Elsevier Editorial System(tm) for The Lancet Infectious Diseases Manuscript Draft Manuscript Number: Title: Relative abundance

of extended-spectrum beta-lactamases-producing Escherichia coli among faecal enterobacteria and occurrence of urinary-tract infections in women Article Type: Articles (Original Research) Keywords: concentration, density of colonisation, faeces, antibiotic Corresponding Author: Dr ETIENNE RUPPE, Corresponding Author's Institution: AP-HP, Hpitaux Paris Nord - Val de Seine First Author: ETIENNE RUPPE Order of Authors: ETIENNE RUPPE; BRANDUSA LIXANDRU; RADU COJOCARU; CAGRI BUKE; ELISABETH PARAMYTHIOTOU; CECILE ANGEBAULT; CLAIRE VISSEAUX; INGRID DJUIKOUE; OLGA BURDUNIUC; ASSIYA EL MNIAI; CANDICE MARCEL; THOMAS KESTEMAN; LAURENCE ARMANDLEFEVRE; ANTOINE ANDREMONT Manuscript Region of Origin: FRANCE Abstract: Background. Escherichia coli, commonly found in the intestinal tract, can cause urinary tract infections (UTIs). Moreover, infections caused by extended spectrum beta-lactamase (ESBL)-E. coli are of major concern because few antibiotics remain active. We investigated whether the relative abundance of ESBL-E. coli (ESBL-RA) among total faecal enterobacteria is associated with occurrence of ESBL-E. coli UTIs. Methods. We analyzed the stool of 310 women in whom E. coli-causing UTIs were diagnosed using a urine sample. The first stool since the urine sample was taken was sampled and tested for ESBL-RA by culture on selective agar. The relationship between ESBL-RA and occurrence of ESBL-E. coli-causing UTI was analyzed in patients who were not exposed to antibiotics when the stool was passed. ESBL-E. coli was characterised by ESBL type, phylogroup, relatedness, and virulence factors. Findings. The prevalence of ESBL-E. coli faecal carriage was 20*3%, and ESBL-E. coli-causing UTIs were found in 12*3% women. Antibiotic exposure at sampling increased the mean ESBL-RA by 13-fold (14*3% vs. 1*1%, respectively; p < 0*001). Mean ESBL-RA was 18-fold higher in women with ESBL-E. coli-causing UTI than in those with a UTI caused by a different E. coli strain (10% vs. 0*56%, respectively; p < 0*05). Phylogroup and virulence factors were not significantly different irrespective of whether the ESBL-E. coli strain from faeces was found in urine. ESBL-RA prevalence < 0*1% was highly predictive of a non-ESBL-E. coli UTI. Interpretation. ESBL-RA plays key role in the occurrence of ESBL-E. coli in UTI. Funding. This work was supported in part by EGIDE, by the French National Reference Centre and by OSEO.

Manuscript

Relative abundance of extended-spectrum beta-lactamases-producing Escherichia coli among faecal enterobacteria and occurrence of urinary-tract infections in women Etienne Rupp (PharmD)1,2*, Brandusa Lixandru (MSc)3, Radu Cojocaru (MD)4, ar Bke (MD) 5, Elisabeth Paramythiotou (MD)6, Ccile Angebault (PharmD)1,2, Claire Visseaux (PharmD)1,2, Ingrid Djuikoue (MSc)1,2, Olga Burduniuc (MD)4, Assiya El Mniai (Engineer)2, Candice Marcel (Technician)1,2*, Thomas Kesteman (MD)2, Laurence Armand-Lefvre (PharmD)1,2, Antoine Andremont (MD, PhD)1,2

EA3964, Facult de Mdecine Paris Diderot, Paris, France Laboratoire de Bactriologie, Hpital Bichat-Claude Bernard, AP-HP, Paris, France Laboratory Nosocomial Infections and Antibiotic Resistance, Cantacuzino Institute,

Bucarest, Romania
4

National Centre for Preventive Medicine, Chiinu, Moldova Infectious Diseases Polyclinic, Ege University, Izmir, Turkey Fourth Internal Medicine Department, Attikon Hospital, Athens, Greece The authors equally contributed to the study (co-second authors)

Running head: Relative abundance of and infections caused by ESBL-E. coli Keywords: concentration, density of colonisation, faeces, antibiotic Corresponding author: Etienne Rupp Laboratoire de Bactriologie, Hpital Bichat-Claude Bernard, 46 rue Henri Huchard, 75018 Paris, France Email: etienne.ruppe@bch.aphp.fr Tel. (+33) (0)1 40 25 85 07 Fax. (+33) (0)1 40 25 85 81

Abstract (262 words)

Background. Escherichia coli, commonly found in the intestinal tract, can cause urinary tract infections (UTIs). Moreover, infections caused by extended spectrum beta-lactamase (ESBL)E. coli are of major concern because few antibiotics remain active. We investigated whether the relative abundance of ESBL-E. coli (ESBL-RA) among total faecal enterobacteria is associated with occurrence of ESBL-E. coli UTIs.

Methods. We analyzed the stool of 310 women in whom E. coli-causing UTIs were diagnosed using a urine sample. The first stool since the urine sample was taken was sampled and tested for ESBL-RA by culture on selective agar. The relationship between ESBL-RA and occurrence of ESBL-E. coli-causing UTI was analyzed in patients who were not exposed to antibiotics when the stool was passed. ESBL-E. coli was characterised by ESBL type, phylogroup, relatedness, and virulence factors.

Findings. The prevalence of ESBL-E. coli faecal carriage was 203%, and ESBL-E. colicausing UTIs were found in 123% women. Antibiotic exposure at sampling increased the mean ESBL-RA by 13-fold (143% vs. 11%, respectively; p < 0001). Mean ESBL-RA was 18-fold higher in women with ESBL-E. coli-causing UTI than in those with a UTI caused by a different E. coli strain (10% vs. 056%, respectively; p < 005). Phylogroup and virulence factors were not significantly different irrespective of whether the ESBL-E. coli strain from faeces was found in urine. ESBL-RA prevalence < 01% was highly predictive of a nonESBL-E. coli UTI.

Interpretation. ESBL-RA plays key role in the occurrence of ESBL-E. coli in UTI.

Funding. This work was supported in part by EGIDE, by the French National Reference Centre and by OSEO.

Introduction

Escherichia coli is a commensal of the human intestinal microbiota with a colonisation density of 107 to 108 colony-forming units (CFU) per gram of faeces. The E. coli intestinal population comprises one to several clones, and the relative abundances (RAs) of these clones vary.1 Usually, E. coli members of the dominant population are susceptible to antibiotics, and resistant E. coli are subdominant.2 However, the proportion of the resistant clones increases with antibiotic exposure.3-4 E. coli is also a major pathogen and the leading cause of urinarytract infections (UTI).5 It is commonly accepted that E. coli strains that cause UTIs originate from the intestine;6 nonetheless, the precise physiopathological pathway remains unclear.5 It is has been suggested that prominent E. coli clones colonize the urethra more often than subdominant clones,7 but subdominant E. coli may overcome this disadvantage when carrying so-called virulence factors such as adhesins or siderophores which may help bacteria to survive and multiply in the urinary tract.8 This hypothesis was previously untested due to the difficulty of quantifying infecting clones within total intestinal E. coli when the clone is subdominant. However, this issue can be resolved by focusing on antibiotic-resistant E. coli, which is easily detectable in the faeces by using antibiotic selective agar, even when the organisms are present in low counts.9

Third-generation cephalosporins (3GC) are often used for the treatment of UTIs.10 However, resistance to 3GC has recently increased among E. coli strains, both in hospitals and in the community, as a result of a worldwide dissemination of extended spectrum beta-lactamase (ESBL)-producing strains, particularly those of the CTX-M variety.11-12 This increase has been fuelled by intestinal colonization, which occurs more frequently than actual infections.4 In our study, we assessed the relationship between faecal RA of ESBL E. coli (ESBL-RA) and the occurrence of ESBL-E. coli-causing UTIs.

Material and methods Patients and strains We included patients from 5 centres located in countries with a high prevalence of ESBL E. coli (http://www.ecdc.europa.eu/en/activities/surveillance/EARS-Net), including the National Centre for Preventive Medicine (Chiinu, Moldova; May 2009June 2010), Modus Vivendi community-based laboratory (Chiinu, Moldova: February 2009September 2010), the Cantacuzino Institute (Bucarest, Romania: October 2008July 2009), the Infectious Diseases Polyclinic of the Ege University Teaching Hospital (Izmir, Turkey: September 2010March 2011), and the Department of the Attikon University Teaching Hospital (Athens, Greece: January 2010February 2011). A local investigator from each centre was trained in our central laboratory (Bichat-Claude Bernard Hospital, Paris, France) in order to ensure experimental homogeneity among sites. Female outpatients consulting these centres for UTI over the study period were considered for enrolment and were asked (i) to store the first stool passed after their initial visit in a specifically provided container at 4 C and (ii) to bring the stool sample when returning for definitive urine test results. Patients returning after 72 h were excluded from the study. All ethical and informed consent rules from each country were followed. Patients diagnosed with an E. coli-causing UTI upon return were further included in the study. Approximately 100 mg of the stool sample was diluted in 1mL of brain-heart infusion broth supplemented with 10% glycerol and maintained at 80C by local investigators. Stool samples and UTI strains preserved in dry ice were transported by air to the central laboratory and kept frozen until they were ready to be harvested in batches. UTI strains were re-isolated and tested for antibiotic susceptibility. The presence of ESBL was detected by the disk diffusion method, as recommended by the French Society for Microbiology (http://www.sfm-microbiologie.org). Stool samples were defrosted in batches at

4C, and 100 L of the broths were plated on Drigalski agar plates supplemented either with or without 1 mg/L of cefotaxime and cultured for 48 h at 37C. All CFU with distinct morphotypes were further identified by Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometer (MALDI-TOFF; Bruker Daltonics, Bremen, Germany) and tested for antibiotic susceptibility and the presence of ESBL. E. coli counts were determined by plating serial dilutions of the broth on Drigalski agar with or without 1 mg/L cefotaxime. CFU were counted in decimal logarithms at the dilution in which 1100 CFU were visible. ESBLRA was calculated as the ratio of counts of ESBL E. coli over total enterobacteria, expressed as a percentage.

Antibiotic exposure at time of faecal sampling Patients were categorized as having antibiotic exposure at the time of faecal sampling if (i) they informed that they had taken antibiotics for the current infection, or (ii) antibiotic activity was detected in their faeces, even if they did not declare it. Detection was achieved using a simple microbiological assay performed as described.13 Briefly, 10 L of defrosted stool was dropped on antibiotic-free, sterile 6-mm diameter paper discs (Dutscher, Brumath, France), which were spotted on Mueller-Hinton agar inoculated with a suspension of 105 CFU/mL of the fully antibiotic susceptible strain E. coli ATCC 25922. Stools for which an inhibition zone was observed around the disc following overnight incubation at 37C were determined as having antibacterial activity. Studies in which volunteers are experimentally exposed to antibiotics have shown that this test is always negative in samples taken before exposition begins.14

Molecular characterization of ESBL strains

All faecal and UTI ESBL-producing strains were further characterised. DNA was extracted using a triton-based lysis buffer combined with microbeads and further quantified with a NanoDrop quantifier (Thermo Scientific). Group 1 and 9 blaCTX-M were amplified and sequenced using previously described primers.15 Phylogroups were determined by triplex PCR, as previously described.16 Virulence factors PapC, Sfa, IroN, Cnf1, HlyC, Aer, FyuA, Irp2, IreA, Iha, and Usp were detected by PCR, as described,18 and virulence scores were calculated.19

Concordance between E. coli strains from urine and faeces in ESBL-E. coli carriers Relatedness between ESBL-E. coli strains from urines and faeces was assessed by rep-PCR using primers REP1R 5-NNNGCGCCGNCATCAGGC-3 and REP2R 5ACGTCTTATCAGGCCTAC-3.17 Rep-PCR products were migrated on an Agilent 2100 Bioanalyzer with DiversiLab software (bioMrieux). Concordant women referred to women with ESBL-E. coli strains from urine and faeces that displayed 95% similarity. Discordant women referred to women in which (i) ESBL-E. coli strains from urine and faeces displayed <95% similarity patterns or (ii) ESBL-E. coli from faeces was not found in urine (i.e. urine E. coli were ESBL negative).

Statistical analysis Data were analyzed using Epi-Info v32 (www.cdc.gov/epiinfo). Qualitative variables were tested by or Fishers exact test. Associations between continuous variables and qualitative variables were tested by the Students or ANOVA tests. The significance level was set at 005.

Role of the funding source

This work was supported in part by Egide (ECO-NET program) that provided funding for local expenses in Romania and Moldova, by the French National Reference Center Resistance in commensal flora, and by OSEO (Nosobio program) that provided funding for additional expenses.

Results ESBL-E. coli prevalence in urine and faeces In total, 310 women with E. coli UTIs returned a stool sample within 72 h after their first visit, including 184, 39, 76, and 11 from Moldova, Romania, Turkey, and Greece, respectively. The median age was 35 years (range, 1792). Totally, 247 women (797%), including 14 (45%) with ESBL-UTI, were not found to be ESBL faecal carriers (Figure 1). The average prevalence of faecal carriage among all sites was high at 203% (63/310) and varying between 98% (18/184) in Moldova, 179% (7/39) in Romania, 474% (36/76) in Turkey, and 182% (2/11) in Greece. Among the 63 carriers, 58 (921%) and 5 (79%) carried 1 and 2 ESBL-E. coli strains, respectively. Regardless of the origin (urine or stool), CTX-M15 was the predominant allele (689%) (Table 2). ESBL-E. coli phylogroups were categorized according to their origin (urine or stool) as follows: B2 (respectively 368% and 176%), A (316% and 691%), and D (316% and 132%) (p < 005). Among ESBL-E. coli carriers, 516% (32/63) were exposed to antibiotics at the time of sampling, either because they admitted to having taken antibiotics (n = 30) or because antibacterial activity was detected in their faeces (n = 2) without declaration.

Associations with ESBL-RA Mean ESBL-RA (95% CI) was 13-fold higher in patients exposed to antibiotics when the stool was passed compared to those not exposed to antibiotics (143% [56%369%] vs. 11%

[032%36%], respectively, p < 0001) (Figure 2). This value did not vary significantly as a function of day the stool was returned. Day 0 (n = 5), 1 (n = 5), 2 (n = 17), or 3 (n = 17) showed mean ESBL-RAs of 25% [010%654%], 10% [0017%570%], 10% [016% 62%], and 056% [00011%341%], respectively (p = NS). Neither did the mean ESBL-RA vary among carriers of CTX-M-15-E. coli (n = 19) or any type of CTX-M (n = 12) (079% [018%34%] and 18% [017%-185%], respectively, NS). Similarly, mean ESBL-RA was not correlated with the virulence factor score (056% [0050%64%], 32% [051%195%], and 052% [0036%75%] for virulence scores of 01, 23, and 46, NS). We analyzed the relationship between ESBL-RA and occurrence of ESBL-E. coli UTI among the 31 patients (11 with ESBL-E. coli UTI and 20 with no ESBL-E. coli UTI) (Figure 1) who were not exposed to antibiotics when the stool was passed. A total of 24 women were discordant either (i) because they had a non-ESBL-E. coli UTI (n = 20) or (ii) because the ESBL-E. coli from faeces and urine displayed dissimilar rep-PCR patterns. Conversely, seven women were found to be concordant. Mean ESBL-RA was 18-fold higher in the seven concordant women than the 24 discordant ones (100% [054%100%] vs. 056% [015% 21%], respectively, p < 005). In contrast, mean VF scores of the faecal ESBL-E. coli strains were not significantly different between concordant and discordant women (17 [0050 34]vs. 23 [1431], NS). The sensitivity, specificity, and predictive values of ESBL-RA for the presence of ESBL-E. coli in urine are shown in Table 2. When the ESBL-RA was <01%, the negative predictive value for ESBL-E. coli UTI was 100%. In contrast, positive predictive values reached a maximum of 57% when ESBL-RA was 10%.

Discussion In our study, we found that faecal ESBL-RA was significantly higher in women with a UTI caused by ESBL-E. coli than those who were ESBL faecal carriers but had non-ESBL-E. coli

in their urine. This finding stresses the importance of ESBL-RA as a predictor of ESBL-E. coli UTI occurrence. It has been previously suggested that the predominant faecal E. coli had more chances to cause a UTI5 or to translocate to the bloodstream20 than subdominant faecal E. coli; nonetheless, the precise quantification of the phenomenon has not been reported thus far. Another striking result of our study is the 13-fold increase of ESBL-RA in women exposed to antibiotics. The increase in intestinal density of resistant gram-negative bacilli following antibiotics has been observed previously with drugs such as cotrimoxazole.3 However, to the best of our knowledge, increases were never quantified for ESBL. Percentages of ESBL-E. coli in the faeces of food chain animals have been studied recently and were shown to be variable. However, the role of antibiotic exposure was not explored.21 It is also known that previous antibiotic treatment is a risk factor for the occurrence of UTI with resistant bacteria.22-24 This evidence, together with our results, suggests that the occurrence of ESBL-E. coli UTI results from a two-step phenomenon, which includes (1) the increase of faecal ESBL-RA following antibiotic exposure in low count carriers and (2) the development of ESBL-E. coli UTI in those with already high ESBL-RA. Our results suggest that maintaining low ESBL-RA in the faeces might help to minimize the probability of developing a ESBL-E. coli UTI, even if complete eradication is not obtained. Indeed, the negative predictive value for ESBL-E. coli UTI was 100% when ESBL-RA was <01%. If this proves accurate, it should be investigated whether digestive decontamination in women at risk of repeated UTI can be useful if they are carriers with high ESBL-RA25 or whether it might prove efficient to prevent the increase of ESBL-RA during antibiotic treatments.26 To characterize the level of ESBL-E. coli faecal carriage, we chose to measure RA instead of density of colonization (DC, i.e. concentrations of bacteria) for two major reasons. First, despite efforts to standardize the faeces process (amount of sampling, transportation, and thaw

cycles), RA is less subject to individual variations compared with DC. Second, when DC of the global E. coli population increases, as it was observed after exposure to anti-anaerobic antibiotics such as clindamycin,27 RA of resistant E. coli does not change. It is therefore possible that DC and RA do not reflect the same phenomena. Additionally, because RA is a ratio, it can be determined using rectal swabs for which the precise quantity of the faecal material is unknown, which is not possible with DC. This may be relevant for clinical research because swabs are easier to obtain than faecal samples. We did not observe any influence of virulence factors in faecal ESBL-E. coli on their propensity to cause UTI. Thus, our results suggest a major role of dominance over virulence in the pathogenesis of UTI in women, at least for ESBL-E. coli strains. Additionally, no correlation was found between phylogroups and ESBL-RA, suggesting that other traits might determine the level of ESBL-E. coli within E. coli populations. Our study has some limitations. Despite recommendations, the returned stool samples may not have been the first one passed after the urine sample had been taken. This may explain why 14 women had an ESBL-E. coli-causing UTI even though they were not ESBL-E. coli carriers. In these women, an unreported and undetected antibiotic exposure may have eliminated carriage. We attempted to determine whether women had been exposed to antibiotics when the stool was passed by performing a wide-range antibiotic detection assay in their stools. This strategy is widely used for eliminating antibiotic-containing milk in the dairy industry.13 A limitation of this type of assay, when performed for faeces, is that antibiotics such as beta-lactam may be hydrolysed by commensal Bacteroides and lead to false negative results.28 However, in spite of this incertitude, our results were significant. In conclusion, the level of ESBL-E coli intestinal carriage, as measured by ESBL-RA, was linked to the presence of an ESBL-E. coli in the urine of infected patients. New perspectives in the clinical management of ESBL-E. coli UTI could emerge from this observation.

10

Conflict of interest We declare that we have no conflict of interest.

Acknowledgments The authors wish to thank Irina Codita, Alexandru Decusar, Iurii Roscin, Ala Marina, Iulia Marusicenco, Oleg Benes, Garyfallia Poulakou, Kiriaki Kanellakopoulou, Stavroula Kanellaki and Florentia Pournou for their contribution to the inclusion and sampling processes. We thank Olivier Clermont and Erick Denamur for helpful discussion and Editage for providing editorial assistance. Results have partly been presented at the European congress of clinical microbiology and infectious diseases (ECCMID 2012, London, United Kingdom).

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References 1. Lidin-Janson G, Kaijser B, Lincoln K, Olling S, Wedel H. The homogeneity of the faecal coliform flora of normal school-girls, characterized by serological and biochemical properties. Med Microbiol Immunol 1978; 164: 24753. 2. Marshall B, Tachibana C, Levy SB. Frequency of tetracycline resistance determinant classes among lactose-fermenting coliforms. Antimicrob Agents Chemother 1983; 24: 83540. 3. Murray BE, Rensimer ER, DuPont HL. Emergence of high-level trimethoprim resistance in fecal Escherichia coli during oral administration of trimethoprim or trimethoprim--sulfamethoxazole. N Engl J Med 1982; 306: 1305. 4. Tosh PK, McDonald LC. Infection control in the multidrug-resistant era: tending the human microbiome. Clin Infect Dis 2012; 54: 70713. 5. Hooton T. Uncomplicated Urinary Tract Infection. N Engl J Med 2012; 366: 1028-37. 6. Yamamoto S, Tsukamoto T, Terai A, Kurazono H, Takeda Y, Yoshida O. Genetic evidence supporting the fecal-perineal-urethral hypothesis in cystitis caused by Escherichia coli. J Urol 1997; 157: 11279. 7. Moreno E, Andreu A, Pigrau C, Kuskowski M, Johnson J, Prats G. Relationships between Escherichia coli strains causing acute cystitis in women and the fecal E. coli population of the host. J Clin Microbiol 2008; 46: 252934. 8. Plos K, Connell H, Jodal U, et al. Intestinal carriage of P fimbriated Escherichia coli and the susceptibility to urinary tract infection in young children. J Infect Dis 1995; 171: 625 31. 9. Bhalla A, Pultz NJ, Ray AJ, Hoyen CK, Eckstein EC, Donskey CJ. Antianaerobic antibiotic therapy promotes overgrowth of antibiotic-resistant, gram-negative bacilli and vancomycin-resistant enterococci in the stool of colonized patients. Infect Control Hosp Epidemiol 2003; 24: 6449. 10. Gupta A, Hooton TM, Naber KG, et al. International Clinical Practice Guidelines for the Treatment of Acute Uncomplicated Cystitis and Pyelonephritis in Women: A 2010 Update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases Clin Infect Dis 2011; 52: 10320. 11. Bush K, Fisher JF. Epidemiological expansion, structural studies, and clinical challenges of new beta-lactamases from gram-negative bacteria. Annu Rev Microbiol 2011; 65: 45578. 12. Pitout JD, Laupland KB. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008; 8: 15966. 13. Messer JW, Leslie JE, Houghtby GA, Peeler JT, Barnett JE. Bacillus stearothermophilus disc assay for detection of inhibitors in milk: collaborative study. J Assoc Off Anal Chem 1982; 65: 120814. 14. Pletz MW, Rau M, Bulitta J, et al. Ertapenem pharmacokinetics and impact on intestinal microflora, in comparison to those of ceftriaxone, after multiple dosing in male and female volunteers. Antimicrobial Agents & Chemotherapy 2004; 48: 376572. 15. Saladin M, Cao VT, Lambert T, et al. Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett 2002; 209: 1618. 16. Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 2000; 66: 45558. 17. Ruimy R, Genauzeau E, Barnabe C, Beaulieu A, Tibayrenc M, Andremont A. Genetic diversity of Pseudomonas aeruginosa strains isolated from ventilated patients with nosocomial pneumonia, cancer patients with bacteremia, and environmental water. Infect Immun 2001; 69: 5848.

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18. Johnson JR, Clermont O, Menard M, Kuskowski MA, Picard B, Denamur E. Experimental mouse lethality of Escherichia coli isolates, in relation to accessory traits, phylogenetic group, and ecological source. J Infect Dis 2006; 194: 114150. 19. Lefort A, Panhard X, Clermont O, et al. Host factors and portal of entry outweigh bacterial determinants to predict the severity of Escherichia coli bacteremia. J Clin Microbiol 2011; 49: 77783. 20. Berg RD. Promotion of the translocation of enteric bacteria from the gastrointestinal tracts of mice by oral treatment with penicillin, clindamycin, or metronidazole. Infect Immun 1981; 33: 85461. 21. Horton RA, Randall LP, Snary EL, et al. Fecal carriage and shedding density of CTXM extended-spectrum {beta}-lactamase-producing escherichia coli in cattle, chickens, and pigs: implications for environmental contamination and food production. Appl Environ Microbiol 2011; 77: 37159. 22. Rodriguez-Bano J, Navarro MD, Romero L, et al. Epidemiology and clinical features of infections caused by extended-spectrum beta-lactamase-producing Escherichia coli in nonhospitalized patients. J Clin Microbiol 2004; 42: 108994. 23. Calbo E, Romani V, Xercavins M, et al. Risk factors for community-onset urinary tract infections due to Escherichia coli harbouring extended-spectrum beta-lactamases. J Antimicrob Chemother 2006; 57: 7803. 24. Colodner R, Rock W, Chazan B, et al. Risk factors for the development of extendedspectrum beta-lactamase-producing bacteria in nonhospitalized patients. Eur J Clin Microbiol Infect Dis 2004; 23: 1637. 25. de Smet AM, Kluytmans JA, Blok HE, et al. Selective digestive tract decontamination and selective oropharyngeal decontamination and antibiotic resistance in patients in intensivecare units: an open-label, clustered group-randomised, crossover study. The Lancet Infectious Diseases 2011; 11: 37280. 26. Tarkkanen AM, Heinonen T, Jogi R, et al. P1A recombinant beta-lactamase prevents emergence of antimicrobial resistance in gut microflora of healthy subjects during intravenous administration of ampicillin. Antimicrob Agents Chemother 2009; 53: 245562. 27. Donskey CJ. Antibiotic regimens and intestinal colonization with antibiotic-resistant gram-negative bacilli. Clin Infect Dis 2006; 43 Suppl 2: S629. 28. Leonard FC, Andremont AO, Tancrede CH. In vivo influence of three B-lactam antibiotics on the intestinal microflora of man. A preliminary study in gnotobiotic mice. Prog Clin Biol Res 1985; 181: 27982. 29. Shlaes DM, Gerding DN, John JF, Jr., et al. Society for Healthcare Epidemiology of America and Infectious Diseases Society of America Joint Committee on the Prevention of Antimicrobial Resistance: guidelines for the prevention of antimicrobial resistance in hospitals. Infect Control Hosp Epidemiol 1997; 18: 27591. 30. Donskey CJ, Chowdhry TK, Hecker MT, et al. Effect of antibiotic therapy on the density of vancomycin-resistant enterococci in the stool of colonized patients. N Engl J Med 2000; 343: 192532.

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Table 1: Characterisation of ESBL-E. coli from urine and stool

ESBL name Group 1 CTX-M CTX-M-15 CTX-M-1 CTX-M-3 Group 9 CTX-M CTX-M-14 CTX-M-16 Total Origine of the strain Urines (n = 38) Stool (n = 63)* 28 1 5 3 1 38 45 4 7 12 68 Total 73 5 12 15 1 106

* including 5 women with 2 different ESBL-E. coli strains producing the respective CTX-M alleles (1 women each): 15 and 1, 15 and 3, 15 and 14, 15 and 15, and 1 and 1

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Table 2: Faecal ESBL-RA as a predictor of an ESBL-E. coli-causing UTI. ESBL-RA: extended-spectrum beta-lactamase relative abundance; Sens: sensitivity; Spec: specificity; PPV: positive predictive value; NPV: negative predictive value

ESBL-RA 10% RA < 100% 1% RA < 10% 01% RA < 1% 001% RA < 01% 0001% RA < 001%

Same ESBL-E coli in urine Sens Spec PPV NPV 057 077 057 088 057 061 033 084 086 045 035 093 100 010 026 100 100 003 023 100

15

Figure

Figure 1: Flow-chart of the study. ESBL: extended-spectrum beta-lactamases; UTI: urinary-tract infection Dashed-bold boxes represent discordant women, and the straight-bold box represents concordant women (see methods)
373 women with E. coli UTI 310 women with stool returned in 72 h or less 63 women with stool returned after 72 h

63 women with ESBL-E. coli in faeces

247 women with no ESBL-E. coli in faeces

32 women exposed to antibiotics

31 women not exposed to antibiotics

14 women with ESBL-E. coli in UTI

233 women without ESBL-E. coli in UTI

20 women with a non-ESBL E. coli UTI

11 women with ESBL-E. coli in UTI

4 women with different ESBL-E. coli in UTI and stool

7 women with the same ESBL-E. coli in UTI and stool

Figure 2: Intestinal ESBL-E. coli relative abundance (ESBL-RA) in 63 women carrying ESBL-E. coli in the faeces according to antibiotic exposure ATB: antibiotics Main horizontal bar represents mean. Error bars represent 95% confidence interval

P < 0001

ESBL-RA (log)

-2

-4

-6

With ATB (n = 32)

Without ATB (n = 31)

Figure 3: Intestinal ESBL-E. coli relative abundance (ESBL-RA) in 31 women without antibiotic exposure according to concordance status (see methods) Main horizontal bar represents mean. Error bars represent 95% confidence interval

P < 005
0

ESBL-RA (log)

-2

-4

-6

Concordant women (n = 7)

Discordant women (n = 24)