Effect of antibiotic and healthcare exposure on long-term dynamics of extended-spectrum beta-lactamase producing (ESBL) Enterobacterales colonisation in Cambodian children

Effect of antibiotic and healthcare exposure on long-term dynamics of extended-spectrum beta-lactamase producing (ESBL) Enterobacterales colonisation in Cambodian children | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of antibiotic and healthcare exposure on long-term dynamics of extended-spectrum beta-lactamase producing (ESBL) Enterobacterales colonisation in Cambodian children Cristina Ardura-Garcia, Sambou Bran, Poda Sar, Keang Suy, Sreymom Pol, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7229248/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The gastrointestinal tract is a major reservoir of potentially pathogenic and antimicrobial resistant (AMR) bacteria, such as extended-spectrum-beta-lactamase-producing Enterobacterales (ESBL-E). Healthcare and antibiotic exposure are key determinants for ESBL-E colonisation, but little is known about the long-term temporal dynamics of carriage after exposure. Methods: COMRU-META was a clinical cohort of 1–59-month-olds seen at Angkor Hospital for Children, Cambodia, in 2021-22. Metadata (sociodemographic, clinical and environmental) and rectal swabs (RS) were collected at presentation to healthcare and at 1, 3 and 6-month follow-up visits. To detect ESBL and carbapenem resistant Enterobacterales (CRE), we cultured RS on selective chromogenic media, performed MALDI-TOF mass-spectrometry for isolate identification and determined antimicrobial susceptibilities by disk diffusion and automated minimum inhibitory concentration testing. We performed multivariable logistic regression to assess factors associated with ESBL-E and CRE colonisation at baseline and Cox regression to assess the effect of healthcare and antibiotic exposure on the time-to-gain and time-to-loss of ESBL-E colonisation during follow-up. Results: Among 605 children (median age 1.4 years, 47% female), colonisation at each visit over the 6 months by ESBL Escherichia coli was 85-88% and by ESBL Klebsiella pneumoniae, 27-29%. CRE colonisation proportion range was 1-2%. At baseline, most common risk factors for ESBL-E and CRE colonisation were previous healthcare or antibiotic exposure, while ESBL and CRE K. pneumoniae carriers were also younger and malnourished. For ESBL E. coli, there were 160 colonisation ‘gain’ episodes and 145 ‘loss’ episodes during the 6-month follow-up, not associated with antibiotic and healthcare exposure. For ESBL K. pneumoniae, children with any antibiotic (HR: 1.40, 95% CI: 1.05-1.89), 3rd generation-cephalosporin (HR: 2.06, 95% CI: 1.12-3.79) and inpatient exposures (HR: 1.42, 95% CI: 1.04-1.96) were more likely to have colonisation ‘gain’ episodes (N=250); children with healthcare exposure (HR: 0.70, 95% CI: 0.49-1.00) and who were inpatients (HR: 0.66, 95% CI: 0.43-1.00) were less likely to have colonisation ‘loss’ episodes (N=235). Conclusion: ESBL-E carriage is prevalent in Cambodian children. Persistent healthcare and antibiotic exposure were associated with changes in colonisation dynamics for ESBL K. pneumoniae, but not for ESBL E. coli. These factors should be prioritised in AMR prevention strategies Antimicrobial resistance colonisation Cambodia paediatrics Escherichia coli Klebsiella pneumoniae Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION Antimicrobial resistance (AMR) is one of the major current global public health threats and a leading cause of death worldwide ( 1 ). The gastrointestinal tract (GI) is a major reservoir of potentially pathogenic and AMR bacteria. Colonisation with antimicrobial resistant (AMR) bacteria increases the risk of AMR infection ( 2 ). Although AMR affects all regions of the world, the highest burdens are in low- and middle-income countries (LMICs) ( 1 ), and South-East Asia is estimated to be the region with the highest risk of emergence and spread of AMR ( 3 ). In Cambodia, high colonisation prevalence of extended-spectrum beta-lactamase producing Enterobacterales (ESBL-E) have been documented in children: 55% Escherichia coli / Klebsiella pneumoniae combined in older children ( 4 ); 92% E. coli and 36% K. pneumoniae in children post-discharge ( 5 ). Hospitalisation and broad-spectrum antibiotic use are the main determinants for acquisition of AMR E. coli and K. pneumoniae ( 6 – 8 ). Travel to high prevalence AMR areas, chronic diseases, previous surgeries, invasive procedures, dietary habits and intrafamilial transmission have also been identified as risk factors for AMR bacterial colonisation ( 6 , 9 , 10 ). However, risk factors may vary by region, especially in LMICs. In Cambodian children, hospital admission and intestinal parasites were identified as independent risk factors for AMR E. coli and K. pneumoniae colonisation ( 4 ). Little is known about the long-term temporal dynamics of carriage after exposure, especially in children. Studies on adults returning to Europe after international travel have shown a high proportion of gut colonisation with multidrug-resistant Enterobacterales (24–51%), especially when returning from India (72% in a meta-analysis) ( 11 – 15 ). Persistence of colonisation occurred in 36% of the colonised travellers 1 month later ( 12 ), and in 5–28% 6 months later ( 11 , 14 , 15 ), associated with a vegetarian diet, travel to Asia, high relative abundance of AMR Enterobacterales, gut microbiota composition and cat ownership ( 11 , 12 , 14 , 15 ). Although genotypically-verified data are relatively sparse, GI duration of colonisation by AMR Enterobacterales in children may be prolonged ( 17 – 20 ). Understanding the temporal dynamics of AMR colonisation is crucial for designing future strategies to reduce its burden. Details of the long-term temporal dynamics of AMR bacterial colonisation and the factors that drive them remain relatively scarce in high disease burden LMIC populations, where effective interventions to prevent infections are most needed. We aimed to define the proportion of Cambodian children under 5 years old who were colonised by AMR Enterobacterales on presentation for healthcare, identify risk factors for colonisation at presentation, describe the colonisation dynamics over time, and the roles of healthcare and antibiotic exposure for the gain or loss of colonisation over time. 2. METHODS 2.1 Study design and setting COMRU-META was a prospective clinical cohort study of children seen at Angkor Hospital for Children (AHC), a non-governmental paediatric referral hospital based in Siem Reap, Cambodia. The study was approved by the AHC Executive Committee (reference 0102 − 21 AHC), Cambodia National Ethics Committee for Health Research (NECHR, references 079-NECHR (23/04/2021) / 090-NECHR (18/04/2022) / 089-NECHR (17/03/2023)), and Oxford Tropical Research Ethics Committee (OxTREC, reference 514 − 21). Findings were reported in line with Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) ( 16 ) and the Microbiology Investigation Criteria for Reporting Objectively (MICRO) ( 17 ) guidelines. 2.2 Participants We included children 1–59 months old visiting the outpatient (OPD) and inpatient (IPD) departments for non-elective healthcare during 2021–2022, whose parents or legal representatives consented to the child’s participation. Exclusion criteria were: residing outside Siem Reap district, systemic antibiotic use for current illness, chronic medical condition (immunosuppression, active chronic infections, active cardiorespiratory conditions, or upper respiratory tract or intestinal tract abnormalities) or AHC prescribed antibiotic started more than 1 hour before enrolment. 2.3 Study procedures Baseline clinical, treatment, and environmental data were collected at enrolment along with nasopharyngeal and rectal swabs (RS), either prior to departure from the OPD or on the day of IPD admission, though only RS were used for this analysis. Hospital admission and antibiotic treatment data were obtained from medical records and the AHC hospital information system. Household and environmental data were captured via administration of a questionnaire to the participant’s parent/legal representative. Children were followed up for 6 months with clinical visits and RS at 1, 3 and 6 months and telephone follow-up at 2, 4 and 5 months. At each follow-up a short questionnaire on time-varying exposures was completed (acute infections, healthcare and antibiotic exposure). 2.4 Sample handling RS were cultured onto selective chromogenic agar plates and incubated aerobically at 37°C for 24 hours to detect colonisation by ESBL-E and carbapenem resistant Enterobacterales (CRE) E. coli and K. pneumoniae (CHROMagar ESBL and KPC media). Target species were identified by MALDI-TOF mass-spectrometry (bioMerieux VITEK MS; Knowledge Base V3.2.0) and standard microbiological techniques. Antimicrobial susceptibilities were determined by VITEK 2 (bioMerieux) testing using AST-GN84 cards and interpreted using current Clinical and Laboratory Standard Institute (CLSI) Antimicrobial Susceptibility Testing guidelines ( 18 ). Multidrug-resistance (MDR) was defined as non-susceptibility to ≥ 1 agent in ≥ 3 antimicrobial classes tested, excluding intrinsic resistance ( 18 ). 2.5 Definitions of risk factors and outcomes Potential risk factors for colonisation with AMR bacteria were selected based on previous literature. These included data from the questionnaires: sociodemographic information (age, sex), environmental exposures (pets and farm animals, number of people in the household, access to a toilet inside the house and handwashing basin within 2 meters of the toilet, school or day care attendance), current breastfeeding, comorbidities, healthcare exposure in the previous 3 months (any healthcare consultation and hospital admission), and antibiotic use in the previous 4 weeks. Also included were weight and height to estimate body mass index (BMI), that were transformed into Z-scores using World Health Organisation references values ( 19 ). For the Cox regression analysis, antibiotic and healthcare exposure extracted from follow-up visit forms were defined as ‘since previous visit’. Outcomes of interest were the presence of ESBL and CRE E. coli and K. pneumoniae in RS, and the time-to-gain or -loss of ESBL E. coli and K. pneumoniae colonisation. A ‘gain’ episode was defined as a visit when a child non-colonised in the previous visit became colonised. A ‘loss’ episode was defined as a visit when a child colonised in the previous visit became non-colonised. 2.6 Statistical analyses Demographic, clinical, treatment and colonisation data were summarised using descriptive statistics. GI colonisation proportions by AMR bacteria were estimated at each time point and dynamics shown with Sankey diagrams. Risk factors for ESBL and CRE E. coli and K. pneumoniae colonisation at baseline were identified by univariable and multivariable logistic regression models (described further in the Supplementary Materials). Kaplan Meier curves were used to represent time-to-gain and time-to-loss of ESBL E. coli and K. pneumoniae colonisation. Cox regression analysis was performed to study the effect of healthcare (any or inpatient) and antibiotic (any or 3rd generation cephalosporin) on the time-to-gain and time-to-loss of ESBL E. coli and K. pneumoniae colonisation. For colonisation gain or loss, time to the event was defined as the mid-point between the two time points at which colonisation differed (from non-colonised to colonised or vice versa). Periods with no gain or loss were censored at the time of the last visit. Healthcare and antibiotic exposure were included as time-varying covariates. These models were adjusted for sex, age and relevant risk factors identified at baseline (toilet inside the house, available hand washing basin, weight-for-age Z score and current breastfeeding). Children with missing values for included variables were excluded from the analyses. Estimates of effect (odds ratios, OR and hazard ratios, HR) are reported together with 95% confidence intervals (CI). The R statistical programme version 4.3.0 was used for computation. 3. RESULTS During the study period, 661 children were eligible and invited to participate, of whom 605 (92%) agreed to participate (Fig. 1 ). The study cohort consisted of 460 outpatients and 145 inpatients. The study population characteristics at enrolment are documented in Table 1 and Supplementary Table 1. Median age was 1.4 years (interquartile range, IQR, 0.8–2.4 years), 47% were female, and few (21, 4%) attended day-care or school. Mean BMI and weight-for-age Z-scores were less than 0 for outpatients, and less than − 1 among those admitted to hospital. One third (202, 33%, mostly < 1-year olds) were currently breastfeeding. Most children (511, 85%) reported recent healthcare contact and 41 (7%) reported antibiotic intake in the last month. Common diagnoses were upper respiratory infection (258, 43%) and gastroenteritis (115, 19%) (Supplementary Table 1). Among outpatients, 50/460 (11%) were prescribed an antibiotic, and among inpatients 17/145 (12%) were treated empirically with ceftriaxone or cefotaxime and 1 (0.7%) with meropenem. Table 1 Characteristics at enrolment of children under 5 years old presenting at the Angkor Hospital for Children for healthcare during 2021 to 2022 N (%) N All (N = 605) Outpatient (N = 460) Inpatient (N = 145) Sociodemographics: Female sex 605 285 (47) 213 ( 46 ) 72 (50) Age (median, IQR) (years) 605 1.4 (0.8–2.4) 1.6 (0.9–2.5) 1.0 (0.6-2) Environment : People in household (median, IQR) 605 5 ( 4 – 7 ) 5 ( 4 – 7 ) 5 ( 4 – 6 ) Any animal in household 605 376 (62) 289 (63) 87 (60) Pets (dogs/cats) 605 298 (49) 226 (49) 72 (50) Farm animals 605 225 ( 37 ) 175 ( 38 ) 50 ( 34 ) Toilet in house Inside 598 334 (56) 257 (56) 77 (54) With basin for handwashing 605 494 (83) 372 (82) 122 (86) School/day care attendance 605 21 ( 4 ) 17 (3.7) 4 (2.8) Personal history : BMI Z score (mean, SD) 538 -0.66 (1.19) -0.57 (1.09) -1.05 (1.50) Weight for age Z score (mean, SD) 605 -0.93 (1.32) -0.83 (1.25) -1.24 (1.49) Breastfeeding currently 605 202 ( 33 ) 156 ( 34 ) 46 ( 32 ) Any vaccine 599 598 (99) 455 (100) 143 (99) Hib/PCV13 at least 2 doses 599 566 (94) 439 (97) 127 (88) Comorbidities 605 15 ( 2 ) 8 (1.7) 7 (4.8) Any health care previous 3 months 605 511 (85) 377 (82) 134 (92) Inpatient 604 58 ( 10 ) 26 (5.7) 32 ( 22 ) Surgery 602 3 (0.5) 0 (0) 3 (2.1) Healthcare visit 605 352 (58) 264 (57) 88 (61) Pharmacy consultation 605 318 (53) 232 (50) 86 (59) Traditional healer 604 19 ( 3 ) 10 (2.2) 9 (6.2) Antibiotics previous 4 weeks 549 41 ( 7 ) 30 (7.1) 11 (8.8) Amoxicillin (% of those who took abx) 8 ( 20 ) 7 ( 23 ) 1 (9.1) Ceftriaxone / cefotaxime 4 ( 9 ) 2 (6.6) 2 ( 18 ) Amoxicillin-clavulanate 1 ( 2 ) 1 (3.3) 0 (0) Ofloxacin 1 ( 2 ) 1 (3.3) 0 (0) Metronidazole 1 ( 2 ) 1 (3.3) 0 (0) Meropenem 1 ( 2 ) 0 (0) 1 (9.1) Unknown 25 (61) 18 (60) 7 (64) Colonisation status 605 ESBL E. coli 512 (85) 391 (85) 121 (83) CRE E. coli 13 ( 2 ) 8 ( 2 ) 5 ( 3 ) ESBL K. pneumoniae 162 ( 27 ) 108 ( 23 ) 54 ( 37 ) CRE K. pneumoniae 8 ( 1 ) 4 ( 1 ) 4 ( 3 ) 3.1 Bacterial colonisation and antimicrobial resistance at enrolment From 605 baseline RS, 531 (88%) children were colonised with an ESBL-E (512 E. coli , 162 K. pneumoniae , 143 both) and 21 (3%) by CRE (13 E. coli , 8 K. pneumoniae ) at enrolment (Table 1 ). Four and three children were colonised, respectively, with both an ESBL and a CRE E. coli or K. pneumoniae . ESBL and CRE K. pneumoniae colonisation decreased with age, being most prevalent in < 1-year olds admitted to hospital (Supplementary Fig. 1), whilst ESBL E. coli colonisation peaked in 1-year olds and CRE E. coli carriage increased with age. Of the 525 isolated E. coli at enrolment, 129 (25%) were ampicillin-gentamicin-resistant, 232 (44%) fluoroquinolone-resistant, and 359 (68%) were MDR. The respective results for the 170 isolated K. pneumoniae were: 37 (21%), 96 (55%) and 106 (61%) (Supplementary Table 2). AMR data are summarised in Supplementary Table 2. 3.2 Risk factors for AMR bacterial colonisation at enrolment Independent risk factors for ESBL E. coli colonisation at enrolment were male sex (Adjusted OR, AOR, 0.56 for female sex, 95% CI 0.36–0.88) and any healthcare exposure in the previous 3 months (AOR 1.99, 95% CI 1.14–3.39) (Fig. 2 , Supplementary Table 3). For ESBL K. pneumoniae colonisation, younger age (AOR 0.59 per 1 year increase, 95% CI 0.47–0.73), lower weight for age Z-score (AOR 0.82 per 1 Z-score increase, 95% CI 0.71–0.95), and inpatient care in the previous 3 months (AOR 2.40, 95% CI 1.32–4.35) increased the odds while current breastfeeding was shown to be protective (AOR 0.51, 95% CI 0.32–0.79) for children under 1 year old (Fig. 2 , Supplementary Table 4). For CRE, antibiotic exposure in the previous 4 weeks increased the risk of CRE E. coli colonisation (AOR 9.04, 95% CI 2.56–29.2), while younger age (OR 0.17 per 1 year increase, 95% CI 0.03–0.58) and inpatient exposure (OR 5.90, 95% CI 1.19–24.7) were associated to CRE K. pneumoniae colonisation at enrolment (Fig. 2 , Supplementary Tables 5 & 6). 3.3 Long-term dynamics of ESBL-E and CRE colonisation At the 6-month visit, 108/605 (18%) children were lost-to-follow-up (Fig. 1 ). ESBL-E colonisation proportions remained quite stable over the 6-month follow-up, ranging between 84–88% and for E. coli and 25–30% for K. pneumoniae (of available samples) (Fig. 3 , Supplementary Table 2). However, there were changes in colonisation status for individual children, especially for ESBL K. pneumoniae colonisation. For ESBL E. coli , at each visit, 86–92% of those previously colonised remained colonised and 71–80% of those non-colonised became colonised. For ESBL K. pneumoniae , at each visit, 38–50% of those previously colonised remained colonised and 19–24% of those non-colonised became colonised. Median time to colonisation gain was 15 days (IQR: 15–15 days) and 135 days (IQR: 135-NA days), respectively, for ESBL E. coli and K. pneumoniae , while mean time to colonisation loss was undefined for ESBL E. coli (Kaplan-Meier curve does not cross 50%) and 60 days (IQR: 15–60 days) for ESBL K. pneumoniae (Fig. 4 ). Overall antibiotic susceptibilities were similar to baseline results (Supplementary Table 2), however there was considerable intra-individual temporal variation (Supplementary Fig. 2). 3.4 Effect of antibiotic and healthcare exposure on ESBL-E colonisation dynamics There were 160 ESBL E. coli new colonisations (‘gains’) out of 218 potential events and 145 de-colonisations (‘losses’) out of 1325 potential events during the 6 months follow-up (Table 2 , Fig. 4 ). Overall, per visit, 377 children (18%) reported previous antibiotic use, 39 (1.8%) 3rd generation cephalosporin exposure, 1918 (89%) previous healthcare exposure and 287 (13%) inpatient exposure. Neither healthcare nor antibiotic exposure during this time were associated with time-to-gain or time-to-loss for ESBL E. coli colonisation (Table 2 ). For ESBL K. pneumoniae , there were 250 colonisation gains out of 1118 potential events and 235 colonisation losses out of 425 potential events during the 6 months follow-up (Table 2 , Fig. 4 ). Reported previous exposures were similar to ESBL E. coli (Supplementary Table 7). Inpatient (Hazard Ratio, HR: 1.42, 95% CI: 1.03–1.96), any antibiotic (HR: 1.40, 95% CI: 1.05–1.89), and 3rd generation cephalosporin exposure (HR: 2.06, 95% CI: 1.12–3.79) were associated with a shorter time to colonisation gain, while any healthcare (HR: 0.68, 95% CI: 0.48–0.94), and inpatient exposure (HR: 0.66, 95% CI: 0.43-1.00) increased the time to colonisation loss, after adjusting for potential baseline confounders (Table 2 ). Table 2 Cox proportional hazard regression analysis for effect of healthcare and antibiotic exposure on time-to-gain and time-to-loss of extended spectrum-beta-lactamase producing (ESBL) Escherichia coli and Klebsiella pneumoniae gastrointestinal colonisation during 6-month follow-up in children under 5 years old seen at Angkor Hospital for Children. Time to gain Time to loss aHR* 95% CI p-value aHR* 95% CI p-value ESBL - E. coli (N = 218 records, 160 events) (N = 1325 records, 145 events) Any healthcare exposure 0.80 0.54–1.17 0.246 0.93 0.54–1.60 0.788 Inpatient exposure 1.02 0.68–1.53 0.905 0.79 0.45–1.38 0.409 Any antibiotic exposure 0.76 0.52–1.11 0.157 0.91 0.60–1.38 0.653 3rd gen cephalosporin exposure 0.73 0.20–2.70 0.643 0.73 0.20–2.70 0.643 ESBL - K. pneumoniae (N = 1118 records, 250 events) (N = 425 records, 235 events) Any healthcare exposure 1.40 0.83–2.38 0.210 0.68 0.48–0.94 0.022 Inpatient exposure 1.42 1.03–1.96 0.031 0.66 0.43-1.00 0.048 Any antibiotic exposure 1.40 1.05–1.89 0.023 0.79 0.59–1.07 0.132 3rd gen cephalosporin exposure 2.06 1.12–3.79 0.019 0.70 0.30–1.59 0.388 * Adjusted for age, sex, toilet inside the house, hand washing basin in toilet, weight-for-age Z score and current breastfeeding. CI: confidence interval; ESBL: extended-spectrum beta-lactamase producers; aHR: adjusted hazard ratio. 4. DISCUSSION This prospective cohort study has detected high AMR Enterobacterales carriage proportions in the GI tract of Cambodian children attending a healthcare facility. Risk factors for AMR Enterobacterales carriage at presentation to healthcare included male sex (ESBL E. coli ), younger age, and malnourishment (ESBL and CRE K. pneumoniae ), not currently breastfeeding (ESBL K. pneumoniae ), healthcare exposure (ESBL E. coli and K. pneumoniae ) and antibiotic exposure (CRE E. coli ). ESBL-E colonisation proportions remained stable during the 6-month-follow-up, despite changes in colonisation status for individual children. Persistent healthcare and antibiotic exposure (particularly 3rd generation cephalosporins and hospitalisations) were associated with changes in colonisation dynamics for ESBL K. pneumoniae , but not for ESBL E. coli . 4.1 Strengths and limitations This study included a relatively large sample size and collected extensive information on potential factors associated with AMR bacterial carriage. We included 1, 3 and 6-month follow-ups with both colonisation and exposure dynamic data, with a good retention proportion (82%). We used high standard microbiological techniques for bacteria and AMR identification including culture on chromogenic media, MALDI-TOF MS, and automated antimicrobial susceptibility testing. However, this study also presents some limitations. First, information on environmental and previous healthcare and antibiotic exposure were collected through parental questionnaires, which may introduce information bias. Second, we only included 1–59 months-old children attending a single centre in Cambodia. This may limit the generalisability of our findings to other regions and age groups. Third, we only cultured E. coli and K. pneumoniae using selective media, and therefore could not estimate the number of children colonised by non-ESBL-E. This should not be an issue for E. coli , as colonisation occurs in nearly all children, but it would have been informative to assess the proportion of children colonised by non-ESBL K. pneumoniae . Finally, only one colony per plate was selected so multiple strains with different resistance profiles would not have been detected. In addition, we did not perform whole genome sequencing, which could help determine if the isolates identified at two different timepoints in the same child were the same strain or differed, increasing the accuracy of colonisation gain or loss detection. 4.2 Findings in relation to other studies Current ESBL-E colonisation rates in our study (88%) were even higher than previously reported in this setting (55% for children < 16 years old in 2012) ( 4 ). In Sub-Saharan Africa, a 2019 meta-analysis reported a much lower pooled ESBL-E colonisation prevalence of 10% (95% CI 1–32%, range 10–60%) in children ( 20 ). However, South-East Asia is the region with the highest estimated risk for AMR spread ( 3 ). Most other studies on ESBL-E colonisation rates in children in Asia have focused on hospitalised patients, especially neonates, hampering the comparison with our estimates from children presenting to healthcare, as carriage rates of ESBL-E increase rapidly after admission ( 20 ). A meta-analysis of clinically-relevant specimens from children’s infections in Asian studies reported a pooled prevalence of 3rd -generation cephalosporins and carbapenem resistance, respectively, of 73% (95% CI 50–86%) and 15% (95% CI 1–33%) among E. coli , and 76% (95% CI 40–92%) and 13% (95% CI 0–46%) among K. pneumoniae isolates ( 21 ). Prevalence of CRE colonisation (3%) was low in our setting, though it is a growing problem in neighbouring countries, such as Vietnam, where 13% of children were colonised with CRE at admission ( 22 ). This is aggravated by the lack of access to more extended spectrum and novel antibiotics in LMICs, such as Cambodia ( 23 , 24 ). These data emphasise the serious and urgent public health problem of AMR Enterobacterales in Asia. Most identified risk factors for AMR Enterobacterales colonisation are related with gut microbiome health. Reported antibiotic and healthcare exposure, identified as key risk factors for AMR colonisation in children in previous studies ( 2 , 6 – 8 , 20 , 25 – 27 ), were associated to colonisation with ESBL E. coli and K. pneumoniae , and CRE E. coli in our analysis. In Cambodia there is a high prevalence of inappropriate antibiotic prescription and use in the community, including broad-spectrum antibiotics ( 28 , 29 ), and this can result in diminished gut microbiome diversity ( 30 ). Undernutrition appeared to increase the risk of ESBL and CRE K. pneumoniae colonisation, even after adjusting for previous healthcare or antibiotic exposure. Undernutrition may affect the gut microbiome composition and is associated with reduced host immunity, therefore increasing the risk of colonisation by pathogenic bacteria ( 31 – 33 ). Breastfeeding was associated with lower ESBL K. pneumoniae colonisation risk in children under 1 year old, as has already been shown in this setting for hospitalised neonates ( 34 ). As with the other risk factors, breastfeeding has also been shown to play a key role in gut microbiome composition and diversity ( 35 – 37 ). Gut microbiome reduced diversity and altered composition can increase the risk of colonisation with AMR bacteria, which benefit from reduced competition due to the suppression of more robust, antibiotic-sensitive strains ( 38 ). Long term colonisation dynamics of ESBL-E in children following healthcare contact have been scarcely studied. Most studies were performed on adults, either during hospitalisation or among returning travellers and with short follow-up times ( 39 – 42 ). Additionally, some of these studies did not assess ESBL E. coli and K. pneumoniae colonisation separately ( 39 – 42 ). Given our findings at baseline, we believe colonisation dynamics of these two Enterobacterales may differ greatly. In previous studies, the mean colonisation duration for ESBL-E was only 30 days for international travellers upon return (ESBL K .pneumoniae having the shortest decolonisation time) ( 39 ), but was longer for European adults and children who had acquired the colonisation in the community (128 days) ( 42 ). In this study, we showed that ESBL E. coli colonisation occurs much faster and remains for longer than K. pneumoniae , with shorter time-to-gain time (38 vs 118 days) and longer time-to-loss time (148 vs 58 days). As a consequence, most of the children already colonised with ESBL E. coli at baseline remained colonised during the follow-up and the few that lost colonisation (8–14%), were replaced by children newly colonised, to maintain a similar proportion of colonisation at each timepoint. For ESBL K. pneumoniae , however, a larger proportion of those colonised at baseline became decolonised (50–62%), but similar to E. coli , the proportion of colonisation at each follow-up visit remained stable. These differences in long term ESBL-E colonisation dynamics in children have not been previously described. The impact of repeated antibiotic and healthcare exposure on the long term ESBL-E colonisation dynamics in children is largely unknown. A previous Cambodian study reported that any antibiotic use increased the daily acquisition risk of 3rd generation cephalosporin resistant K. pneumoniae among neonates during hospitalisation ( 34 ). We have now shown that persistent antibiotic exposure and repeated hospitalisations increase the risk of new ESBL K. pneumoniae colonisation episodes over time after a first presentation to healthcare, while repeated healthcare exposures increase the time to colonisation loss. A similar study in Malawian adults also showed that repeated hospitalisations increased ESBL-E colonisation risk over time, and that persistent antibiotic exposure prolonged colonisation by reducing colonisation loss ( 41 ). However, in the present study, repeated healthcare and antibiotic exposure were not associated with long term ESBL E. coli colonisation gain or loss. This may be due to the higher proportion of children in the community in our setting that are colonised with 3rd generation cephalosporin resistant E. coli compared to K. pneumoniae (53 vs 10%) ( 4 ), which would also explain the higher proportion of new colonisation episodes occurring at each visit over time for ESBL E. coli compared to K. pneumoniae in our study (71–80% vs 19–24%). 4.3 Implications for practice and future research The high rate of AMR bacterial gut colonisation found in our setting confirms the urgent need to reduce this global health problem. Some identified risk factors are modifiable, and point towards interventions that may reduce AMR carriage. Antimicrobial stewardship and reducing healthcare contact, at least with large healthcare centres, are two key interventions. Digital health algorithms have shown to reduce antibiotic prescription in children in community settings in Tanzania, with no increased clinical failure ( 43 ). Such tools may not only reduce antibiotic use but also unnecessary referrals to larger hospitals, and their extended use should be now assessed in large multicentre clinical trials. Adding bacterial colonisation analysis to these studies will enable the study of their impact on AMR colonisation. Interventions modifying gut microbiome diversity and composition, may also reduce AMR gut colonisation in children. The use of probiotics use has been associated with reduced 3rd generation cephalosporin resistant bacteria gut colonisation in hospitalised neonates ( 44 ), but this effect has not been studied in clinical trials. Finally, advanced techniques such as targeted metagenomic sequencing from cultured specimens, may increase bacterial colonisation detection ( 45 ) and enable longitudinal changes of specific resistomes to be studied ( 46 ), an avenue we are currently pursuing. 4.4 Conclusion Carriage of ESBL Enterobacterales is prevalent in Cambodian children. This may limit effective antibiotic choices if followed by invasive infections. Healthcare and antibiotic exposure reduction may prevent ESBL K. pneumoniae colonisation with further prevention strategies needed for ESBL E. coli colonisation. Abbreviations AHC: Angkor Hospital for Children AMR: antimicrobial resistance / resistant AOR: adjusted odds ratio BMI: body mass index CRE: carbapenem resistant Enterobacterales ESBL(-E): extended-spectrum beta-lactamase producing (Enterobacterales) GI: gastrointestinal HR: hazard ratio IPD: inpatient IQR: interquartile range LMICs: low- and middle-income countries MDR: multi-drug resistant OPD: outpatient OR: odds ratio RS: rectal swab SE: standard error Declarations Ethics approval and consent to participate The study was approved by the AHC Executive Committee (reference 0102-21 AHC), Cambodia National Ethics Committee for Health Research (NECHR, references 079-NECHR (23/04/2021) / 090-NECHR (18/04/2022) / 089-NECHR (17/03/2023)), and Oxford Tropical Research Ethics Committee (OxTREC, reference 514-21). Parents or legal guardians of participating children consented to participate. The parent or legally acceptable representative of the participant signed or thumb-printed and dated the informed consent form before any study specific procedures were performed. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the Mahidol-Oxford Tropical Medicine Research Unit Data Access Committee on reasonable request (https://www.tropmedres.ac/units/moru-bangkok/bioethics-engagement/data-sharing). Competing interests The authors have no competing interests to declare. Funding This research was funded in whole, or in part, by the Wellcome Trust (grant numbers 206194 and 220211). CAG was funded by a Postdoctoral Mobility Fellowship from the Swiss National Science Foundation (grant number P500PM_217605), JC was funded by the ERC (grant number 742154) and by Norwegian Research Council FRIPRO (grant number 299941). 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Pol S, Kallonen T, Mäklin T, Sar P, Hopkins J, Soeng S, et al. Exploring the pediatric nasopharyngeal bacterial microbiota with culture-based MALDI-TOF mass spectrometry and targeted metagenomic sequencing. mBio. 2024;0(0):e00784-24. Manenzhe RI, Dube FS, Wright M, Lennard K, Zar HJ, Mounaud S, et al. Longitudinal changes in the nasopharyngeal resistome of South African infants using shotgun metagenomic sequencing. PLoS One. 2020;15(4):e0231887. Additional Declarations No competing interests reported. Supplementary Files COMRUMETAColonisationESBLCRESupplementary02.07.25.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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09:54:36","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26656,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7229248/v1/1701afc610fcabff5328f209.png"},{"id":93030007,"identity":"0e189b89-bfdb-4b59-ade1-f72370fb2d97","added_by":"auto","created_at":"2025-10-08 10:02:35","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":37243,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7229248/v1/96f3312e8c5d06fd37188c9b.png"},{"id":93029280,"identity":"3d316269-9798-4d35-833d-016534b102a5","added_by":"auto","created_at":"2025-10-08 09:54:35","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":150616,"visible":true,"origin":"","legend":"","description":"","filename":"ca1d3e9889fc47368510ceda034820511structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7229248/v1/6df0a98827f298c333bac2d4.xml"},{"id":93030008,"identity":"c4c35b2b-93fe-43ad-90f2-dc61414ba2a0","added_by":"auto","created_at":"2025-10-08 10:02:36","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165514,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7229248/v1/74e0c75eee40a4ad7f1db359.html"},{"id":93029256,"identity":"870c4a7f-16db-4357-a386-cf0932934fdc","added_by":"auto","created_at":"2025-10-08 09:54:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39758,"visible":true,"origin":"","legend":"\u003cp\u003eStudy population flow\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7229248/v1/b32e847ec45811925e5dad40.png"},{"id":93029257,"identity":"053ec891-b855-40ec-bd56-5977fc81346c","added_by":"auto","created_at":"2025-10-08 09:54:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":211578,"visible":true,"origin":"","legend":"\u003cp\u003eAssociation between sociodemographic, personal history and environmental factors with gastrointestinal colonisation of extended-spectrum-beta-lactamase producing (ESBL) and carbapenem-resistant (CRE) \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003ein children under 5 years old seen at Angkor Hospital for Children, at enrolment. N=605\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7229248/v1/e791d94002221917a9c46b1b.png"},{"id":93029997,"identity":"3a76b757-de9b-489d-9aba-d2ebb9237356","added_by":"auto","created_at":"2025-10-08 10:02:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":188521,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal variation of extended-spectrum-beta-lactamase producing (ESBL) and carbapenem-resistant (CRE) \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e gastrointestinal colonisation in children under 5 years old seen at Angkor Hospital for Children, at enrolment and during 6 months follow-up.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7229248/v1/f445efb2c6ea0abbbadcbfff.png"},{"id":93029259,"identity":"7265e4c3-41f0-4ab9-a76f-14ac090c2c68","added_by":"auto","created_at":"2025-10-08 09:54:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":83213,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan Meier curves for A)\u003cstrong\u003e \u003c/strong\u003etime-to-gain and B) time-to loss for gastrointestinal colonisation of extended-spectrum-beta-lactamase (ESBL) producing \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, in children under 5 years old seen at Angkor Hospital for Children, at enrolment and during 6 months follow-up.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7229248/v1/833650948744e47f11971e66.png"},{"id":98434325,"identity":"38349dcd-0642-4226-8e79-9a2c2cc33d77","added_by":"auto","created_at":"2025-12-17 16:51:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1607437,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7229248/v1/da448d73-4341-4ccb-bcaa-4df5b9322b81.pdf"},{"id":93029998,"identity":"d8542b95-675f-48e9-97a9-8fd421b51732","added_by":"auto","created_at":"2025-10-08 10:02:35","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1237472,"visible":true,"origin":"","legend":"","description":"","filename":"COMRUMETAColonisationESBLCRESupplementary02.07.25.docx","url":"https://assets-eu.researchsquare.com/files/rs-7229248/v1/5c46ca314c4242219373c167.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of antibiotic and healthcare exposure on long-term dynamics of extended-spectrum beta-lactamase producing (ESBL) Enterobacterales colonisation in Cambodian children","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eAntimicrobial resistance (AMR) is one of the major current global public health threats and a leading cause of death worldwide (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The gastrointestinal tract (GI) is a major reservoir of potentially pathogenic and AMR bacteria. Colonisation with antimicrobial resistant (AMR) bacteria increases the risk of AMR infection (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Although AMR affects all regions of the world, the highest burdens are in low- and middle-income countries (LMICs) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), and South-East Asia is estimated to be the region with the highest risk of emergence and spread of AMR (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In Cambodia, high colonisation prevalence of extended-spectrum beta-lactamase producing Enterobacterales (ESBL-E) have been documented in children: 55% \u003cem\u003eEscherichia coli / Klebsiella pneumoniae\u003c/em\u003e combined in older children (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e); 92% \u003cem\u003eE. coli\u003c/em\u003e and 36% \u003cem\u003eK. pneumoniae\u003c/em\u003e in children post-discharge (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHospitalisation and broad-spectrum antibiotic use are the main determinants for acquisition of AMR \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Travel to high prevalence AMR areas, chronic diseases, previous surgeries, invasive procedures, dietary habits and intrafamilial transmission have also been identified as risk factors for AMR bacterial colonisation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). However, risk factors may vary by region, especially in LMICs. In Cambodian children, hospital admission and intestinal parasites were identified as independent risk factors for AMR \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Little is known about the long-term temporal dynamics of carriage after exposure, especially in children. Studies on adults returning to Europe after international travel have shown a high proportion of gut colonisation with multidrug-resistant Enterobacterales (24\u0026ndash;51%), especially when returning from India (72% in a meta-analysis) (\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Persistence of colonisation occurred in 36% of the colonised travellers 1 month later (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and in 5\u0026ndash;28% 6 months later (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), associated with a vegetarian diet, travel to Asia, high relative abundance of AMR Enterobacterales, gut microbiota composition and cat ownership (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Although genotypically-verified data are relatively sparse, GI duration of colonisation by AMR Enterobacterales in children may be prolonged (\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnderstanding the temporal dynamics of AMR colonisation is crucial for designing future strategies to reduce its burden. Details of the long-term temporal dynamics of AMR bacterial colonisation and the factors that drive them remain relatively scarce in high disease burden LMIC populations, where effective interventions to prevent infections are most needed. We aimed to define the proportion of Cambodian children under 5 years old who were colonised by AMR Enterobacterales on presentation for healthcare, identify risk factors for colonisation at presentation, describe the colonisation dynamics over time, and the roles of healthcare and antibiotic exposure for the gain or loss of colonisation over time.\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Study design and setting\u003c/h2\u003e\u003cp\u003eCOMRU-META was a prospective clinical cohort study of children seen at Angkor Hospital for Children (AHC), a non-governmental paediatric referral hospital based in Siem Reap, Cambodia. The study was approved by the AHC Executive Committee (reference 0102\u0026thinsp;\u0026minus;\u0026thinsp;21 AHC), Cambodia National Ethics Committee for Health Research (NECHR, references 079-NECHR (23/04/2021) / 090-NECHR (18/04/2022) / 089-NECHR (17/03/2023)), and Oxford Tropical Research Ethics Committee (OxTREC, reference 514\u0026thinsp;\u0026minus;\u0026thinsp;21). Findings were reported in line with Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) and the Microbiology Investigation Criteria for Reporting Objectively (MICRO) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Participants\u003c/h2\u003e\u003cp\u003e We included children 1\u0026ndash;59 months old visiting the outpatient (OPD) and inpatient (IPD) departments for non-elective healthcare during 2021\u0026ndash;2022, whose parents or legal representatives consented to the child\u0026rsquo;s participation. Exclusion criteria were: residing outside Siem Reap district, systemic antibiotic use for current illness, chronic medical condition (immunosuppression, active chronic infections, active cardiorespiratory conditions, or upper respiratory tract or intestinal tract abnormalities) or AHC prescribed antibiotic started more than 1 hour before enrolment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Study procedures\u003c/h2\u003e\u003cp\u003eBaseline clinical, treatment, and environmental data were collected at enrolment along with nasopharyngeal and rectal swabs (RS), either prior to departure from the OPD or on the day of IPD admission, though only RS were used for this analysis. Hospital admission and antibiotic treatment data were obtained from medical records and the AHC hospital information system. Household and environmental data were captured via administration of a questionnaire to the participant\u0026rsquo;s parent/legal representative. Children were followed up for 6 months with clinical visits and RS at 1, 3 and 6 months and telephone follow-up at 2, 4 and 5 months. At each follow-up a short questionnaire on time-varying exposures was completed (acute infections, healthcare and antibiotic exposure).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Sample handling\u003c/h2\u003e\u003cp\u003eRS were cultured onto selective chromogenic agar plates and incubated aerobically at 37\u0026deg;C for 24 hours to detect colonisation by ESBL-E and carbapenem resistant Enterobacterales (CRE) \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e (CHROMagar ESBL and KPC media). Target species were identified by MALDI-TOF mass-spectrometry (bioMerieux VITEK MS; Knowledge Base V3.2.0) and standard microbiological techniques. Antimicrobial susceptibilities were determined by VITEK 2 (bioMerieux) testing using AST-GN84 cards and interpreted using current Clinical and Laboratory Standard Institute (CLSI) Antimicrobial Susceptibility Testing guidelines (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Multidrug-resistance (MDR) was defined as non-susceptibility to \u0026ge;\u0026thinsp;1 agent in \u0026ge;\u0026thinsp;3 antimicrobial classes tested, excluding intrinsic resistance (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Definitions of risk factors and outcomes\u003c/h2\u003e\u003cp\u003ePotential risk factors for colonisation with AMR bacteria were selected based on previous literature. These included data from the questionnaires: sociodemographic information (age, sex), environmental exposures (pets and farm animals, number of people in the household, access to a toilet inside the house and handwashing basin within 2 meters of the toilet, school or day care attendance), current breastfeeding, comorbidities, healthcare exposure in the previous 3 months (any healthcare consultation and hospital admission), and antibiotic use in the previous 4 weeks. Also included were weight and height to estimate body mass index (BMI), that were transformed into Z-scores using World Health Organisation references values (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). For the Cox regression analysis, antibiotic and healthcare exposure extracted from follow-up visit forms were defined as \u0026lsquo;since previous visit\u0026rsquo;. Outcomes of interest were the presence of ESBL and CRE \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e in RS, and the time-to-gain or -loss of ESBL \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation. A \u0026lsquo;gain\u0026rsquo; episode was defined as a visit when a child non-colonised in the previous visit became colonised. A \u0026lsquo;loss\u0026rsquo; episode was defined as a visit when a child colonised in the previous visit became non-colonised.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical analyses\u003c/h2\u003e\u003cp\u003eDemographic, clinical, treatment and colonisation data were summarised using descriptive statistics. GI colonisation proportions by AMR bacteria were estimated at each time point and dynamics shown with Sankey diagrams. Risk factors for ESBL and CRE \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation at baseline were identified by univariable and multivariable logistic regression models (described further in the Supplementary Materials). Kaplan Meier curves were used to represent time-to-gain and time-to-loss of ESBL \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation. Cox regression analysis was performed to study the effect of healthcare (any or inpatient) and antibiotic (any or 3rd generation cephalosporin) on the time-to-gain and time-to-loss of ESBL \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation. For colonisation gain or loss, time to the event was defined as the mid-point between the two time points at which colonisation differed (from non-colonised to colonised or vice versa). Periods with no gain or loss were censored at the time of the last visit. Healthcare and antibiotic exposure were included as time-varying covariates. These models were adjusted for sex, age and relevant risk factors identified at baseline (toilet inside the house, available hand washing basin, weight-for-age Z score and current breastfeeding). Children with missing values for included variables were excluded from the analyses. Estimates of effect (odds ratios, OR and hazard ratios, HR) are reported together with 95% confidence intervals (CI). The R statistical programme version 4.3.0 was used for computation.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003eDuring the study period, 661 children were eligible and invited to participate, of whom 605 (92%) agreed to participate (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The study cohort consisted of 460 outpatients and 145 inpatients. The study population characteristics at enrolment are documented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Table 1. Median age was 1.4 years (interquartile range, IQR, 0.8\u0026ndash;2.4 years), 47% were female, and few (21, 4%) attended day-care or school. Mean BMI and weight-for-age Z-scores were less than 0 for outpatients, and less than \u0026minus;\u0026thinsp;1 among those admitted to hospital. One third (202, 33%, mostly\u0026thinsp;\u0026lt;\u0026thinsp;1-year olds) were currently breastfeeding. Most children (511, 85%) reported recent healthcare contact and 41 (7%) reported antibiotic intake in the last month. Common diagnoses were upper respiratory infection (258, 43%) and gastroenteritis (115, 19%) (Supplementary Table 1). Among outpatients, 50/460 (11%) were prescribed an antibiotic, and among inpatients 17/145 (12%) were treated empirically with ceftriaxone or cefotaxime and 1 (0.7%) with meropenem.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCharacteristics at enrolment of children under 5 years old presenting at the Angkor Hospital for Children for healthcare during 2021 to 2022\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eN (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAll\u003c/p\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;605)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOutpatient\u003c/p\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;460)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInpatient\u003c/p\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;145)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSociodemographics:\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale sex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e285 (47)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e213 (\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (median, IQR) (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.4 (0.8\u0026ndash;2.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.6 (0.9\u0026ndash;2.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0 (0.6-2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnvironment\u003c/strong\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePeople in household (median, IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAny animal in household\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e376 (62)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e289 (63)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87 (60)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePets (dogs/cats)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e298 (49)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e226 (49)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFarm animals\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e225 (\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e175 (\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50 (\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eToilet in house\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e598\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e334 (56)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e257 (56)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77 (54)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWith basin for handwashing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e494 (83)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e372 (82)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e122 (86)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSchool/day care attendance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21 (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17 (3.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePersonal history\u003c/strong\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBMI Z score (mean, SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.66 (1.19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.57 (1.09)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.05 (1.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWeight for age Z score (mean, SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.93 (1.32)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.83 (1.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.24 (1.49)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBreastfeeding currently\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e202 (\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156 (\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46 (\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAny vaccine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e598 (99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e455 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e143 (99)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHib/PCV13 at least 2 doses\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e566 (94)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e439 (97)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e127 (88)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eComorbidities\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15 (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (1.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 (4.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAny health care previous 3 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e511 (85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e377 (82)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e134 (92)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInpatient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e604\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58 (\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26 (5.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32 (\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSurgery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e602\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (0.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (2.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHealthcare visit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e352 (58)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e264 (57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88 (61)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePharmacy consultation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e318 (53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e232 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86 (59)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTraditional healer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e604\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19 (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (2.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (6.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAntibiotics previous 4 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e549\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41 (\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 (7.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11 (8.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmoxicillin (% of those who took abx)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 (\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (9.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCeftriaxone / cefotaxime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (6.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmoxicillin-clavulanate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (3.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOfloxacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (3.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMetronidazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (3.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMeropenem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (9.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnknown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25 (61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18 (60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 (64)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eColonisation status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eESBL \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e512 (85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e391 (85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e121 (83)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCRE \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13 (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e162 (\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e108 (\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54 (\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCRE \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Bacterial colonisation and antimicrobial resistance at enrolment\u003c/h2\u003e\n \u003cp\u003eFrom 605 baseline RS, 531 (88%) children were colonised with an ESBL-E (512 \u003cem\u003eE. coli\u003c/em\u003e, 162 \u003cem\u003eK. pneumoniae\u003c/em\u003e, 143 both) and 21 (3%) by CRE (13 \u003cem\u003eE. coli\u003c/em\u003e, 8 \u003cem\u003eK. pneumoniae\u003c/em\u003e) at enrolment (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Four and three children were colonised, respectively, with both an ESBL and a CRE \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eK. pneumoniae\u003c/em\u003e. ESBL and CRE \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation decreased with age, being most prevalent in \u0026lt;\u0026thinsp;1-year olds admitted to hospital (Supplementary Fig. 1), whilst ESBL \u003cem\u003eE. coli\u003c/em\u003e colonisation peaked in 1-year olds and CRE \u003cem\u003eE. coli\u003c/em\u003e carriage increased with age. Of the 525 isolated \u003cem\u003eE. coli\u003c/em\u003e at enrolment, 129 (25%) were ampicillin-gentamicin-resistant, 232 (44%) fluoroquinolone-resistant, and 359 (68%) were MDR. The respective results for the 170 isolated \u003cem\u003eK. pneumoniae\u003c/em\u003e were: 37 (21%), 96 (55%) and 106 (61%) (Supplementary Table 2). AMR data are summarised in Supplementary Table 2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Risk factors for AMR bacterial colonisation at enrolment\u003c/h2\u003e\n \u003cp\u003eIndependent risk factors for ESBL \u003cem\u003eE. coli\u003c/em\u003e colonisation at enrolment were male sex (Adjusted OR, AOR, 0.56 for female sex, 95% CI 0.36\u0026ndash;0.88) and any healthcare exposure in the previous 3 months (AOR 1.99, 95% CI 1.14\u0026ndash;3.39) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Table 3). For ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation, younger age (AOR 0.59 per 1 year increase, 95% CI 0.47\u0026ndash;0.73), lower weight for age Z-score (AOR 0.82 per 1 Z-score increase, 95% CI 0.71\u0026ndash;0.95), and inpatient care in the previous 3 months (AOR 2.40, 95% CI 1.32\u0026ndash;4.35) increased the odds while current breastfeeding was shown to be protective (AOR 0.51, 95% CI 0.32\u0026ndash;0.79) for children under 1 year old (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Table 4). For CRE, antibiotic exposure in the previous 4 weeks increased the risk of CRE \u003cem\u003eE. coli\u003c/em\u003e colonisation (AOR 9.04, 95% CI 2.56\u0026ndash;29.2), while younger age (OR 0.17 per 1 year increase, 95% CI 0.03\u0026ndash;0.58) and inpatient exposure (OR 5.90, 95% CI 1.19\u0026ndash;24.7) were associated to CRE \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation at enrolment (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Tables 5 \u0026amp; 6).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Long-term dynamics of ESBL-E and CRE colonisation\u003c/h2\u003e\n \u003cp\u003eAt the 6-month visit, 108/605 (18%) children were lost-to-follow-up (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). ESBL-E colonisation proportions remained quite stable over the 6-month follow-up, ranging between 84\u0026ndash;88% and for \u003cem\u003eE. coli\u003c/em\u003e and 25\u0026ndash;30% for \u003cem\u003eK. pneumoniae\u003c/em\u003e (of available samples) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Table 2). However, there were changes in colonisation status for individual children, especially for ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation. For ESBL \u003cem\u003eE. coli\u003c/em\u003e, at each visit, 86\u0026ndash;92% of those previously colonised remained colonised and 71\u0026ndash;80% of those non-colonised became colonised. For ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e, at each visit, 38\u0026ndash;50% of those previously colonised remained colonised and 19\u0026ndash;24% of those non-colonised became colonised. Median time to colonisation gain was 15 days (IQR: 15\u0026ndash;15 days) and 135 days (IQR: 135-NA days), respectively, for ESBL \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e, while mean time to colonisation loss was undefined for ESBL \u003cem\u003eE. coli\u003c/em\u003e (Kaplan-Meier curve does not cross 50%) and 60 days (IQR: 15\u0026ndash;60 days) for ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Overall antibiotic susceptibilities were similar to baseline results (Supplementary Table 2), however there was considerable intra-individual temporal variation (Supplementary Fig. 2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Effect of antibiotic and healthcare exposure on ESBL-E colonisation dynamics\u003c/h2\u003e\n \u003cp\u003eThere were 160 ESBL \u003cem\u003eE. coli\u003c/em\u003e new colonisations (\u0026lsquo;gains\u0026rsquo;) out of 218 potential events and 145 de-colonisations (\u0026lsquo;losses\u0026rsquo;) out of 1325 potential events during the 6 months follow-up (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Overall, per visit, 377 children (18%) reported previous antibiotic use, 39 (1.8%) 3rd generation cephalosporin exposure, 1918 (89%) previous healthcare exposure and 287 (13%) inpatient exposure. Neither healthcare nor antibiotic exposure during this time were associated with time-to-gain or time-to-loss for ESBL \u003cem\u003eE. coli\u003c/em\u003e colonisation (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). For ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e, there were 250 colonisation gains out of 1118 potential events and 235 colonisation losses out of 425 potential events during the 6 months follow-up (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Reported previous exposures were similar to ESBL \u003cem\u003eE. coli\u003c/em\u003e (Supplementary Table 7). Inpatient (Hazard Ratio, HR: 1.42, 95% CI: 1.03\u0026ndash;1.96), any antibiotic (HR: 1.40, 95% CI: 1.05\u0026ndash;1.89), and 3rd generation cephalosporin exposure (HR: 2.06, 95% CI: 1.12\u0026ndash;3.79) were associated with a shorter time to colonisation gain, while any healthcare (HR: 0.68, 95% CI: 0.48\u0026ndash;0.94), and inpatient exposure (HR: 0.66, 95% CI: 0.43-1.00) increased the time to colonisation loss, after adjusting for potential baseline confounders (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCox proportional hazard regression analysis for effect of healthcare and antibiotic exposure on time-to-gain and time-to-loss of extended spectrum-beta-lactamase producing (ESBL) \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e gastrointestinal colonisation during 6-month follow-up in children under 5 years old seen at Angkor Hospital for Children.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eTime to gain\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTime to loss\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eaHR*\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eaHR*\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eESBL\u003c/strong\u003e \u003cstrong\u003e- E. coli\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;218 records, 160 events)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;1325 records, 145 events)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAny healthcare exposure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.54\u0026ndash;1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.54\u0026ndash;1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.788\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInpatient exposure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.68\u0026ndash;1.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.905\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.45\u0026ndash;1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.409\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAny antibiotic exposure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.52\u0026ndash;1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.60\u0026ndash;1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.653\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3rd gen cephalosporin exposure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.20\u0026ndash;2.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.20\u0026ndash;2.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.643\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eESBL\u003c/strong\u003e \u003cstrong\u003e- K. pneumoniae\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;1118 records, 250 events)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;425 records, 235 events)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAny healthcare exposure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.83\u0026ndash;2.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.68\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.48\u0026ndash;0.94\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.022\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInpatient exposure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.03\u0026ndash;1.96\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.031\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.66\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.43-1.00\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.048\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAny antibiotic exposure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.40\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.05\u0026ndash;1.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.023\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.59\u0026ndash;1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.132\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3rd gen cephalosporin exposure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.06\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.12\u0026ndash;3.79\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.019\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.30\u0026ndash;1.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.388\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e* Adjusted for age, sex, toilet inside the house, hand washing basin in toilet, weight-for-age Z score and current breastfeeding.\u003c/p\u003e\n \u003cp\u003eCI: confidence interval; ESBL: extended-spectrum beta-lactamase producers; aHR: adjusted hazard ratio.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThis prospective cohort study has detected high AMR Enterobacterales carriage proportions in the GI tract of Cambodian children attending a healthcare facility. Risk factors for AMR Enterobacterales carriage at presentation to healthcare included male sex (ESBL \u003cem\u003eE. coli\u003c/em\u003e), younger age, and malnourishment (ESBL and CRE \u003cem\u003eK. pneumoniae\u003c/em\u003e), not currently breastfeeding (ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e), healthcare exposure (ESBL \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e) and antibiotic exposure (CRE \u003cem\u003eE. coli\u003c/em\u003e). ESBL-E colonisation proportions remained stable during the 6-month-follow-up, despite changes in colonisation status for individual children. Persistent healthcare and antibiotic exposure (particularly 3rd generation cephalosporins and hospitalisations) were associated with changes in colonisation dynamics for ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e, but not for ESBL \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Strengths and limitations\u003c/h2\u003e\u003cp\u003eThis study included a relatively large sample size and collected extensive information on potential factors associated with AMR bacterial carriage. We included 1, 3 and 6-month follow-ups with both colonisation and exposure dynamic data, with a good retention proportion (82%). We used high standard microbiological techniques for bacteria and AMR identification including culture on chromogenic media, MALDI-TOF MS, and automated antimicrobial susceptibility testing. However, this study also presents some limitations. First, information on environmental and previous healthcare and antibiotic exposure were collected through parental questionnaires, which may introduce information bias. Second, we only included 1\u0026ndash;59 months-old children attending a single centre in Cambodia. This may limit the generalisability of our findings to other regions and age groups. Third, we only cultured \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e using selective media, and therefore could not estimate the number of children colonised by non-ESBL-E. This should not be an issue for \u003cem\u003eE. coli\u003c/em\u003e, as colonisation occurs in nearly all children, but it would have been informative to assess the proportion of children colonised by non-ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e. Finally, only one colony per plate was selected so multiple strains with different resistance profiles would not have been detected. In addition, we did not perform whole genome sequencing, which could help determine if the isolates identified at two different timepoints in the same child were the same strain or differed, increasing the accuracy of colonisation gain or loss detection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Findings in relation to other studies\u003c/h2\u003e\u003cp\u003eCurrent ESBL-E colonisation rates in our study (88%) were even higher than previously reported in this setting (55% for children\u0026thinsp;\u0026lt;\u0026thinsp;16 years old in 2012) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In Sub-Saharan Africa, a 2019 meta-analysis reported a much lower pooled ESBL-E colonisation prevalence of 10% (95% CI 1\u0026ndash;32%, range 10\u0026ndash;60%) in children (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, South-East Asia is the region with the highest estimated risk for AMR spread (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Most other studies on ESBL-E colonisation rates in children in Asia have focused on hospitalised patients, especially neonates, hampering the comparison with our estimates from children presenting to healthcare, as carriage rates of ESBL-E increase rapidly after admission (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). A meta-analysis of clinically-relevant specimens from children\u0026rsquo;s infections in Asian studies reported a pooled prevalence of 3rd -generation cephalosporins and carbapenem resistance, respectively, of 73% (95% CI 50\u0026ndash;86%) and 15% (95% CI 1\u0026ndash;33%) among \u003cem\u003eE. coli\u003c/em\u003e, and 76% (95% CI 40\u0026ndash;92%) and 13% (95% CI 0\u0026ndash;46%) among \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Prevalence of CRE colonisation (3%) was low in our setting, though it is a growing problem in neighbouring countries, such as Vietnam, where 13% of children were colonised with CRE at admission (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). This is aggravated by the lack of access to more extended spectrum and novel antibiotics in LMICs, such as Cambodia (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). These data emphasise the serious and urgent public health problem of AMR Enterobacterales in Asia.\u003c/p\u003e\u003cp\u003eMost identified risk factors for AMR Enterobacterales colonisation are related with gut microbiome health. Reported antibiotic and healthcare exposure, identified as key risk factors for AMR colonisation in children in previous studies (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), were associated to colonisation with ESBL \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e, and CRE \u003cem\u003eE. coli\u003c/em\u003e in our analysis. In Cambodia there is a high prevalence of inappropriate antibiotic prescription and use in the community, including broad-spectrum antibiotics (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), and this can result in diminished gut microbiome diversity (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Undernutrition appeared to increase the risk of ESBL and CRE \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation, even after adjusting for previous healthcare or antibiotic exposure. Undernutrition may affect the gut microbiome composition and is associated with reduced host immunity, therefore increasing the risk of colonisation by pathogenic bacteria (\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Breastfeeding was associated with lower ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation risk in children under 1 year old, as has already been shown in this setting for hospitalised neonates (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). As with the other risk factors, breastfeeding has also been shown to play a key role in gut microbiome composition and diversity (\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Gut microbiome reduced diversity and altered composition can increase the risk of colonisation with AMR bacteria, which benefit from reduced competition due to the suppression of more robust, antibiotic-sensitive strains (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLong term colonisation dynamics of ESBL-E in children following healthcare contact have been scarcely studied. Most studies were performed on adults, either during hospitalisation or among returning travellers and with short follow-up times (\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Additionally, some of these studies did not assess ESBL \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation separately (\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Given our findings at baseline, we believe colonisation dynamics of these two Enterobacterales may differ greatly. In previous studies, the mean colonisation duration for ESBL-E was only 30 days for international travellers upon return (ESBL \u003cem\u003eK .pneumoniae\u003c/em\u003e having the shortest decolonisation time) (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), but was longer for European adults and children who had acquired the colonisation in the community (128 days) (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). In this study, we showed that ESBL \u003cem\u003eE. coli\u003c/em\u003e colonisation occurs much faster and remains for longer than \u003cem\u003eK. pneumoniae\u003c/em\u003e, with shorter time-to-gain time (38 vs 118 days) and longer time-to-loss time (148 vs 58 days). As a consequence, most of the children already colonised with ESBL \u003cem\u003eE. coli\u003c/em\u003e at baseline remained colonised during the follow-up and the few that lost colonisation (8\u0026ndash;14%), were replaced by children newly colonised, to maintain a similar proportion of colonisation at each timepoint. For ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e, however, a larger proportion of those colonised at baseline became decolonised (50\u0026ndash;62%), but similar to \u003cem\u003eE. coli\u003c/em\u003e, the proportion of colonisation at each follow-up visit remained stable. These differences in long term ESBL-E colonisation dynamics in children have not been previously described.\u003c/p\u003e\u003cp\u003eThe impact of repeated antibiotic and healthcare exposure on the long term ESBL-E colonisation dynamics in children is largely unknown. A previous Cambodian study reported that any antibiotic use increased the daily acquisition risk of 3rd generation cephalosporin resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e among neonates during hospitalisation (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). We have now shown that persistent antibiotic exposure and repeated hospitalisations increase the risk of new ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation episodes over time after a first presentation to healthcare, while repeated healthcare exposures increase the time to colonisation loss. A similar study in Malawian adults also showed that repeated hospitalisations increased ESBL-E colonisation risk over time, and that persistent antibiotic exposure prolonged colonisation by reducing colonisation loss (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). However, in the present study, repeated healthcare and antibiotic exposure were not associated with long term ESBL \u003cem\u003eE. coli\u003c/em\u003e colonisation gain or loss. This may be due to the higher proportion of children in the community in our setting that are colonised with 3rd generation cephalosporin resistant \u003cem\u003eE. coli\u003c/em\u003e compared to \u003cem\u003eK. pneumoniae\u003c/em\u003e (53 vs 10%) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), which would also explain the higher proportion of new colonisation episodes occurring at each visit over time for ESBL \u003cem\u003eE. coli\u003c/em\u003e compared to \u003cem\u003eK. pneumoniae\u003c/em\u003e in our study (71\u0026ndash;80% vs 19\u0026ndash;24%).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Implications for practice and future research\u003c/h2\u003e\u003cp\u003eThe high rate of AMR bacterial gut colonisation found in our setting confirms the urgent need to reduce this global health problem. Some identified risk factors are modifiable, and point towards interventions that may reduce AMR carriage. Antimicrobial stewardship and reducing healthcare contact, at least with large healthcare centres, are two key interventions. Digital health algorithms have shown to reduce antibiotic prescription in children in community settings in Tanzania, with no increased clinical failure (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Such tools may not only reduce antibiotic use but also unnecessary referrals to larger hospitals, and their extended use should be now assessed in large multicentre clinical trials. Adding bacterial colonisation analysis to these studies will enable the study of their impact on AMR colonisation. Interventions modifying gut microbiome diversity and composition, may also reduce AMR gut colonisation in children. The use of probiotics use has been associated with reduced 3rd generation cephalosporin resistant bacteria gut colonisation in hospitalised neonates (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), but this effect has not been studied in clinical trials. Finally, advanced techniques such as targeted metagenomic sequencing from cultured specimens, may increase bacterial colonisation detection (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) and enable longitudinal changes of specific resistomes to be studied (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), an avenue we are currently pursuing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Conclusion\u003c/h2\u003e\u003cp\u003eCarriage of ESBL Enterobacterales is prevalent in Cambodian children. This may limit effective antibiotic choices if followed by invasive infections. Healthcare and antibiotic exposure reduction may prevent ESBL \u003cem\u003eK. pneumoniae\u003c/em\u003e colonisation with further prevention strategies needed for ESBL \u003cem\u003eE. coli\u003c/em\u003e colonisation.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAHC: Angkor Hospital for Children\u003c/p\u003e\n\u003cp\u003eAMR: antimicrobial resistance / resistant\u003c/p\u003e\n\u003cp\u003eAOR: adjusted odds ratio\u003c/p\u003e\n\u003cp\u003eBMI:\u0026nbsp;body mass index\u003c/p\u003e\n\u003cp\u003eCRE: carbapenem resistant Enterobacterales\u003c/p\u003e\n\u003cp\u003eESBL(-E): extended-spectrum beta-lactamase producing (Enterobacterales)\u003c/p\u003e\n\u003cp\u003eGI: gastrointestinal\u003c/p\u003e\n\u003cp\u003eHR: hazard ratio\u003c/p\u003e\n\u003cp\u003eIPD: inpatient\u003c/p\u003e\n\u003cp\u003eIQR:\u0026nbsp;interquartile range\u003c/p\u003e\n\u003cp\u003eLMICs: low- and middle-income countries\u003c/p\u003e\n\u003cp\u003eMDR: multi-drug resistant\u003c/p\u003e\n\u003cp\u003eOPD: outpatient\u003c/p\u003e\n\u003cp\u003eOR: odds ratio\u003c/p\u003e\n\u003cp\u003eRS: rectal swab\u003c/p\u003e\n\u003cp\u003eSE: standard error\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the AHC Executive Committee (reference 0102-21 AHC), Cambodia National Ethics Committee for Health Research (NECHR, references 079-NECHR (23/04/2021) / 090-NECHR (18/04/2022) / 089-NECHR (17/03/2023)), and Oxford Tropical Research Ethics Committee (OxTREC, reference 514-21). Parents or legal guardians of participating children consented to participate. The parent or legally acceptable representative of the participant signed or thumb-printed and dated the informed consent form before any study specific procedures were performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the Mahidol-Oxford Tropical Medicine Research Unit Data Access Committee on reasonable request (https://www.tropmedres.ac/units/moru-bangkok/bioethics-engagement/data-sharing). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded in whole, or in part, by the Wellcome Trust (grant numbers 206194 and 220211). CAG was funded by a Postdoctoral Mobility Fellowship from the Swiss National Science Foundation (grant number P500PM_217605), JC was funded by the ERC (grant number 742154) and by Norwegian Research Council FRIPRO (grant number 299941). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePT and JC designed the study and obtained funding. SB performed the patient recruitment, study procedures and data collection. PS performed the microbiology laboratory analyses. CAG performed the data cleaning, data analysis and first draft of the manuscript. SK and SP supervised the field work, CLL the microbiology laboratory work, SJL the statistical analysis and PT the overall work. All authors critically revised successive drafts and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMurray CJ, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022;399(10325):629-55.\u003c/li\u003e\n\u003cli\u003eFlokas ME, Karageorgos SA, Detsis M, Alevizakos M, Mylonakis E. Vancomycin-resistant enterococci colonisation, risk factors and risk for infection among hospitalised paediatric patients: a systematic review and meta-analysis. Int J Antimicrob Agents. 2017;49(5):565-72.\u003c/li\u003e\n\u003cli\u003eChereau F, Opatowski L, Tourdjman M, Vong S. Risk assessment for antibiotic resistance in South East Asia. BMJ. 2017;358.\u003c/li\u003e\n\u003cli\u003evan Aartsen JJ, Moore CE, Parry CM, Turner P, Phot N, Mao S, et al. 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Journal of Antimicrobial Chemotherapy. 2023;79(1):1-10.\u003c/li\u003e\n\u003cli\u003eArnold KE, Leggiadro RJ, Breiman RF, Lipman HB, Schwartz B, Appleton MA, et al. Risk factors for carriage of drug-resistant Streptococcus pneumoniae among children in Memphis, Tennessee. J Pediatr. 1996;128(6):757-64.\u003c/li\u003e\n\u003cli\u003eLevy SS, Mello MJ, Gusmao-Filho FA, Correia JB. Colonisation by extended-spectrum beta-lactamase-producing \u003cem\u003eKlebsiella\u003c/em\u003e spp. in a paediatric intensive care unit. J Hosp Infect. 2010 Sep;76(1):66-9.\u003c/li\u003e\n\u003cli\u003eTfifha M, Ferjani A, Mallouli M, Mlika N, Abroug S, Boukadida J. Carriage of multidrug-resistant bacteria among pediatric patients before and during their hospitalization in a tertiary pediatric unit in Tunisia. 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Nat Med. 2024 2024/01/01;30(1):76-84.\u003c/li\u003e\n\u003cli\u003eTurner P, Pol S, Soeng S, Sar P, Neou L, Chea P, et al. High Prevalence of Antimicrobial-resistant Gram-negative Colonization in Hospitalized Cambodian Infants. The Pediatric Infectious Disease Journal. 2016 Aug;35(8):856-61.\u003c/li\u003e\n\u003cli\u003ePol S, Kallonen T, M\u0026auml;klin T, Sar P, Hopkins J, Soeng S, et al. Exploring the pediatric nasopharyngeal bacterial microbiota with culture-based MALDI-TOF mass spectrometry and targeted metagenomic sequencing. mBio. 2024;0(0):e00784-24.\u003c/li\u003e\n\u003cli\u003eManenzhe RI, Dube FS, Wright M, Lennard K, Zar HJ, Mounaud S, et al. Longitudinal changes in the nasopharyngeal resistome of South African infants using shotgun metagenomic sequencing. PLoS One. 2020;15(4):e0231887.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial resistance, colonisation, Cambodia, paediatrics, Escherichia coli, Klebsiella pneumoniae","lastPublishedDoi":"10.21203/rs.3.rs-7229248/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7229248/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background: The gastrointestinal tract is a major reservoir of potentially pathogenic and antimicrobial resistant (AMR) bacteria, such as extended-spectrum-beta-lactamase-producing Enterobacterales (ESBL-E). Healthcare and antibiotic exposure are key determinants for ESBL-E colonisation, but little is known about the long-term temporal dynamics of carriage after exposure.\nMethods: COMRU-META was a clinical cohort of 1–59-month-olds seen at Angkor Hospital for Children, Cambodia, in 2021-22. Metadata (sociodemographic, clinical and environmental) and rectal swabs (RS) were collected at presentation to healthcare and at 1, 3 and 6-month follow-up visits. To detect ESBL and carbapenem resistant Enterobacterales (CRE), we cultured RS on selective chromogenic media, performed MALDI-TOF mass-spectrometry for isolate identification and determined antimicrobial susceptibilities by disk diffusion and automated minimum inhibitory concentration testing. We performed multivariable logistic regression to assess factors associated with ESBL-E and CRE colonisation at baseline and Cox regression to assess the effect of healthcare and antibiotic exposure on the time-to-gain and time-to-loss of ESBL-E colonisation during follow-up.\nResults: Among 605 children (median age 1.4 years, 47% female), colonisation at each visit over the 6 months by ESBL Escherichia coli was 85-88% and by ESBL Klebsiella pneumoniae, 27-29%. CRE colonisation proportion range was 1-2%. At baseline, most common risk factors for ESBL-E and CRE colonisation were previous healthcare or antibiotic exposure, while ESBL and CRE K. pneumoniae carriers were also younger and malnourished. For ESBL E. coli, there were 160 colonisation ‘gain’ episodes and 145 ‘loss’ episodes during the 6-month follow-up, not associated with antibiotic and healthcare exposure. For ESBL K. pneumoniae, children with any antibiotic (HR: 1.40, 95% CI: 1.05-1.89), 3rd generation-cephalosporin (HR: 2.06, 95% CI: 1.12-3.79) and inpatient exposures (HR: 1.42, 95% CI: 1.04-1.96) were more likely to have colonisation ‘gain’ episodes (N=250); children with healthcare exposure (HR: 0.70, 95% CI: 0.49-1.00) and who were inpatients (HR: 0.66, 95% CI: 0.43-1.00) were less likely to have colonisation ‘loss’ episodes (N=235).\nConclusion: ESBL-E carriage is prevalent in Cambodian children. Persistent healthcare and antibiotic exposure were associated with changes in colonisation dynamics for ESBL K. pneumoniae, but not for ESBL E. coli. 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