Transmission dynamics of carbapenemase-producing bacteria and mobile genetic elements between hospital and household settings in Benin

doi:10.21203/rs.3.rs-7220165/v1

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Research Article

Transmission dynamics of carbapenemase-producing bacteria and mobile genetic elements between hospital and household settings in Benin

https://doi.org/10.21203/rs.3.rs-7220165/v1

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Background

Patients can acquire resistance to critical carbapenemase-producing bacteria during hospitalization and may transmit them to household contacts after discharge. The present study aimed to assess the transmission of carbapenemase-producing bacteria from hospitals to households through patients and their caregivers.

Methods

A total of 248 participants were included in this study across 5 hospitals, comprising 81 index patients, 81 caregivers, 58 healthcare workers, and 28 household members. Rectal swabs samples were collected from the participants from admission to discharge and during follow-up at home. Samples were plated on mSuperCARBA™, and obtained isolates were tested for carbapenem resistance. A subset of 41 carbapenem resistant isolates were selected and subjected to Whole Genome Sequencing (WGS) and hybrid assembly analysis to determine genetic relatedness and resistance determinants across sampling networks.

Results

A total of 412 isolates were recovered from hospital and household settings. Among these, 141 isolates were resistant to carbapenem, predominantly E. coli (40.42%), K. pneumoniae (19.14%), and A. baumannii (10.63%). For those enrolled at hospital, the prevalence of carbapenem-resistant bacterial colonization increased from 7.3% (16/220) at admission to 22.7% (10/44) by day 8 of hospitalization. Among patients or caregivers that were still positive at discharge, the prevalence at home rose from 8.0% (7/87) on day 0 of discharge to 57.1% (8/14) by day 14 of follow-up. Long- and short-read sequencing identified the carbapenem resistance genes blaNDM-5 and blaNDM-1 as the main determinants found in Escherichia coli ST410 and Klebsiella pneumoniae ST147, respectively. Several strains, including Klebsiella pneumoniae (ST147) and Escherichia coli (ST410), showed genetic relatedness and potential transmission within households and between healthcare and home settings. IncFII and IncFI conjugative plasmids carried most carbapenemase genes in healthcare and community settings representing another layer of resistance spread via horizontal gene transfer.

Conclusion

This study reveals, for the first time in Benin, the role of patients and their caregivers in facilitating the spread of antimicrobial resistance between hospitals and the community.

Carbapenemase producing bacteria

Healthcare

Community

Benin

Hospitals serve as hotspots for the emergence and transmission of multidrug-resistant organisms (MDROs), which can colonize patients before or during hospitalization. This colonization increases the risk of subsequent infections and facilitates the dissemination of antimicrobial resistance into the community post-discharge [1]. Healthcare workers and the hospital environments contribute to the spread of antibiotic-resistant bacteria, leading to hospital-acquired infections (HAIs) [2]. Contaminated hands, inadequately disinfected medical instruments, and frequently touched surfaces act as key reservoirs for MDR bacteria, posing risks to patients, staff, and the broader community [3].

Beyond hospital settings, colonized patients act as reservoirs for for MDROs including carbapenem-resistant bacteria (CRB), facilitating household transmission and sustaining the cycle of spread and infection [4]. While carbapenemase-producing bacteria have been detected in the community, data on their transmission dynamics and risk factors in household settings remain limited [5, 6]. Discharged patients and their caregivers, if still colonised, can transmit CRB, particularly in households where close contact is frequent (7). Notably, even short hospital stays (1–5 days) can lead to persistent colonization lasting months or years in the absence of antimicrobial therapy, further increasing the risk of community dissemination [8, 9].

In West Africa, systematic CRB surveillance is lacking, but regional prevalence averages 4.6%, with reported rates ranging from 1.6–18.6% [10]. In Benin, CRB prevalence was estimated 8.1% (64/790) in patients, yet comprehensive surveillance systems for HAIs are absent, and data on CRB transmission dynamics remain sparse [11]. Existing studies in Benin are small-scale and do not examine hospital-to-household transmission of bacterial resistance. Addressing this knowledge gap is essential for designing effective containment strategies in both healthcare and community settings.

This study investigates the role of patients and caregivers in CRB transmission from hospitals to households. It further analyses the genetic relatedness of carbapenem-resistant bacterial isolates and identifies mobile genetic elements involved in resistance spread.

Study design and sampling procedure

This study was conducted from January to March 2023 in five hospitals in Benin. All newly admitted patients in surgical or pediatric wards who provided informed consent were included in the study. These wards were targeted because those patients, children and others with limited mobility capacity, mostly require a caregiver (a relative, who stayed in the patient’s room, assisted with personal care, and prepared meals) to assist them during hospitalization. Patients in other wards or those who refused consent were excluded. Healthcare workers (nurses and medical doctors) directly involved in patient care and consenting relatives in contact with CRB-positive patients or caregivers were also included.

Rectal swabs were systematically collected from patients, caregivers, and healthcare workers at five time points: on admission (D0), and then every 48 hours (D2, D4, D6, and D8) until discharge. For Patients who tested positive at least once during hospitalization and consented to be followed-up, additional samples were collected at their household level from index patients, caregivers and their household contacts (Fig. 1). Sampling in the household was conducted over two weeks, with swabs collected on the day of return (D0), 48 hours later (D2), on day 7 (D7), day 9 (D9), and day 14 (D14) post-discharge. All follow-up sampling was performed directly by the research team under strict aseptic conditions.

Informed written consent was obtained from all participants. The study was approved by the Ethics and Research Committee of the Institute of Applied Biomedical Sciences (CER-ISBA) under reference number 154.

Microbiological analysis

Rectal swabs were plated on mSuperCARBATM™ (CHROMagar™, Paris, France) and incubated at 37°C for 18–24 hours. Based on colony color and morphology, distinct bacterial colonies were subcultured on MacConkey agar and incubated under the same conditions, repeating until pure cultures were obtained. A total of 981 samples were collected from 248 participants, yielding 412 pure culture isolates on mSuperCARBATM™ selective media. All isolates underwent species identification using MALDI-TOF and antimicrobial susceptibility testing (AST) with two carbapenem discs: imipenem (10 µg) and ertapenem (10 µg) following the guidelines of the European Committee on Antimicrobial Susceptibility Testing [12]. At this stage, 141 isolates were found to be resistant to at least one of the tested carbapenems. From this pool, 41 isolates were selected for whole genome sequencing based on species identity, collection time points, and participant origin criteria that could help infer potential transmission events and map possible resistance networks among individuals. These selected isolates were subsequently tested for the carbapenem inactivation method (CIM) using meropenem discs (10 µg) [13] to confirm carbapenemase production prior to sequencing. E. coli ATCC 29522 served as a negative control, while a clinical strain of E. coli harboring the bla_NDM−1 gene was used as a positive control.

Whole Genome Sequencing and Bioinformatic Analysis

DNA was extracted from the isolates using the Qiagen DNeasy kit, following the manufacturer’s instructions. Short-read whole genome sequencing (WGS) was performed on the Illumina NovaSeq platform, as previously described [14]. Sequence reads were quality-checked with FastQC (v0.11.9) [15], trimmed with Fastq [16], and assembled using SPAdes (v3.15.5) [17]. The quality of assembled genomes was assessed using QUAST [18], while taxonomic classification was performed with Kraken2 (v2.14) [19], and cross-verified with GTDB-Tk (v2.4.0) [20]. Multi-locus sequence typing (MLST) was conducted using pubMLST (v.2.23) [21] after species confirmation. Antimicrobial resistance (AMR) genes were identified using Abricate (v1.0.1) [22] with the ResFinder 4.1 [23] database for acquired resistance, while the web version of ResFinder was used to detect resistance conferred by chromosomal mutations. Plasmid replicons were identified using Abricate with the PlasmidFinder 2.1 database [24].

For each species group of interest, core-genome SNP-based phylogenetic analysis was conducted to assess genetic relatedness between isolates obtained at hospital and household levels at different type points. We applied a SNP threshold of ≤ 30 SNPs to infer close genetic relatedness and potential transmission events, aligning with thresholds commonly reported in the literature for Enterobacteriaceae and Acinetobacter. Genome alignment and variant calling were performed using Snippy (v4.6.0) [25], followed by recombinant removal with Gubbins (v2.4.1) [26]. Phylogenetic trees were reconstructed using RAxML (v8.2.12) [27] with 200 bootstrap replicates and visualized in iTOL 3 [28].

After initial short-read analysis, six isolates (E. coli, A. baumannii and K. pneumoniae) suspected to be part of a transmission network were selected for long-read sequencing using Oxford Nanopore technology to obtain fully assembled genomes for comprehensive analysis of mobile genetic elements. These isolates originated from both hospital and community settings and were recovered across different time points. Genomic analysis revealed that they shared key genotypic characteristics, including resistance gene profiles, plasmid content, and sequence types, with other isolates suspected to be part of the same transmission cluster. Hybrid assembly and plasmid reconstruction were performed using both long- and short-read data, as previously described [14]. The genetic context of carbapenemase-encoding genes on the reconstructed plasmids was examined in all complete genomes, with contigs containing these genes following our previously described protocol [14].

To put our strains into a regional context, (West Africa) phylogenetic analyses were performed focusing on three key species: Acinetobacter baumannii bla_NDM−1, Escherichia coli ST410 bla_NDM−5, and Klebsiella pneumoniae ST147 bla_NDM−5. These strains were compared with publicly available genomes from West Africa, incorporating metadata on country of origin, year of isolation, and sample type to evaluate genetic relatedness and evolutionary trends.

All bioinformatic analysis was performed using customized scripts on the Danish National Life Science high performance computer cluster, Computerome2.

Distribution of Carbapenem-Resistant Bacteria across hospital and household setting

A total of 248 participants were included in this study comprising 81 index patients (65 admitted to pediatric wards and 16 to surgical wards) and 81 matched caregivers. Additionally, 58 healthcare workers and 28 index household members participated. Combining hospital and household phases of the study, we obtained a total of 412 isolates from the msupercarba plates, with 179 from patients, 140 from caregivers, 50 from healthcare workers and 43 from household members during follow-ups. The presumptive species from the selective plate culturing were Escherichia coli (n = 161 isolates), Klebsiella pneumoniae (n = 87 isolates), Acinetobacter baumannii (n = 58 isolates), Stenotrophomonas maltophilia (n = 47 isolates), Enterobacter cloacae (n = 28 isolates), Pseudomonas putida (n = 23 isolates), Delftia acidovorans (n = 6 isolates) and Achromobacter xyloxidans (n = 2 isolates).

The prevalence of carbapenem-resistant bacteria (CRB) in this study is calculated with the number of isolates that grew on selective CRB plates and confirmed resistant to at least one of the carbapenem drugs tested. CRB colonization was very high in the studied population. A total of 141 isolates were found to be resistant to at least one of the tested carbapenems. The predominant CRB species were Escherichia coli (40.42%), Klebsiella pneumoniae (19.14%) and Acinetobacter baumannii (10. 63%).

Prevalence of carbapenem-resistant bacteria colonisation in hospital

Of the 220 participants enrolled at hospitals, on their day of admission (D0), only 62 remained under follow-up by day 4, as the majority were discharged early. The overall prevalence of CRB at the hospital, increased from 7.3% on D0 to 22.7% by D8 (Table 1). A total of 81 carbapenem resistant isolates were found in samples collecting in hospital setting. The predominant CRB species were Escherichia coli (47.03%), Klebsiella pneumoniae (26.04%) and Acinetobacter baumannii (7.40%).

Specifically, the prevalence of CRB among patients ranged from 11.11% (9/81) on admission to 26,31% (5/19) by D8 of hospitalization (Table 1). However, in a few instances like on day 6, some patients were unavailable for sampling but were sampled again on day 8 (Table 1). A total of 45 CRB were isolated from patients during hospitalization. The predominant CRB species were Escherichia coli (n = 20 isolates), Klebsiella pneumoniae (n = 12 isolates) and Acinetobacter baumannii (n = 3 isolates).

Among caregivers CRB colonization evolved from 1.23% (1/81) at admission to 18.75% (3/16) by day 8. Similar to the case of patients, some caregivers skipped sampling on day 6 and were sampled again on day 8 (Table 1). A total of 20 CRB were isolated from caregivers during their stay in hospital. The predominant CRB species were Escherichia coli (n = 11 isolates), Klebsiella pneumoniae (n = 4 isolates) and Acinetobacter baumannii (n = 2 isolates).

The number of sampled healthcare workers dropped over time due to their scheduled shifts. However, based on consistent follow-up of the same healthcare workers that were available from day 0 to day 8, the prevalence of CRB increased from 10.34% (6/58) to 22.22% (2/9) on day 8 (Table 1). A total of 16 CRB were isolated from caregivers during their stay in hospital. The predominant CRB species were Escherichia coli (n = 8 isolates), Klebsiella pneumoniae (n = 6 isolates) and Acinetobacter baumannii (n = 1 isolates).

Table 1
Distribution of participants carrying confirmed carbapenem-resistant bacteria (CRB) by sampling day at the hospital
Days	Patients carrying CRB	Caregivers carrying CRB	Healthcare workers carrying CRB	Number of participants carrying CRB (%)
D0	9/81	1/81	6/58	16/220 (7.3)
D2	12/79	6/78	3/47	21/204 (10.3)
D4	3/22	3/25	0/15	6/62 (9.7)
D6	2/11	3/11	0/12	5/34 (14.7)
D8	5/19	3/16	2/9	10/44 (22.7)

Prevalence of carbapenem-resistant bacteria colonisation in household

At discharge, 30 patients had tested positive at least once during hospitalization. Of these, 29 patients and 28 household contacts consented to home follow-up. The corresponding 30 caregivers were also included in the household follow-up phase. The overall prevalence of CRB colonization at home on D0 of return was 8% increasing to 57.1% by D14 during the follow-up (Table 2). A total of 60 carbapenem resistant isolates were found in samples collecting in household setting. The species at home were consistent with those that were detected in hospital setting i.e. Escherichia coli (30%), Acinetobacter baumannii (15%) and Klebsiella pneumoniae (8.33%).

Specifically, the prevalence of CRB among patients at household ranged from 13.79% (4/29) on admission to 40% (2/5) by D14 (Table 2). It is important to note that some patients were unavailable for sampling at certain time points during the 14-day follow-up. A total of 27 CRB were isolated from patients at household. The predominant CRB species were Escherichia coli (n = 9 isolates), Acinetobacter baumannii (n = 5 isolates) and Klebsiella pneumoniae (n = 2 isolates).

Among caregivers CRB colonization reached 60% (3/5) by day 14. As observed with patients, some caregivers were not available for sampling at certain time points during the 14-day follow-up period (Table 2). A total of 14 CRB were isolated from caregivers at household. The predominant CRB species were Escherichia coli (n = 5 isolates), Klebsiella pneumoniae (n = 2 isolates) and Acinetobacter baumannii (n = 2 isolates).

At the household level, 10.71% (3/28) of household contacts were already colonized with CRB on the day the index patient or caregiver returned home. This proportion increased to 75% (3/4) by day 14 (Table 2). Among the 25 household members who initially tested negative upon the return of a positive patient or caregiver, 6 became colonized with CRB over time. A total of 19 CRB were isolated from household contact the follow up. The predominant CRB species were Escherichia coli (n = 4 isolates), Acinetobacter baumannii (n = 2 isolates) and Klebsiella pneumoniae (n = 1 isolate).

Table 2
Distribution of participants carrying carbapenem-resistant bacteria (CRB) by sampling day in households.
Day	Patients carrying carbapenem resistant bacteria	Patients Guards carrying carbapenem resistant bacteria	Household contacts carrying carbapenem resistant bacteria (total)	Number of participants carrying carbapenem resistant bacteria (total)
D0	4/29	0/30	3/28	7/87 = 8%
D2	6/16	4/14	3/25	13/55 = 23.6%
D7	5/21	4/23	4/21	13/65 = 20%
D9	1/10	1/13	1/5	3/28 = 10.7%
D14	2/5	3/5	3/4	8/14 = 57.1%

Distribution of the bacterial species by whole genome sequencing (WGS)

A total of 141 isolates were resistant to one of the carbapenem tested, from which 41 were selected for short-reads WGS to support the clinical study. The selection was made to represent each sampling group and potential species sharing cases identified above. Before sequencing the 41 isolates were further confirmed by meropenem inactivation test and all tested positive. Among sequenced genomes, Escherichia coli was the most prevalent species (34.1%, n = 14), followed by Klebsiella pneumoniae (29.3%, n = 12) and Acinetobacter baumannii (14.6%, n = 6). Stenotrophomonas maltophilia (14.6%, n = 6) were found to be sequenced and were excluded for further analysis.

Distribution of Resistance genes

All sequenced isolates, except Pseudomonas putida, carried at least one carbapenem or cephalosporin resistant genes (Table 3). Among Escherichia coli isolates, bla_NDM−5 (n = 6) and bla_OXA−181 (n = 6) were the most frequently carbapenemase genes. Klebsiella pneumoniae (n = 10) and Acinetobacter baumannii (n = 5) predominanly harbored bla_NDM−1. The bla_VIM−5 gene was found exclusively in Achromobacter xyloxidans (n = 2). The most commonly detected ESBL gene was bla_CTX−M−15 (n = 16).

Table 3
Distribution of carbapenemase and ESBL resistance genes among sequenced isolates.
Bacterial species (n)	blaNDM-1	blaNDM-5	blaOXA-181	blaOXA-like	blaVIM-5	blaCTX-M-15	CMY-2	blaCTX-M-36
Escherichia coli (14)	0	6	6	0	0	4	11	2
Klebsiella pneumoniae (12)	10	1	0	1	0	12	0	0
Acinetobacter baumannii (6)	5	0	0	3	0	0	0	0
Achromobater xylosoxidans (2)	0	0	0	0	2	0	0	0
Pseudomonas putida (1)	0	0	0	0	0	0	0	0

Genetic characteristics of carbapenem-resistant isolates harbouring plasmids

The most represented sequence types (STs) were Escherichia coli ST410 (n = 11) and Klebsiella pneumoniae ST147 (n = 10). Among Acinetobacter baumanii isolates, ST860 (n = 3) was the most represented lineage (Table 4).

Table 4
Distribution of sequence types among selected sequenced genomes.
Bacterial isolates	ST410	ST167	ST657	ST147	ST967	ST11	ST860	ST1463	ST107
Escherichia coli (14)	11	2	1	0	0	0	0	0	0
Klebsiella pneumoniae (12)	0	0	0	10	1	1	0	0	0
Acinetobacter baumannii (5)	0	0	0	0	0	0	3	1	1

Among the sequenced isolates, Escherichia coli and Klebsiella pneumoniae were the only species harboring plasmids. All Escherichia coli ST410 (n = 11) isolate harboured the IncFII plasmid type. None Acinetobacter baumanii isolates carried plasmids (Table 5).

Table 5
Plasmid replicons present in selected sequenced genomes.
	IncFII	IncX3	IncXI	IncR	IncFII		IncFIB	IncHI1B	IncL/M
Escherichia coli (14)	11	1	1	1		0	0	0	0
Klebsiella pneumoniae (12)	0	0	0	0		2	8	1	1

Genetic context of carbapenem resistance genes

Hybrid assemblies (Fig. 2) illustrate the genetic context of carbapenemase genes in Escherichia coli (panel a); Acinetobacter baumannii (panel b) and Klebsiella pneumoniae (panel c). These analyses reveal that plasmids of the same IncF family are responsible for mobilizing bla_NDM genes in E. coli and K. pneumoniae isolates from both hospitals and household environments. In Acinetobacter baumanii, where carbapenem resistance is chromosomally encoded, bla_NDM genes are located within an insertion sequence (ISAba 125) carrying the composite transposon Tn125 (Fig. 2, Panel C).

Hybrid genome assemblies were further used to reconstruct the genetic context of the two representative carbapenemase-carrying plasmids (Fig. 3). The predominant plasmid types mediating dissemination in both healthcare and community settings were IncF types (IncFII for bla_NDM−5 and IncFI for bla_NDM−1). These carbapenemase genes were concentrated in genome regions co-localized with multiple antimicrobial resistance determinants.

Genomic transmission networks

We reconstructed the transmission networks of carbapenemase-producing bacterial strains identified in this study (Fig. 4).

Three Acinetobacter baumannii ST860 strains carrying the bla_NDM−1 gene were isolated from a discharged patient (C22), their caregiver (C26, initially negative upon the patient’s return home), and a household contact (C24, also initially negative) (Fig. 4a). The caregiver and household contact tested positive two days after the index patient’s return. However, SNP analysis (≥ 30) did not confirm their genetic relatedness.

Persistent colonization was observed in patient 3 from hospital admission (Day 0) through Day 14 at home (M33, M50, M51, and M69) (Fig. 4b). Additionally, three K. pneumoniae ST147 isolates were recovered from the patient’s caregiver between Day 2 and Day 14 at home (M34, M53, and M70). SNP analysis revealed ≤ 30 differences between these genomes, indicating a closely related genetic background and suggesting that the strain was introduced into the household by the discharged patient.

A potential transmission event was identified from the caregiver of patient 1 on Day 7 in the hospital (L62) to patient 1 on Day 2 at home after discharge (L71) (Fig. 4c). Core-genome SNP analysis revealed approximately 20 SNP differences, confirming a closely related genetic background and supporting the transmission of E. coli ST410 from hospital to household.

A potential transmission event of E. coli ST410 was also observed from the caregiver of patient 3 on Day 4 at home (T18), persisting in the caregiver until Day 9 (T34), and subsequently detected in both patient 3 and their household contact on Day 9 (T33, T34) with ≤ 20 SNP differences (Fig. 4d). Patient 3 was found colonized with this strain on Day 2 of hospitalization, and the isolate was confirmed via long-read sequencing. Given the timeline, this event suggests community-level transmission of a multidrug-resistant clone.

Whole genome-based phylogenetic tree

Figure 5 presents core-genome phylogenetic analyses of Acinetobacter baumannii (Fig. 5a), Escherichia coli ST410 (Fig. 5b), and Klebsiella pneumoniae ST147 (Fig. 5c). These analyses illustrate phylogenetic clustering based on country of origin, year of isolation, and sample type. Newly sequenced strains are compared with publicly available genomes carrying similar genetic markers (bla_NDM−1 or bla_NDM−5), providing insights into their genetic relationships and potential transmission pathways. The blue shading in Fig. 5 indicates the clade of isolates from this study.

Two distinct phylogenetic clusters were observed for A. baumannii carrying bla_NDM−1. One cluster represented strains from Benin, which were genetically distinct from Nigerian isolates, suggesting localized evolutionary divergence (Fig. 5a). These strains were isolated from diverse clinical samples (blood, rectal swabs, and wound samples), indicating a consistent infection source within the region but clear geographical differentiation.

E. coli ST410 carrying bla_NDM−5 demonstrated phylogenetic closeness between strains from this study and those from Nigeria, forming a tightly clustered lineage (Fig. 5b). This suggests regional dissemination of a shared genetic lineage. Samples were obtained from urine, rectal swabs, and wounds across multiple years (2020–2023), indicating the sustained circulation of this lineage.

K. pneumoniae ST147 carrying bla_NDM−5 formed a distinct cluster positioned between Nigerian and Ghanaian isolates, suggesting intermediate evolutionary traits (Fig. 5c). Compared to older regional isolates, the strains in this study demonstrated closer phylogenetic relationships for strains, indicating potential recent transmission events.

The overall prevalence of carbapenem-resistant bacteria (CRB) colonization in the study population was 7.3% at hospital admission (D0), rising to 22.7% by D8. These findings align with studies conducted in Turkey and Kenya, where the prevalence of CRB carriage at admission was 12.5% [4, 29]. Similarly, in India, 6.6% of hospitalized patients were colonized with carbapenem-resistant bacteria [29]. However, our results contrast with the significantly higher prevalence (52%) reported among patients in Vietnamese hospitals [30], which may be attributed to differences in sample size, study design, and local infection control measures. The increasing prevalence of CRB colonization during hospitalization suggests nosocomial transmission, likely facilitated by direct patient-to-patient contact, healthcare worker-to-patient interactions, or contaminated fomites, particularly in settings with suboptimal infection prevention and control practices [31]. Furthermore, this study observed both acquisition and loss of CRB colonization over time, consistent with previous findings [7]. In the household setting, CRB colonization was detected in 8% of participants at D0, increasing to 57.1% by D14. This is notably higher than the 14.4% prevalence reported in a community study from Rawalpindi, Pakistan [32]. These findings suggest that community CRB carriage contributes to resistant strain spread in healthcare, highlighting the need for broader infection control strategies.

Based on presumptive species identification from selective mSuperCarba plates, a diverse range of Gram-negative bacterial species was recovered across hospital and household settings, totaling 412 isolates. The most frequently detected species were Escherichia coli (n = 161), Klebsiella pneumoniae (n = 87), and Acinetobacter baumannii (n = 58). Among the 141 isolates confirmed to be resistant to at least one of the tested carbapenems, the predominant species were E. coli (40.42%), K. pneumoniae (19.14%), and A. baumannii (10.63%). These proportions suggest that these three species were the major contributors to carbapenem resistance in the studied population. A subset of 41 isolates comprising predominantly Escherichia coli (34.1%), Klebsiella pneumoniae (29.3%), and Acinetobacter baumannii (14.6%) was selected for whole-genome sequencing. This subset was chosen to facilitate the investigation of potential transmission events and to characterize resistance dissemination networks across hospital and household environments. E. coli isolates harboring bla_NDM-5 and bla_OXA-181 were detected for the first time in Benin. The spread of bla_NDM-5 and bla_OXA-181-producing E. coli has also been reported in Chad [33]. Among K. pneumoniae and A. baumannii isolates, bla_NDM-1 was the most frequently detected carbapenemase gene, consistent with findings from South Africa [34]. Similarly, A. baumannii producing New Delhi metallo-β-lactamase-1 (NDM-1) had previously been identified in a clinical setting in Benin [35]. Furthermore, this study is the first to report the presence of the bla_VIM-5 gene in Achromobacter xylosoxidans isolates in Benin. Previously, carbapenem-resistant A. xylosoxidans carrying the blaVIM-2 gene had been reported only in a European setting [36]. The growing Africa-Europe travel likely spreads resistant strains beyond their original epidemiological contexts.

The transmission networks of carbapenemase-producing bacteria reveal complex dynamics, with both direct and indirect transmission occurring in close-contact settings. While some strains, such as Klebsiella pneumoniae ST147 and Escherichia coli ST410, exhibit clear genetic links indicative of household transmission, others, like Acinetobacter baumannii ST860, show high genetic diversity, suggesting multiple introduction events or independent transmission routes. These findings highlight the importance of genomic surveillance in detecting hidden carbapenem-resistant bacteria transmission, aiding outbreak investigations, and improving infection control [37].

Consistent with previous studies, this study confirms the persistence of colonization by resistant organisms even after patient discharge and highlights the occurrence of transmission in household settings [38]. The predominance of E. coli clone ST410 (n = 11) in this study, along with its detection in Ghana, suggests a broader clonal dissemination across West Africa [39]. Among K. pneumoniae isolates, ST147 (n = 10) was the most represented. The spread of NDM-1-producing K. pneumoniae ST147 has also been reported in Tunisian hospitals [40], emphasizing its potential role in hospital outbreaks in Benin. The widespread distribution of IncF-type plasmids harboring carbapenemase genes among hospitalized patients has been extensively documented [41, 42]. Previous studies have demonstrated a conserved genetic arrangement among NDM-producing isolates [43], with similar configurations reported across multiple countries and continents. This widespread conservation suggests that these plasmids, particularly in E. coli, contribute significantly to global antimicrobial resistance and predict resistance to a broad range of antibiotics [44].

A major limitation of this study lies in the substantial loss to follow-up, both within the hospital setting and after discharge at the household level. The primary reason for this loss is linked to the structure of patient care in the pediatric ward. Out of 81 patients enrolled, 65 were admitted to the pediatric unit where the average duration of stay was relatively short typically two to three days. As a result, many caregivers and family members who accompanied the children also left shortly after hospital day two. Furthermore, some samples could not be collected during hospitalization due to the patients' clinical conditions, leading to gaps in daily follow-up (for instance Day 6 and Day 8). In this study, Day 0 (D0) in the household context was defined as the day of hospital discharge. On that day, it was relatively easy to collect samples from the patients who had tested positive, as they were still available and accessible. However, follow-up at home proved more challenging. Household-level follow-up was dependent on the availability and willingness of the discharged patient and their household contacts. To mitigate this limitation, the follow-up period was extended from the initially planned 7 days to 14 days, in an attempt to improve the chances of reaching and sampling household contacts. Despite this adjustment, the follow-up coverage remained insufficient, and data completeness was not achieved. Finaly, for the household phase, the study focuses on patient and caregiver transmission, omitting healthcare workers as potential vectors of transmission from heathcare to community settings. This was because we did not have healthcare workers consent to follow them up at household levels and this should be further considered in future studies.

The findings highlight the role of patients in facilitating the spread of carbapenem-resistant bacteria between healthcare settings and the community. Genomic surveillance is crucial to mapping transmission and understanding resistance dynamics. Ongoing monitoring and targeted interventions are important for control and public health.

AST: Antimicrobial Susceptibility Testing

CMY: Cephamycinase

CRB: Carbapenem-Resistant Bacteria

CTX-M: Cefotaximase-Munich

HAIs: Hospital-Acquired Infections

ISAba125: Insertion Sequence Aba125

MDROs: Multidrug-Resistant Organisms

NDM: New Delhi Metallo-β-lactamase

Oxa: Oxacillinase

STs: Sequence Types

Tn125: Transposon 125

VIM: Verona Integron-encoded Metallo-β-lactamase

Acknowledgments

The authors would like to express their sincere gratitude to Arielle Kounou, Lydie Comlan, Lidwine Aidodo, Belvida Houndonougbo, Claudiane Adigbonon for their invaluable assistance during the implementation of this study. We also thank all the staff of the participating hospitals for their cooperation and support in ensuring the best possible care for the patients. Lastly, we appreciate the participation of all study participants.

Author Contributions

V.D., Y.M.G.H., and K.S wrote the protocol. K.S., B.L., B.H., K.F and H.K., collected and processed the samples. Y.M.G.H., K.S., B.H and B.L., did the bioinformatics analyses. K.S., Y.M.G.H., V.D., and A.D wrote the draft of the manuscript, Y.M.G.H, V.D and A.D. provided funding.

Funding

This study received support from existing funding at the University of Copenhagen, including the following projects: The Danish International Development Assistance (DANIDA) funded projects “Health and Antibiotics in Vietnamese Pig Production” (grant DFC file 17-M06-KU) and “Salmonella Control in the Colombian Pig Industry” (grant DFC file 18-M07-KU); and the Innovation Fund Denmark (IFD) under the umbrella of the JPIAMR (Joint Programming Initiative on Antimicrobial Resistance, Research Project: 2022-02-15 - 2025-02-14).

Data availability

The sequenced genomes (both Illumina and Nanopore) that support the findings of this study have been deposited in the European Nucleotide Archive under the project accession number PRJEB85980.

Ethical approval and Consent to Participate

The study was approved by the Ethics and Research Committee of the Institute of Applied Biomedical Sciences (CER-ISBA) under reference number 154. The research work (sampling from hospitalized patients, sample processing and data analysis) in our study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient or their parent/guardian before participation, accompanied by a concise explanation of the study’s objective. Patients were provided the option to decline participation (opt-out) if they chose.

Consent to publish

All authors have read and agreed to the published version of the manuscript.

Competing Interest

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Transmission dynamics of carbapenemase-producing bacteria and mobile genetic elements between hospital and household settings in Benin

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