1. Index cases with fecal carriage of ESBL-producing organism and their corresponding household contacts Index cases: 40 Index case carriage (-): 12 Index case carriage (+): 28 With household contacts: 10 Household contacts carriage (+): 1 Without household contacts: 2 Household contacts carriage (-): 15 With household contacts: 19 Household contacts carriage (+): 8 Household contact: 54 Index case: Patient with ESBL-Producing Organism Infection Without household contacts: 9 Household contacts carriage (-): Index Case Carriage (+) : Patient with ESBL-Producing Organism Infection and fecal positive ESBL culture Household contacts Carriage (+) : Household contacts with positive ESBL-Producing Organism in fecal sample 15