# Early neonatal acquisition of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in Madagascar and Cambodia: clonal and plasmid-level contributions of maternal gut carriage

Anne-Lise Beaumont, Agathe de Lauzanne, Alexis Criscuolo, Laetitia Fabre, Mamitiana Rabenandrasana, Norohasina Randriamanga, Sandrine Bernabeu, Aina Harimanana, Rindra Randremanana, Perlinot Herindrainy, et al.

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Anne-Lise Beaumont, Agathe de Lauzanne, Alexis Criscuolo, Laetitia Fabre, Mamitiana Rabenandrasana, et al. Early neonatal acquisition of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in Madagascar and Cambodia: clonal and plasmid-level contributions of maternal gut carriage. 2025. hal-05059815

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# Early neonatal acquisition of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in Madagascar and Cambodia: clonal and plasmid-level contributions of maternal gut carriage

Anne-Lise Beaumont

annelisebeaumont@gmail.com

Institut Pasteur Paris https://orcid.org/0000-0003-2475-8847

Agathe de Lauzanne

Institut Pasteur du Cambodge

Alexis Criscuolo

Institut Pasteur https://orcid.org/0000-0002-8212-5215

Laetitia Fabre

Institut Pasteur

Mamitiana Rabenandrasana

Institut Pasteur de Madagascar

Norohasina Randriamanga

Institut Pasteur de Madagascar

Sandrine Bernabeu

Bicêtre Hospital, Assistance Publique - Hôpitaux de Paris

Aina Harimanana

Institut Pasteur de Madagascar

Rindra Randremanana

Institut Pasteur de Madagascar

Perlinot Herindrainy

Institut Pasteur de Madagascar

Jean-Marc Collard

Institut Pasteur Paris

Long Pring

Institut Pasteur du Cambodge

Navin Sreng

Institut Pasteur du Cambodge

Sokleaph Cheng

Institut Pasteur du Cambodge
**Laurence Borand**

Johns Hopkins University School of Medicine

**Elsa Kermorvant**

APHP- Necker hospital https://orcid.org/0000-0002-0668-7945

**Tania Crucitti**

Institut Pasteur de Madagascar

**Didier Guillemot**

Institut Pasteur, Versailles Saint Quentin University, Inserm

**Bich-Tram Huynh**

Epidemiology and Modelling of Antibiotic Evasion, Institut Pasteur; UMR 1018, team "Anti-infective Evasion and Pharmacoepidemiology", Université Paris-Saclay, UVSQ, Inserm

**BIRDY Study Group**

Pasteur Network

**Article**

**Keywords:** antimicrobial resistance, newborn, beta-lactam resistance, low- and middle-income countries

**Posted Date:** April 15th, 2025

**DOI:** https://doi.org/10.21203/rs.3.rs-6271227/v1

**License:** © Ⓟ This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** There is **NO** Competing Interest.
**Abstract**

Early neonatal infections caused by extended-spectrum-beta-lactamase-producing Enterobacterales (ESBL-PE) are prevalent in low- and middle-income countries, posing significant treatment challenges.

This study investigated ESBL-PE colonization in 499 mother-neonate pairs from Madagascar and Cambodia, with stool samples collected at delivery and from neonates before day three. Using short- and long-read sequencing, identical isolates or plasmids were identified within each pair to assess neonatal acquisition from maternal source. Multinomial regression identified associated risk factors.

Maternal gut carriage accounted for only 16.5% of neonatal ESBL-PE acquisition, suggesting that most cases originated from other sources. Primiparity and wet season were associated with an increased risk of acquisition from the mother, while cesarean delivery, neonatal resuscitation, and wet season were associated with acquisition from other sources.

These findings challenge the paradigm that maternal carriage is the primary source of early neonatal ESBL-PE colonization, highlighting the need for further research into alternative sources to inform targeted interventions.

**Introduction**

Neonatal bacterial sepsis represents a critical health challenge, particularly in low- and middle-income countries (LMICs)¹,². Most of these infections occur during the first week of life, particularly within the first three days³, the timeframe defined as early-onset neonatal sepsis⁴.

In LMICs, Enterobacterales are the primary bacteria driving neonatal sepsis⁵⁻⁷, with a significant proportion producing extended-spectrum beta-lactamases (ESBL-PE)⁸,⁹. Consequently, antibiotic treatment options are limited to molecules such as carbapenems¹⁰, which are either unavailable or prohibitively expensive in many LMICs. This contributes to an estimated 214,000 neonatal deaths annually linked to antimicrobial resistance¹¹.

Early-onset neonatal sepsis is primarily attributed to the maternal transmission of pathogens during pregnancy, labor, or delivery¹²,¹³. In the case of ESBL-PE infections, this assumption is supported by the high prevalence of ESBL-PE gut colonization among pregnant women in LMICs, reaching up to 80% in Cambodia¹⁴ and 18.5% in Madagascar¹⁵. While studies on hospitalized neonates have documented instances of ESBL-PE acquisition from maternal gut strains¹⁶, the overall predominance of this source, particularly in community settings, has yet to be confirmed. Newborns are also exposed to Enterobacterales from other sources, including surfaces, or caregiver's hands. Distinguishing and quantifying the maternal contribution to acquisition is critical to design prevention strategies, but molecular studies tracking ESBL-PE transmission remain limited¹⁷. Most rely on comparing maternal
and neonatal strains using low-resolution techniques, such as pulsed-field gel electrophoresis (PFGE)¹⁸,¹⁹, which may overestimate the role of maternal source.

The source of bacterial acquisition is most often inferred from the detection of identical isolates, indicating clonal transmission. However, if the focus is on resistance acquisition, the identification of shared plasmids carrying resistance genes, even among genetically distinct strains, is equally important. Analyzing plasmid content is beyond the scope of traditional typing methods and short-read sequencing, as these approaches lack the required resolution to accurately reconstruct plasmids from fragmented data²⁰. In contrast, long-read sequencing enables complete plasmid sequence assembly and accurate comparison of plasmid contents²¹. However, to our knowledge, the detection of shared plasmids carrying ESBL-encoding genes has not yet been explored in studies investigating neonatal sources of ESBL-PE acquisition.

Community-level studies combining whole-genome sequencing with plasmid analysis to elucidate neonatal ESBL-PE transmission pathways are yet to be conducted in LMICs, limiting the development of effective prevention strategies. By identifying both identical isolates and plasmids, this study aims to quantify the contribution of the maternal gut to early ESBL-PE acquisition in newborns from two mother-newborn cohorts in Madagascar and Cambodia.

# Results

## Population characteristics and ESBL-PE colonization prevalence

Overall, 147 mother-neonate pairs (hereafter referred to as pairs) in Cambodia and 377 in Madagascar, were screened for this ancillary study of two community-based mother and child cohorts called BIRDY³ (Bacterial Infections and antibiotic Resistant Diseases among Young children in low income countries) and NeoLIC²² (Neonatal acquisition of ESBL-PE in the community of a Low-Income Country). A total of 131 pairs from Cambodia and 368 from Madagascar, including two twin pairs, were ultimately included (Fig. 1), resulting in a study population of 499 pairs (996 individuals). In Madagascar, 299 and 67 pairs were included through BIRDY and NeoLIC studies, respectively. Population characteristics are detailed in **Table 1**.

The overall prevalence of ESBL-PE colonization was 48.9% [95% CI: 44.4–53.4%] in mothers and 34.1% [29.9–38.4%] in neonates. Maternal colonization prevalence was 79.4% [71.3–85.8%] (104/131) in Cambodia compared to 38.0% [33.0–43.2%] (139/366) in Madagascar (*p* < 0.01). Neonatal colonization prevalence was also higher in Cambodia, at 53.4% [44.5–62.1%] (70/131), versus 27.2% [22.8–32.1%] (100/368) in Madagascar (*p* < 0.01). In colonized mothers, *Escherichia coli* was the most frequently identified species, detected in 91.3% (95/104) of cases in Cambodia and 75.5% (105/139) in Madagascar. *Klebsiella* spp. were the second most common, found in 8.7% (9/104) of colonized
mothers in Cambodia and 28.1% (39/139) in Madagascar, followed by *Enterobacter* spp., which were absent in Cambodia (0%) but present in 7.2% (10/139) of cases in Madagascar. Among colonized newborns, the species distribution differed significantly. *E. coli* prevalence was notably lower, detected in 64.3% (45/70) of cases in Cambodia and 33.0% (33/100) in Madagascar (p < 0.01). In contrast, *Klebsiella* spp. were identified in 18.6% (13/70) of colonized neonates in Cambodia and 36.0% (36/100) in Madagascar, while *Enterobacter* spp. were found in 20.0% (14/70) and 30.0% (30/100), respectively.

**Contribution of maternal versus other sources to ESBL-PE acquisition**

A total of 170 neonates were colonized, among whom 72 were born to non-colonized mothers, while 98 had mothers colonized with an ESBL-PE (Fig. 1). Among the 98 pairs where both the mother and the newborn were colonized, two pairs were excluded from the analysis because of the insufficient sequence quality of one of the isolates. Out of the 96 remaining pairs, 59 shared at least one isolate of the same bacterial species (i.e., *Escherichia coli*, *Klebsiella quasipneumoniae*, *Enterobacter kobei* and *"Enterobacter intestinihominis"*). Phylogenetic classifications showed 23 cases of identical isolates within pairs (Fig. 2): 10 in Cambodia (all *E. coli*) and 13 in Madagascar, comprising 10 *E. coli* pairs, one *Klebsiella quasipneumoniae* pair, one *Enterobacter kobei* pair, and one *"Enterobacter intestinihominis"* pair. Notably, fewer than 10 Single Nucleotide Polymorphisms (SNPs, **Supplementary Fig. 1**) were observed between genomes of mother-neonate pairs forming a cherry in the phylogenetic trees, therefore confirming the suitability of this cut-off to determine identical isolates²³.

For the remaining 75 ESBL-PE positive pairs, 30 pairs exhibited at least one ESBL-encoding gene in common, including 17 with a common plasmid replicon. After plasmid reconstruction, five pairs with identical plasmids were identified (three in Cambodia and two in Madagascar). Aligned plasmid sequences are displayed in Fig. 3. A total of 28 neonatal acquisition cases were attributed to maternal gut carriage. Of these, 17.8% [3.7–32.0%] (5/28) of cases linked to maternal source were identified by plasmid content analysis.

Overall, combining cases with identical isolates or identical plasmids, 16.5% [11.4–23.1%] (28/170) of neonatal colonization cases were attributable to maternal source. As a result, the remaining 83.5% [76.9–88.6%] (142/170) were attributable to other sources. The proportion of cases attributed to maternal source rate was similar in Cambodia (18.5%, 13/70) and Madagascar (15%, 15/100, p = 0.68).

In Madagascar, a significant difference was observed between the two studies through which mothers were included, with a lower proportion of acquisition attributed to the mother in BIRDY (7.6%, 6/79) compared to NeoLIC (42.8%, 9/21, *p* < 0.01). In the latter study, maternal colonization was more prevalent (34% versus 55%, *p* < 0.01), antibiotic administration at delivery was significantly more frequent (10% versus 60%, *p* < 0.01), and only vaginally delivered infants were included (Table 1).

**Risk factor analysis**
After univariate analysis, a multinomial regression model (Fig. 4) was fitted to identify maternal, neonatal and delivery features associated with a three-level categorical variable: (1) ESBL-PE acquisition attributed to maternal source, (2) ESBL-PE acquisition attributed to other sources, and (3) no acquisition (used as the reference group). The model was applied on complete cases, including 472 pairs. With adjustment on the study project, factors significantly associated with acquisition from maternal source compared to no acquisition were primiparity (adjusted odds ratio aOR 2.76 [1.18–6.47]) and delivery during wet compared to dry season (aOR 2.59 [1.02–6.55]). Factors significantly associated with acquisition from other sources compared to no acquisition were cesarean section (aOR 3.71 [1.90–7.22]), neonatal resuscitation (aOR 2.32 [1.22–4.42]) and delivery during wet season (aOR 1.73 [1.08–2.77]). The multinomial regression models performed on imputed datasets yielded very similar results (Supplementary table 1).

## Phenotypic characteristics of isolates

A total of 515 ESBL-producing isolates were obtained. More than one colonizing isolate was identified in 17.9% (42/243) of ESBL-colonized mothers and 25.4% (43/170) of ESBL-colonized neonates.

Antibiotic resistance profiles were established for 513 isolates (Supplementary Fig. 2). Up to 44% (226/513) of isolates were resistant to both amoxicillin and gentamicin, which is the recommended combination by World Health Organization (WHO)²⁴ for neonatal sepsis. Up to 91.4% (469/513) of isolates were multidrug-resistant, defined as showing resistance to ≥ 1 agent(s) in ≥ 3 antimicrobial categories²⁵. The majority of isolates remained susceptible to carbapenems, with only 2.1% (11/513) showing resistance to ertapenem and none resistant to imipenem.

## Genomic characteristics of isolates

Fourteen samples were excluded due to poor sequencing quality, leaving 501 sequences suitable for genotypic analysis (Fig. 5). Taxonomic distribution was diverse with 10 identified genera: *Atlantibacter*, *Citrobacter*, *Enterobacter*, *Escherichia*, *Klebsiella*, *Kluyvera*, *Leclercia*, *Lelliottia*, *Morganella and Phytobacter* (Supplementary Table 2). The predominant species was *E. coli*, with isolates spread across its overall population structure (Fig. 3, Supplementary Fig. 3), though species distribution substantially differed between mothers and neonates, as previously described (Fig. 2). Diversity among species was high, with notably 105 distinct sequence types (ST) identified among 299 *Escherichia* isolates, and 53 distinct ST among 95 isolates belonging to the *Klebsiella pneumoniae* species complex (Supplementary Table 2).

The *bla*<sub>CTX-M</sub> family accounted for the majority of ESBL genes (89.3%, 469/525), with *bla*<sub>CTX-M-15</sub> being the most prevalent (62.1%, 326/525). It was more common in Madagascar (76.7%, 254/331) than in Cambodia (37.1%, 72/194, *p* < 0.001), where *bla*<sub>CTX-M-55</sub> (18.6%, 36/194) and *bla*<sub>CTX-M-27</sub> (17.5%, 34/194) were also frequently detected. Genotypic co-resistance was found in 98.2% (492/501) of
isolates, mainly to sulfonamides (sul2, 62.6%, 314/501), aminoglycosides (aph(3")-lb and/or aph(6)-Id,
59.6%, 299/501), and tetracyclines (tet(A), 57.4%, 288/501, **Supplementary Fig. 4**). Carbapenemase
genes were detected in two isolates, both from Cambodian mothers (blaNDM-5 and blaNDM-1). The
number of identified resistance genes was significantly higher in isolates from neonates (median 11
[interquartile range IQR 8–15]) compared to isolates from mothers (median 9 [IQR6–12], p < 0.01).
Plasmid replicons were found in 90.6% (454/501) of isolates, with no statistical difference between
isolates from mothers (90.9%, 251/276) and from neonates (90.2%, 203/225, *p* = 0.9). The most frequent
plasmids were from the IncF incompatibility group (63.7%, 319/501), followed by Col (47%, 239/501).

**Discussion**

In a community-based, multicentric mother-child cohort with high rates of maternal and early neonatal ESBL-PE colonization, maternal gut carriage contributed for 16.5% of neonatal acquisition, even when both identical clones and plasmids were considered. Our findings challenge the prevailing assumption that the maternal gut is the primary source of neonatal ESBL-PE colonization. This study is the first to apply high-resolution genomic analyses (including plasmid reconstruction) in a community cohort to assess the role of maternal gut in early neonatal ESBL-PE acquisition. These findings highlight the critical role of other sources of acquisition, and are essential for developing more effective prevention strategies.

Our result underscore the need to reconsider existing paradigms in neonatal sepsis, especially in
reexamining sources of acquisition of antibiotic-resistant bacteria. This aligns with a recent critical
review that advocated for rethinking the traditional classification of neonatal sepsis into early- and late-
onset categories²⁶, highlighting in particular its limited utility for predicting the pathogen spectrum in
LMICs. Several studies conducted in high-income countries attributed neonatal early ESBL-PE
acquisition to maternal source. In Israel, studies have reported that between 72%²⁷,²⁸ and 100%²⁹ of
neonatal colonization cases were linked to maternal gut carriage, while a study in Germany estimated
this proportion at 42%³⁰. Other studies¹⁸,¹⁹ could not quantify the role of different sources due to
analyses limited to subsets of isolates. Additionally, these conclusions often rely on low-resolution
comparison methods such as PFGE¹⁸,³⁰ or AST profiles²⁷,³¹. Their relevance to LMICs is further limited
by the difference in ESBL-E prevalence and epidemiology compared to HICs³².

An hospital-based Cambodian study¹⁶ found that only eight out of 25 neonatal ESBL-PE colonization
cases were attributable to maternal gut carriage, aligning with our findings. However, plasmid content
comparison between different strains was not assessed. In the large-scale BARNARDS study³³ across
seven LMICs, neonatal and maternal microbiota were screened for ESBL-encoding genes. While
systematic mother-neonate pairwise analyses were absent, they identified one indistinguishable isolate
between a mother and her neonate, but also between unrelated neonates at the same clinical site,
pointing to acquisition from the hospital environment. Studies using other methodologies also reinforce
this conclusion. In Gambia, Okomo et al.³⁴ compared blood culture isolates from hospitalized newborns
with sepsis alongside maternal rectovaginal samples. They identified identical isolates in five out of 36 cases (all *Staphylococcus aureus*), none for the 15 sepsis cases caused by *Enterobacterales*. This is further corroborated by studies on the neonatal resistome³⁵. Metagenomic sequencing has revealed that neonatal microbiota harbor more antibiotic resistance genes and mobile genetic elements than the maternal ones³⁶,³⁷, even without antimicrobial exposure.

To the best of our knowledge, our study is the first to identify identical plasmids carrying ESBL genes between distinct maternal and neonatal bacterial isolates. In our cohort, approximately 20% of ESBL acquisitions from maternal source were detected through plasmid content analysis, emphasizing the need to explore resistance transmission mechanisms beyond clonal transfer to fully understand neonatal ESBL-PE acquisition. Plasmids play a major role in the spread of antimicrobial resistance³⁸. The human microbiome is a hotspot for horizontal gene transfers (HGT), with rates of HGT between human-associated bacteria 25 times higher than in non-human isolates³⁹. Childbirth represents a unique moment of microbiome interaction between mother and neonate. Although the newborn gut plasmidome remains underexplored, recent studies show that plasmids are disproportionately abundant in the infant gut early in life⁴⁰. Our findings underscore the importance of tracking plasmids to understand neonatal ESBL-PE acquisition and stress the need to incorporate plasmid analysis in transmission studies more broadly⁴¹.

The limited role of maternal source was observed despite a notably high prevalence of ESBL-PE colonization, both among mothers and neonates. The prevalence rate aligns with previous studies on maternal colonization at delivery¹⁴,⁴² and neonatal colonization⁴³,⁴⁴. Our results suggest that maternal ESBL-PE colonization does not necessarily lead to transmission to the newborn. Our multivariate analysis identified primiparity as a risk factor of acquisition from the mother. Longer labor duration in primiparas⁴⁵ may increase the time of exposure of the neonate to the maternal gut microbiota, potentially heightening the risk of transmission. Additionally, immunological variations between first and subsequent pregnancies⁴⁶ may also play a role. Microbiota composition of primiparous mothers differs from that of multiparous ones⁴⁷⁻⁴⁹ and multiparous women typically show a gestational decline in inflammatory cytokines (TNF-α, IL-10, IFN-γ), absent in first pregnancies⁵⁰. This heightened inflammatory state may promote transmission through mechanisms such as oxidative stress or dysbiosis.

Conversely, the limited role of maternal gut carriage in neonatal acquisition underscores the importance of exploring alternative transmission pathways. Although our study was not designed to identify these pathways, cesarean section and resuscitation were statistically associated with non-maternal sources of acquisition. Cesarean section delivery, previously reported as a risk factor for ESBL-PE acquisition⁴⁴,⁵¹, may increase neonatal exposure to nosocomial ESBL-PE sources (e.g., healthcare workers' hands, prolonged hospitalization) and disrupt microbiome development, impairing colonization resistance⁵²,⁵³. Similarly, neonatal resuscitation may reflect higher exposure to invasive procedures or fetal distress during childbirth, which could increase vulnerability to colonization. The BARNARDS study also identified
similar influencing factors, with complicated births and cesarean sections significantly associated with
the presence of ESBL genes in the neonatal gut³³.

Being born during the wet season was also significantly associated with a higher risk of acquisition,
either from maternal or from other sources. The association between the wet season and an increased
prevalence of ESBL-E. coli colonization has been previously documented in pregnant women¹⁵,²³.

Conditions of higher humidity and temperature during the wet season may promote HGT⁵⁴ and bacterial
growth⁵⁵. Similarly, an increase in infection rates by Gram-negative bacteria during warmer months has
been observed in neonatal intensive care units in Canada⁵⁶. These findings highlight the importance of
considering seasonality in surveillance and intervention studies on neonatal sepsis, notably when
evaluating future vaccines targeting this condition. Stratification and/or adjustment for the season of
birth should therefore be incorporated in statistical analyses.

Our study has several notable strengths. First, it was community-based, with recruitment including
deliveries in various settings, such as home births, which helps reduce the potential for selection bias
compared to previous hospital-based studies¹⁶. Second, samples were systematically collected from
mothers at the time of delivery and from neonates within the first three days of life. This approach has
rarely been implemented, especially in LMICs, where studies often collect samples only when neonates
show signs of sepsis, and not necessarily just after birth¹⁶,³³,³⁴. In addition, we employed cutting-edge
methodologies, combining short- and long-read sequencing, therefore providing a higher resolution
compared to techniques used in the majority of previous studies¹⁸,¹⁹,²⁷,²⁹⁻³¹.

However, our study also has some limitations. First, by focusing on morphologically distinct colonies
from each sample, we may have missed some clonally distinct isolates. These likely represent low-
inoculum strains with lower transmission risk, minimizing bias; however, the role of the maternal source
is probably still underestimated. Second, while we aimed to trace transmission, it remains impossible to
definitively determine whether the observed events were due to direct maternal transmission or
exposure to a third common source. This latter scenario appears nevertheless unlikely given the narrow
timeframe during which the samples were collected. Furthermore, it is still unknown how often identical
plasmids could occur without a transmission, as "core plasmids" of some lineages⁵⁷. Finally, we
observed variations in the proportion of neonatal acquisition cases attributable to maternal sources
between the two studies through which mothers were recruited in Madagascar. One key difference
between the two populations is that no cesarean sections were performed in mothers recruited through
NeoLIC, whereas 12% of mothers in the BIRDY study underwent cesarean sections, which could have
significantly influenced neonatal acquisition. This highlights the need for caution when extrapolating
findings to other settings, as transmission pathways may vary depending on population characteristics,
particularly the prevalence of cesarean deliveries.

Our study has significant implications for policy and future research. Non-maternal sources were the
predominant in neonatal ESBL-PE acquisition, statistically associated to cesarean deliveries and birth
resuscitations in our setting. The neonatal period, even in community settings, involves substantial interactions with the healthcare environment. While nosocomial risks for hospitalized neonates are well-recognized, the role of routine hospital deliveries in neonatal ESBL-PE acquisition remains underexplored. Strengthening maternity ward hygiene is crucial, yet optimal interventions are unclear. Future studies should map transmission pathways across diverse sources, including, among others, baby wipes, healthcare workers and formula bottles. Alternative maternal sources like breastmilk, should also be investigated. While breastfeeding is known to offer protective effects against antimicrobial resistance⁵⁸, it has also been shown to carry antimicrobial resistance genes (ARGs), with studies reporting that 70% of ARGs detected in breastmilk are also found in infant feces³⁶. Such research could guide strategies to delay or reduce neonatal ESBL-PE colonization, a key goal given the heightened vulnerability in the first three days of life, when most severe sepsis cases occur. Reducing maternal colonization, for example by curbing antibiotic use in pregnancy (estimated at 45.3% in low-income countries⁵⁹), could be an additional measure. However, evaluating the appropriateness of these prescriptions is challenging, and recent antibiotic use has not been consistently linked to colonization in several studies¹⁴,²³. Further investigation is essential to better understand the contributing factors and to identify more targeted, effective interventions.

## Conclusion

Our study highlights the complexity of neonatal ESBL-PE acquisition in LMICs, showing that maternal gut carriage accounts for less than 20% of neonatal colonization cases, even when considering both identical bacterial isolates and plasmids. By using high-resolution genomic analyses, including plasmid reconstruction, in a community-based cohort, we provide new molecular insights that challenge the assumption that maternal carriage is the primary source of early neonatal ESBL-PE colonization.

## Methods

### Definitions and conceptual framework

'Acquisition' refers to the event in which a neonate was colonized by an ESBL-PE (Supplementary Figure 5). Two scenarios were considered to confirm the maternal gut as the source of acquisition: either sharing an identical isolate or sharing an identical plasmid between the maternal and neonatal gut.

### Study design

This prospective, cross-sectional study was nested within two multicenter, community-based mother and child cohorts: BIRDY (Bacterial Infections and antibiotic Resistant Diseases among Young children in low income countries) and NeoLIC (Neonatal acquisition of ESBL-PE in the community of a Low-Income Country), whose methodologies have already been described³,²². This ancillary study of BIRDY was conducted from 2019 to 2020 in Madagascar, and from 2016 to 2017 in Cambodia (under the name of PEECNIC study, for Producing Extended Spectrum Beta Lactamase Enterobacteria Carriage in Newborns
and Infants in Cambodia). Two sites were involved in each country: one rural (Kampong Speu and Moramanga, respectively) and one urban (Phnom Penh and Antananarivo, respectively). Mothers were recruited during pregnancy through collaboration with community health workers. This design allowed women to be enrolled regardless of their antenatal care status and enabled the inclusion of home births. NeoLIC project took place between 2021 and 2022 in the same rural site as BIRDY in Madagascar (Moramanga), where mothers were recruited at the primary healthcare center. Stool and/or rectal swabs were collected from mothers at delivery and from their respective neonates before 72 hours of life. Socioeconomic data and medical information at recruitment and after delivery were collected.

## Microbiological analysis

After collection, samples were transported on the same day to the Institut Pasteur of the study site for immediate analysis or were stored frozen at -80°C until further analyses. All samples were pre-enriched following the method described by Jazmati et al⁶⁰. Subsequently, they were inoculated onto antibiotic-supplemented media and incubated overnight in an aerobic atmosphere at 36.5±2°C. In Cambodia, Drigalski plates supplemented with 2 mg/L cefotaxime (Bio-Rad, Marnes-La-Coquette, France) were used, while in Madagascar, CHROMagar ESBL (CHROMagar, Paris, France) media were employed. Up to three colonies per plate were selected based on their color and morphological characteristics. Single bacterial colonies were cultured further, until pure isolates were obtained. Species identification was performed using MALDI-ToF (Madagascar) or API 20E system (Cambodia). The antimicrobial susceptibility (AST) of *Enterobacterales* was determined by disc-diffusion antibiotic susceptibility testing on Mueller-Hinton agar (Bio-Rad) using the Kirby-Bauer disc diffusion method with interpretation according to the European Committee on Antimicrobial Susceptibility Testing (2018; www.eucast.org). ESBL production was detected by the double-disc synergy test by placing the disk of cefotaxime (30 µg), ceftazidime (30 µg) and combination of amoxicillin/clavulanic acid (20 µg/10 µg) on a lawn culture of bacteria on Muller-Hinton agar plate, with a 20 mm distance between each disk from center to center.

## Short-read sequencing

Based on DNA extracts from each ESBL-PE, Illumina short-read sequencing of every ESBL-PE isolate was performed on NextSeq 500 instruments using Nextera XT libraries with a 2x150 bp paired-end protocol. Small subunit gene segments coding for 16S rRNA were assembled using ASSU v1.1 (gitlab.pasteur.fr/GIPhy/ASSU), and genome sequences were assembled using fq2dna v24.02 (gitlab.pasteur.fr/GIPhy/fq2dna). Quality controls were performed using various metrics (e.g. coverage depth distribution, N50).

Taxonomic assignment of each isolate was performed in two steps: (i) the assembled 16S rRNA segment was used as a query to perform a BLASTn search against the RefSeq Targeted Loci Project sequences (www.ncbi.nlm.nih.gov/refseq/targetedloci) to assess the genus (i.e. the one associated to the best BLASTn hit); (ii) the Average Nucleotide Identity (ANI) between the assembled genome and every type strain genome from its genus was computed using FastANI⁶¹ v1.33 to assess the species (i.e.
the one of the closest type strain genome with ANI > 96%). Of note, species names between quotes were
not yet validly published (March 2025) according to the International Code of Nomenclature of
Prokaryotes.

Sequence types (ST) were determined using mlst v2.23.0 (github.com/tseemann/mlst). Acquired
resistance genes and plasmids were searched using AMRfinder⁶² v3.12.8 and PlasmidFinder⁶³ v2.1.1.

## Phylogenetic analysis

In pairs where both the mother and newborn were ESBL-PE carriers, insufficient quality of any sample led to an exclusion from analysis. At the genus level, genome-based phylogenetic trees were inferred using JolyTree⁶⁴,⁶⁵ v2.1 (Supplementary Figures 3, 6 and 7). At the species level, phylogenetic trees were inferred using IQ-TREE⁶⁶ v2.3.4 from recombination-purged core genome alignments built using Parsnp⁶⁷ v1.6.2. Species trees were rooted according to the phylogenetic relationships supported by the genus trees. Neonatal bacterial isolate acquisition from a maternal source was determined by the presence of a "cherry", defined as a pair of adjacent tips on the phylogenetic tree representing isolates from the mother-neonate pair.

## Plasmid content analysis

To identify putative neonatal acquisition of an ESBL-carrying plasmid from the mother, additional long-read sequencing was performed for selected pairs. Selection criteria included: (1) pairs both colonized by ESBL-PE, (2) no identified clonal transmission, (3) at least one common ESBL gene and plasmid replicon (same incompatibility group) between neonatal and maternal isolates. For one isolate per selected pair, DNA was extracted with Genomic-tip 100/G columns (Qiagen). DNA integrity and RNA absence were assessed via agarose gel electrophoresis and A<sub>260</sub>/A<sub>230</sub> and NanoDrop™ 2000 spectrophotometer (A<sub>260</sub>/A<sub>230</sub>, A<sub>260</sub>/A<sub>280</sub> ratios). DNA concentrations were measured using Qubit and dsDNA BR Assay Kit (Invitrogen). Libraries were prepared with the SQK-RBK114.24 rapid sequencing kit (Oxford Nanopore Technologies, RBK_9176_v114_revN_2024) and sequenced on a MinION-Mk1C with R10.4.1 flow cells. Hybrid genome assemblies (combining long- and short-reads) were inferred using Unicycler<sup>68</sup>v.0.4.8. Circularized plasmid sequences were analyzed using AMRfinder to select the potential transmitted plasmid. The *bla*-gene carrier served as reference for aligning short reads from the pair's other isolate using bwa-mem2<sup>69</sup> v2.2.1, with >99% sequence similarity as the cut-off.

## Statistical analysis

### Descriptive analysis

The contribution of maternal versus other sources to ESBL-PE acquisition in newborns was estimated as (c+p)/n and 1-(c+p)/n, respectively, where c is the number of pairs with identical isolates, p the number of pairs with identical plasmids, and n the total number of newborns colonized with ESBL-PE.
The wet season was defined as the period from May to October in Cambodia, and from November to April in Madagascar, with the remainder of the year classified as the dry season.

### Risk factor analysis

The outcome variable for risk factor analysis was early **neonatal ESBL-PE acquisition**, a three-level categorical variable: acquisition resulting from a maternal source, acquisition resulting from another source, and no acquisition of ESBL-PE (reference).

First, a univariate analysis was conducted to explore the associations between neonatal ESBL-PE acquisition and key exposures of interest, including potential confounding factors. Categorical variables were compared using Fisher's exact test or chi-squared tests, depending on the distribution of the data. For continuous variables, differences between groups were assessed using t-tests. Next, a multinomial logistic regression was performed to assess the relationship between exposures and the outcome variable. Variables that showed an association with the outcome at a significance level of *p*<0.2 in the univariate analysis were included in the multivariate model. The model was then refined through a backward selection procedure.

Analyses were conducted on complete cases. For validation, we also used a set of ten imputed datasets, where missing data were addressed via multiple imputation. All statistical analyses were conducted in RStudio v2024.04.2+764 with the packages nnet v7.3.20 and mice v3.17.0. Statistical significance was defined at *p*<0.05.

## Declarations

### Ethics

BIRDY study was approved by the ethics committees of Madagascar (N° 068-MSANP/CE), Cambodia (N° 108-NECHR, N° 275-NECHR), and the Institutional Review Board of Institut Pasteur (N° IRB/2016/08/03), France. NeoLIC study was approved by the ethical committee in Madagascar (N° 190-MSANP/SG/AGMED/CERBM) and by the institutional review board (IRB2020-010) of Institut Pasteur, France. Written informed consent was given by every participant.

### Funding and role of the funding source

NeoLIC study was funded through 'transversal research programs' of the Institut Pasteur, Paris, France (PTR 303-20). The research project BIRDY2 was implemented with the financial support of the Monegasque co-operation for development. PEECNIC study was supported by MSDAVENIR. This study has also been funded by a grant 2024 from the European Society of Clinical Microbiology and Infectious Diseases to ALB.

The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.
## Acknowledgments

We sincerely thank all study participants, and all the members of the BIRDY Study Group: Rasoanaivo Fanjalalaina Vololonirina, Andrianonimiadana Lova Maminirina (in memoriam), Rabearitiana Lydos, Rasoloson Dimitri, Volahasina Antsa Tanjona, Volahasina Antso Fenitra, Randriamamonjiarison Aina Nirina, Andriatahina Todisoa.ALB acknowledges the support of the FRM (Fondation pour la Recherche Médicale, FDM202206015393) and the CMIT (Collège des Maladies Infectieuses et Tropicales) for funding her doctorate. We thank Nathalie de Rekeneire and Bunnet Dim for their valuable help, the Mutualized Platform for Microbiology (P2M) at Institut Pasteur for genome sequencing, and the IT department at Institut Pasteur for providing computational and storage services. Special thanks to Myriam Landau for her contribution to the BIRDY study.

## Contributors

BTH supervised the overall project. BTH, DG, EKD, JMC, ADL and MV designed and implemented the study. PH, MANR, NFR, SB, LP, LB, ADL, ALB and TC took part in data collection, microbiological experiments planning, realization and analyses. LF performed long-read sequencing. AC, LF and ALB performed the bioinformatic analyses. ADL and ALB performed data management. ALB performed statistical analysis and drafted the first version of the manuscript. All authors contributed to the final version of the manuscript.

## Declaration of interests

We declare no competing interests.

## Data sharing statement

The raw sequencing reads and genome assemblies have been deposited in the European Nucleotide Archive (ENA) repository under the BioProject accession number PRJEB85868. Detailed run and GenBank/EMBL/DDBJ accession numbers are provided in **Supplementary Table 2**. Researchers wishing to ask further questions are invited to contact the corresponding author.

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## Table

<caption>**Table 1. Population characteristics stratified by study project.**</caption>

Notably, the study included two pairs of twins, leading to a total of two more neonates than mothers. The variable "neonatal systemic antibiotic administration" excludes antibiotics administered via the ophthalmic route.

ESBL-PE: Extended-Spectrum Beta-Lactamase-producing Enterobacterales.
<table><thead><tr><th></th><th>N</th><th>Cambodia<br>(2016-17)<sup>1</sup></th><th>Madagascar<br>(BIRDY 2019-20)<sup>1</sup></th><th>Madagascar<br>(NeoLIC 2021-22)<sup>1</sup></th></tr></thead><tbody><tr><td>- MOTHERS / HOUSEHOLDS -</td><td></td><td>131</td><td>299</td><td>67</td></tr><tr><td>Setting</td><td>497</td><td></td><td></td><td></td></tr><tr><td>Rural</td><td></td><td>76 (58%)</td><td>203 (68%)</td><td>67 (100%)</td></tr><tr><td>Urban</td><td></td><td>55 (42%)</td><td>96 (32%)</td><td>0 (0%)</td></tr><tr><td>Age of the mother at inclusion</td><td>497</td><td>27.0 (24.0, 31.0)</td><td>24.7 (20.4, 29.8)</td><td>25.7 (22.0, 30.6)</td></tr><tr><td>Latrine localisation</td><td>489</td><td></td><td></td><td></td></tr><tr><td>inside</td><td></td><td>67 (51%)</td><td>26 (8.9%)</td><td>3 (4.5%)</td></tr><tr><td>outside</td><td></td><td>64 (49%)</td><td>265 (91%)</td><td>64 (96%)</td></tr><tr><td>Number of household members</td><td>497</td><td>5 (3, 6)</td><td>4 (3, 5)</td><td>3 (3, 4)</td></tr><tr><td>Housing with electricity</td><td>497</td><td>130 (99%)</td><td>222 (74%)</td><td>49 (73%)</td></tr><tr><td>Education of the mother</td><td>497</td><td></td><td></td><td></td></tr><tr><td>primary or none</td><td></td><td>81 (62%)</td><td>53 (18%)</td><td>13 (19%)</td></tr><tr><td>secondary</td><td></td><td>35 (27%)</td><td>219 (73%)</td><td>41 (61%)</td></tr><tr><td>higher</td><td></td><td>15 (11%)</td><td>27 (9.0%)</td><td>13 (19%)</td></tr><tr><td>Profession of the mother</td><td>497</td><td></td><td></td><td></td></tr><tr><td>paid job</td><td></td><td>91 (69%)</td><td>125 (42%)</td><td>34 (51%)</td></tr><tr><td>unemployed or student</td><td></td><td>40 (31%)</td><td>174 (58%)</td><td>33 (49%)</td></tr><tr><td>Primigravidae</td><td>497</td><td>51 (39%)</td><td>106 (35%)</td><td>26 (39%)</td></tr><tr><td>Maternal antibiotic intake during pregnancy</td><td>497</td><td>2 (1.5%)</td><td>31 (10%)</td><td>18 (27%)</td></tr><tr><td>Delivery location</td><td>495</td><td></td><td></td><td></td></tr><tr><td>home</td><td></td><td>3 (2.3%)</td><td>140 (47%)</td><td>0 (0%)</td></tr><tr><td>primary healthcare center</td><td></td><td>21 (16%)</td><td>116 (39%)</td><td>65 (100%)</td></tr><tr><td>hospital/clinic</td><td></td><td>107 (82%)</td><td>43 (14%)</td><td>0 (0%)</td></tr><tr><td>Cesarean section</td><td>497</td><td>17 (13%)</td><td>36 (12%)</td><td>0 (0%)</td></tr></tbody></table>
<table><thead><tr><th></th><th></th><th colspan="3" style="text-align: center;">Madagascar</th></tr><tr><th></th><th style="text-align: center;">N</th><th style="text-align: center;">Cambodia<br>(2016-17)<sup>7</sup></th><th style="text-align: center;">Madagascar<br>(BIRDY 2019-20)<sup>7</sup></th><th style="text-align: center;">Madagascar<br>(NeoLIC 2021-22)<sup>7</sup></th></tr></thead><tbody><tr><td style="text-align: left;">- MOTHERS / HOUSEHOLDS -</td><td style="text-align: center;"></td><td style="text-align: center;">131</td><td style="text-align: center;">299</td><td style="text-align: center;">67</td></tr><tr><td style="text-align: left;">Perpartum antibiotic administration</td><td style="text-align: center;">485</td><td style="text-align: center;">26 (20%)</td><td style="text-align: center;">42 (14%)</td><td style="text-align: center;">40 (60%)</td></tr><tr><td style="text-align: left;">Maternal colonization with ESBL-PE</td><td style="text-align: center;">497</td><td style="text-align: center;">104 (79%)</td><td style="text-align: center;">102 (34%)</td><td style="text-align: center;">37 (55%)</td></tr><tr><td style="text-align: left;">- NEONATES -</td><td style="text-align: center;">499</td><td style="text-align: center;">131</td><td style="text-align: center;">301</td><td style="text-align: center;">67</td></tr><tr><td style="text-align: left;">Sex of the baby</td><td style="text-align: center;">499</td><td style="text-align: center;"></td><td style="text-align: center;"></td><td style="text-align: center;"></td></tr><tr><td style="text-align: left; padding-left: 20px;">Female</td><td style="text-align: center;"></td><td style="text-align: center;">73 (56%)</td><td style="text-align: center;">143 (48%)</td><td style="text-align: center;">34 (51%)</td></tr><tr><td style="text-align: left; padding-left: 20px;">Male</td><td style="text-align: center;"></td><td style="text-align: center;">58 (44%)</td><td style="text-align: center;">158 (52%)</td><td style="text-align: center;">33 (49%)</td></tr><tr><td style="text-align: left;">Low-birth weight (&lt;2500g)</td><td style="text-align: center;">492</td><td style="text-align: center;">4 (3.1%)</td><td style="text-align: center;">17 (5.8%)</td><td style="text-align: center;">3 (4.5%)</td></tr><tr><td style="text-align: left;">Neonatal resuscitation</td><td style="text-align: center;">498</td><td style="text-align: center;">10 (7.6%)</td><td style="text-align: center;">39 (13%)</td><td style="text-align: center;">13 (19%)</td></tr><tr><td style="text-align: left;">Neonatal systemic antibiotic administration</td><td style="text-align: center;">499</td><td style="text-align: center;">6 (4.6%)</td><td style="text-align: center;">12 (4.0%)</td><td style="text-align: center;">5 (7.5%)</td></tr><tr><td style="text-align: left;">Neonatal colonization with ESBL-PE</td><td style="text-align: center;">499</td><td style="text-align: center;">70 (53%)</td><td style="text-align: center;">79 (26%)</td><td style="text-align: center;">21 (31%)</td></tr></tbody></table>

Figures

<footnote>7n (%); Median (Q1, Q3)</footnote>
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

Study flow-chart.

ESBL+: colonized with extended-spectrum beta-lactamase (ESBL-)producing Enterobacterales.
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>

Phylogenetic trees and identified clones within mother-neonate pairs.

Maximum likelihood phylogenetic trees displaying all the isolates belonging to a mother-neonate pair both colonized by a common species, either *Escherichia coli*, *Klebsiella quasipneumoniae*, *Enterobacter kobei* or "*Enterobacter intestinihominis*". Blue and green leaves indicate *Cambodia* and *Madagascar*, respectively. Empty and filled circles correspond to mother and neonate, respectively. Red leaves indicate the type strain of the species, whereas orange leaves indicate the representative strains of the 14 *E. coli* phylogroups A, B1, B2-1, B2-2, C, D1, D2, D3, E1, E2, F, G, *Shig1* and *Shig2*, as identified by Abram et al⁷⁰. Genetically identical isolates within a pair are highlighted in sand color. Bars indicate the number of nucleotide substitutions per character.
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>

Alignment of maternal and neonatal plasmids in five pairs.

Alignments of ESBL-encoding-genes-carrying plasmids in mother-neonate pairs where identical plasmids were observed. Cambodian and Malagasy plasmid pairs are displayed on the upper (a, b, c) and bottom (d, e) panels, respectively. For each pair, inner purple ring represents the fully circularized plasmid sequence of one mate, whereas outer green ring shows the shared nucleotide identity (according to BLASTn) with the assembly of the other mate. Nucleotide identity percentage is indicated at the upper right corner of plasmid pair. ESBL-encoding genes are indicated in red; other antibiotic resistance genes are indicated in black. The pair identification number, plasmid incompatibility group (Inc) and plasmid size (in bp) are indicated at the center of each alignment. The image was prepared using Blast Ring Image Generator (sourceforge.net/projects/brig)
<figure><img src="image_5.png" /><figcaption>Figure 4</figcaption></figure>

Multinomial regression models.

Forest plot displaying adjusted odds ratios (aOR) for ESBL-PE acquisition attributed to maternal source (left panel) or attributed to other sources (right panel) to no acquisition (reference group). Error bars represent 95% confidence intervals. The aOR is shown as a blue-filled dot when significantly different from 1 (with the estimated value displayed above the dot) and as a white-centered dot when not statistically significant. The absence of error bars indicates the reference category for a variable. The model was performed using complete cases, including 472 pairs.
<figure><img src="image_6.png" /><figcaption>Figure 5</figcaption></figure>

Sankey diagram.

For the 501 bacterial isolates included in the genotypic analysis, this diagram represents the link
between the origin of the sample (neonate or mother), the genus of the bacterial species identified in
silico, the ESBL-encoding gene detected on the isolate, and the country. Of note, only one ESBL-encoding
gene per isolate is displayed.

Supplementary Files

This is a list of supplementary files associated with this preprint. Click to download.

* SupplementaryTable2.xlsx

* SupplementaryMaterial.docx