High Rate of Faecal Carriage of Extended-Spectrum-β-Lactamase-Producing Escherichia coli in Healthy Children in Gipuzkoa, northern Spain

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1 AAC Accepts, published online ahead of print on 6 January 2014 Antimicrob. Agents Chemother. doi: /aac Copyright 2014, American Society for Microbiology. All Rights Reserved High Rate of Faecal Carriage of Extended-Spectrum-β-Lactamase-Producing Escherichia coli in Healthy Children in Gipuzkoa, northern Spain Authors: María Fernández-Reyes, 1 Diego Vicente, 1,2 María Gomariz, 1 Olatz Esnal, 2 Joseba Landa, 3,4 Eider Oñate, 3 Emilio Pérez-Trallero 1,2,4 * Affiliations: 1. Microbiology Department, Hospital Universitario Donostia-Instituto Biodonostia, San Sebastián, Spain 2. Biomedical Research Centre Network for Respiratory Diseases (CIBERES), San Sebastián, Spain 3. Pediatric Department, Hospital Universitario Donostia-Instituto Biodonostia, San Sebastián, Spain 4. Faculty of Medicine, University of the Basque Country, UPV/EHU, San Sebastián, Spain * Corresponding author: Emilio Pérez-Trallero Servicio de Microbiología Hospital Universitario Donostia Paseo Dr Beguiristain s/n San Sebastián mail: mikrobiol@terra.com Running title: Carriage of ESBL-producing E. coli in Spanish children Key words: ESBL, CTX-M, SHV-12, faecal carriage, intestinal colonization 1

2 Abstract The prevalence of extended-spectrum-β-lactamase-producing Enterobacteriaceae (ESBLPE) was studied in stool samples from month-old healthy children. Twenty-four percent of them and 10.7% of the 318 faecal samples studied yielded extended-spectrum-β-lactamaseproducing E. coli, being SHV-12, CTX-M-1, CTX-M-14 and TEM-52, the most common type of β-lactamase. This high prevalence of ESBLPE in healthy people -to our knowledge the highest currently reported in Europe- may represent a risk for increased infections by these organisms in a future. Text The spread of extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae (ESBLPE) is an emerging health problem worldwide (1). These enzymes are often encoded by a variety of transmissible genes, which facilitates transfer among bacteria. Furthermore, they are typically accompanied by genes encoding resistance to multiple antimicrobial groups, such as fluoroquinolones, aminoglycosides and folate pathway inhibitors (trimethoprimsulfamethoxazole), leaving few treatment options (2). Enterobacteriaceae are inhabitants of human intestinal flora, and faecal content with these bacteria is the paradigm of normal circulating strains in a particular time and region. Faecal carriers of ESBLPE represent an important reservoir, contributing to person-to-person transmission and strengthening their dissemination. Few studies have evaluated intestinal carriage of ESBLPE in healthy individuals in the community, especially in children. The main aim of this study was to investigate the prevalence, phenotypic resistance patterns and genetic characteristics of ESBLPE in faeces from healthy infants and children. We performed a prospective follow-up study of a cohort of healthy neonates born in Donostia (Gipuzkoa, Basque Country, Spain). The stool samples from 125 healthy children (term birth, normal pregnancy and Apgar score 8-10) at the ages of 8, 12 and 16 months were analyzed. Informed consent was obtained from the parents of participating children. Three samples were taken from 68 children and only two samples from the remaining 57. To screen for ESBLPE, faecal samples were spread on MacConkey agar (BioMérieux) plates, with subsequent placement of 30 µg cefotaxime and ceftazidime disks (BioConnections, Neo-Sensitabs). We 2

3 selected all colonies growing within the inhibition halos and the presence of ESBLPE was confirmed by standard methods (3). Antimicrobial susceptibility was performed using the microdilution method (Sensititre, TREK diagnostic System). All isolates were screened for the genes bla OXA, bla SHV, bla TEM and bla CTX-M by a multiplex PCR assay (4). To confirm and further determine the identities of the β-lactamase genes, amplified genes (5) were sequenced (ABI PRISM 3100 Genetic Analyser, Applied- Biosystems). Finally, sequences were confirmed by comparison with the ESBLPE included in the GenBank database using BLAST. E. coli strains were assigned to one of the four main phylogenetic groups A, B1, B2 and D by using a multiplex PCR-based method (6). MLST was performed according to the University College Cork scheme for E. coli ( ESBLPE was detected in 18/68 (26.5%) children with all three samples and in 12/57 (21.1%) children with only two samples. The overall colonization prevalence was 24.0% (30/125 children). In total, 10.7% of the 318 samples investigated were positive. In 26 children, these resistant isolates were detected in only one sample and in four children in two consecutive samples. All 34 ESBLPE detected were identified as E. coli. The family of these β-lactamases and the age of the children when the ESBLPE were detected is reported in Table 1. Of the 34 ESBL-producing E. coli, 19 were identified as producing CTX-M-type, 12 as SHV-type and 8 as TEM-type β-lactamase. Two of these isolates simultaneously showed genes coding for CTX-M- and TEM-enzymes and three isolates showed genes for CTX-M- and SHV-enzymes. Among 19 CTX-M-producing E. coli: 11 belonged to group 1 (eight CTX-M-1, two CTX-M-15 and one CTX-M-22) and eight to group 9 (six of them CTX-M-14 and two CTX-M-65). Eight TEM-52-, eleven SHV-12- and one SHV-5-producing E. coli were also found. The gene coding for OXA-type enzyme was not observed in any of the isolates. The most prevalent phylogenetic groups were groups A (50%) and group D (38.2%). Only two (5.9%) isolates (numbers 33 and 34) belonged to the phylogenetic group B2, generally associated with urosepsis and bacteremia in immunocompetent hosts (6,7), and both were isolated from the same children in two different periods. Isolate number 33 was identified as ST219 and a new ST was assigned to isolate 34 (ST3909). 3

4 Carriage of ESBLPE was similar at each age studied, being detected in 8.8%, 13.9% and 8.9% of samples at 8, 12 and 16 months, respectively (Table 1). The persistence of carriage of ESBLPE was infrequent. In only one child, the same resistant isolate, a SVH-12-producing E. coli isolate belonged to D phylogenetic group, was detected in two samples (isolates 26 and 27, Table 1). Ampicillin, cefotaxime and ticarcillin non-susceptibility was found in 100% of the isolates, aztreonam non-susceptibility in 73.5%, and ceftazidime non-susceptibility in 61.8%. Nalidixic acid, ciprofloxacin, levofloxacin and trimethoprim-sulfamethoxazole nonsusceptibility were found in 64.7%, 32.4%, 32.4 and 41.2% of isolates, respectively. Multiresistance to beta-lactams, levofloxacin, trimethoprim-sulfamethoxazole and tetracycline was observed in 20.6% (7/34) of isolates (Table 1). All isolates were susceptible to carbapenems. Among ESBLPE, E. coli is the most prevalent in Europe and has been on a continuous rise during the last decade (8,9). Faecal colonization by ESBL-producing E. coli in healthy individuals has been previously detected in Spain, with rates from 3.7% to 7.7% (10,11,12). A higher prevalence was found among the household contacts of patients infected with ESBLPE (12,13). In other European countries the ESBLPE prevalence in healthy children ranged 2.6% to 4.6% (14-16) with a prevalence of 5.2% in French infants with acute diarrhea (17). CTX-M-1, CTX-M-14 and SHV-12, were the most prevalent ESBL-producing E. coli found in chickens and other animal foods in Spain (18,19), suggesting that foods could be the source of acquisition of these resistant isolates in children. Genes coding for ESBLs are often associated with resistance determinants to other antimicrobials, including aminoglycosides (aac(6)-ib), fluoroquinolones (qnr), tetracycline (teta), and trimethoprim-sulfamethoxazole (sul) (2,14). In the present study, 52.4% of the ESBLPE were resistant to three or more antimicrobial classes and 20.6% of isolates were resistant to tetracycline, trimethoprim-sulfamethoxazole and levofloxacin. In the risk factor analysis, only one factor was significantly associated with a higher prevalence of ESBLPE carriage (Table 2). Surprisingly, children not attending nursery school had the highest percentage of ESBLPE carriage, which was in disagreement with other studies (13,16). 4

5 Dissemination of ESBLPE to healthy people has increased dramatically worldwide. Several factors such as human and animal antibiotic overuse, human cross-infection, transmission from pets or other animals via the food chain, among other factors, have contributed to this dissemination outside hospitals (18,20,21). The high circulation of ESBL-producing E. coli in healthy children in our region should alert us to the possibility of an increase of these strains in infected patients. Acknowledgment. This study was supported in part by grants PI/10/01564 from Fondo de Investigaciones Sanitarias, Ministerio de Sanidad, Servicios Sociales e Igualdad, Spain and UPV/EHU: IT from the Education Department of the Basque Country Government. Downloaded from on May 11, 2018 by guest 5

6 References 1. Pitout JD, Laupland KB Extended-spectrum β-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect. Dis. 8: Coque TM, Baquero F, Canton R. 2008, Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro Surveill. 13: Clinical and Laboratory Standards Institute Performance standard for antimicrobial susceptibility testing, 20 th informational supplement, M100-S21, vol 31, no. 1. Clinical and Laboratory Standards Institute, Wayne, PA. 4. Fang H, Ataker F, Hedin G, Dornbusch K Molecular epidemiology of extendedspectrum β-lactamases among Escherichia coli isolates collected in a Swedish hospital and its associated health care facilities from 2001 to J. Clin Microbiol. 46: Schmitt J, Jacobs E, Schmidt H Molecular characterization of extended-spectrum beta-lactamases in Enterobacteriaceae from patients of two hospitals in Saxony, Germany. J. Med. Microbiol. 56(Pt 2): Clermont O, Bonacorsi S, Bingen E Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66: Jauréguy F, Carbonnelle E, Bonacorsi S, Clec'h C, Casassus P, Bingen E, Picard B, Nassif X, Lortholary O Host and bacterial determinants of initial severity and outcome of Escherichia coli sepsis. Clin. Microbiol. Infect. 13: Clermont O, Bonacorsi S, Bingen E CTX-M: changing the face of ESBLs in Europe. J. Antimicrob. Chemother. 59: European Antimicrobial Resistance Surveillance Network (EARS-Net) Proportion of 3rd gen. cephalosporins resistant (R+I) Escherichia coli isolates in participating countries. Net/database/Pages/graph_report.aspx?MasterPage=1 (access date June 2013). 10. Valverde A, Coque TM, Sánchez-Moreno MP, Rollán A, Baquero F, Cantón R Dramatic increase in prevalence of fecal carriage of extended-spectrum beta-lactamaseproducing Enterobacteriaceae during nonoutbreak situations in Spain. J. Clin. Microb. 42: Vinué L, Sáenz Y, Martínez S, Somalo S, Moreno MA, Torres C, Zarazaga M Prevalence and diversity of extended-spectrum beta-lactamases in fecal Escherichia coli isolates from healthy humans in Spain. Clin. Microbiol. Infect. 15:

7 Rodríguez-Baño J, López-Cerero L, Navarro MD, Díaz de Alba P, Pascual A Faecal carriage of extended-spectrum beta-lactamase-producing Escherichia coli: prevalence, risk factors and molecular epidemiology. J. Antimicrob. Chemother. 62: Valverde A, Grill F, Coque TM, Pintado V, Baquero F, Cantón R, Cobo J High rate of intestinal colonization with extended-spectrum-lactamase-producing organisms in household contacts of infected community patients. J. Clin. Microbiol. 46: Guimarães B, Barreto A, Radhouani H, Figueiredo N, Gaspar E, Rodrigues J, Torres C, Igrejas G, Poeta P Genetic detection of extended-spectrum beta-lactamasecontaining Escherichia coli isolates and vancomycin-resistant enterococci in fecal samples of healthy children. Microb. Drug. Resist. 15: Birgy A, Cohen R, Levy C, Bidet P, Courroux C, Benani M, Thollot F, Bingen E Community fecal carriage of extended-spectrum beta-lactamase-producing Enterobacteriaceae in French children. BMC Infect. Dis. 12: Kaarme J, Molin Y, Olsen B, Melhus A Prevalence of extended-spectrum betalactamase-producing Enterobacteriaceae in healthy Swedish preschool children. Acta Paediatr. 102: Boutet-Dubois A, Pantel A, Prère MF, Bellon O, Brieu-Roche N, Lecaillon E, Le Coustumier A, Davin-Regli A, Villeneuve L, Bouziges N, Gleize E, Lamarca R, Dunyach-Remy C, Sotto A, Lavigne JP Faecal carriage of oxyiminocephalosporinresistant Enterobacteriaceae among paediatric units in different hospitals in the south of France. Eur. J. Clin. Microbiol. Infect. Dis. 32: Ojer-Usoz E, González D, Vitas AI, Leiva J, García-Jalón I, Febles-Casquero A, Escolano Mde L Prevalence of extended-spectrum- β-lactamase-producing Enterobacteriaceae in meat products sold in Navarre, Spain. Meat Science. 93: Briñas L, Moreno MA, Zarazaga M, Porrero C, Sáenz Y, García M, Dominguez L, Torres C Detection of CMY-2, CTX-M-14, and SHV-12 β-lactamases in Escherichia coli fecal-sample isolates from healthy chickens. Antimicrob. Agents Chemother. 47: Torres C, Zarazaga M BLEE en animales y su importancia en la transmisión a humanos. Enferm Infecc Microbiol Clin. 25 Supl. 2: Foxman B Editorial commentary: extended-spectrum β-lactamase-producing Escherichia coli in the United States: time to rethink empirical treatment for suspected E. coli infections? Clin Infect Dis. 56:

8 TABLE 1. Age of children at sampling, type and phylogenetic group of extended-spectrum beta- lactamase (ESBL) enzymes, and antimicrobial resistance phenotype of 34 ESBL-producing Escherichia coli. Isolate Children age (months) E. coli enzyme type Phylogenetic group Antimicrobial resistance Beta-lactams Quinolones Other 1 8 CTX-M-1 A AMP, A/S, TIC, FOT NAL, CIP, TET 2 12 CTX-M-1 A AMP, A/S, TIC, FOT, TET 3 12 CTX-M-1 A AMP, A/S, TIC, FOT, FEP, NAL TET 4 12 CTX-M-1 A AMP, A/S, TIC, FOT, TET 5 16 CTX-M-1 A AMP, A/S, TIC, FOT, TET, SXT 6 16 CTX-M-1 A FOT, FOX, AMP, A/S, TIC, TET, SXT 7 16 CTX-M-1 D AMP, A/S, TIC, FOT TET 8 8 CTX-M-1, D AMP, FOX, FOT, TAZ NAL, CIP, TET, SXT TEM-52 AMC, A/S, TIC, 9 12 CTX-M-14 A AMP, A/S, TIC, FOX, FOT NAL, CIP, TET, SXT CTX-M-14 A AMP, A/S, TIC, FOT NAL SXT CTX-M-14 B1 AMP, A/S, TIC, FOT NAL TET, SXT CTX-M-14 D AMP, A/S, TIC, FOT NAL TET, SXT 13 8 CTX-M-14, SHV-12 A AMP, A/S, TIC, FOT, TAZ, CTX-M-14, SHV-5 D TAZ, FOT, AMP, TIC, NAL GEN, TOB, TET CTX-M-15 D AMP, A/S, TIC, FOT, TAZ, FEP, NAL, CIP, GEN, TOB, TET, SXT CTX-M-15, D TAZ, FOT, AMP, A/S, TIC, NAL, CIP, TET, SXT TEM CTX-M -22 D AMP, AMC, A/S, TIC, FOS FOT, CTX-M-65 D AMP, A/S, TIC, FOT, FOX NAL,, CIP GEN, TOB, TET, SXT CTX-M-65 A AMP, A/S, TIC, FOT, TAZ, NAL TET SHV SHV-12 A AMP, TIC, FOT, TAZ, NAL TET, SXT SHV-12 A AMP, TIC, FOT, TAZ, NAL TET 8

9 SHV-12 A AMP, AMC, A/S, TIC, FOT, TAZ, SHV-12 A AMP, A/S, TIC, FOT, TAZ, SHV-12 A AMP, TIC, FOT, TAZ, SHV-12 B1 AMP, TIC, FOT, TAZ, 26 8 SHV-12 D AMP, TIC, FOT, TAZ, SHV-12 D AMP, TIC, FOT, TAZ, 28 8 SHV-12 D AMP, TIC, FOT, TAZ, TEM-52 A AMP, AMC, A/S, TIC, P/T, FOX, FOT, TAZ, TEM-52 A AMP, AMC, A/S, TIC, FOX, FOT, TAZ 31 8 TEM-52 D AMP, AMC, A/S, TIC, FOX, FOT, TAZ TEM-52 D AMP, AMC, A/S, TIC, FOX, FOT, TAZ TEM-52 B2 AMP, AMC, TIC, FOT, TAZ NAL, CIP, TET, SXT NAL, CIP, TET, SXT TET NAL, CIP, NAL NAL NAL, CIP, TET TET NAL GEN, TOB, TET, SXT TET NAL, CIP, TET TEM-52 B2 AMP, TIC, FOT, TAZ, AMP: ampicillin, AMC: amoxicillin-clavulanic acid, A/S: ampicillin-sulbactam, TIC: ticarcillin, P/T: Piperracillin-Tazobactam, FOX: cefoxitin, FOT: cefotaxime, TAZ: ceftazidime, FEP: cefepime, : aztreonam, GEN: gentamycin, TOB: tobramycin, NAL: nalidixic acid, CIP: ciprofloxacin, : levofloxacin, TET: tetracyclin, SXT: trimetropim-sulfamethoxazole, FOS: Fosfomycin. 9

10 TABLE 2. Potential risk factors for children with and without extended-spectrum betalactamases-producing Escherichia coli (ESBL) faecal colonization. ESBL carriers Non-ESBL (n=30) carriers (n=95) Nº Percentage Nº Percentage Sex (male) % % Pets % % Two or more siblings % % Breastfeeding % % Nursery % % p= Previous antibiotherapy 1 During the 16 months of life % % Four months before sampling % 20 21% 1 Third generation cephalosporins were taken by 3 carrier and 3 non-carrier children. Downloaded from on May 11, 2018 by guest 10