Antibiotic resistance genes in the gut microbiota of mothers and linked neonates with or without sepsis from low- and middle-income countries

Abstract

Early development of the microbiome has been shown to affect general health and physical development of the infant and, although some studies have been undertaken in high-income countries, there are few studies from low- and middle-income countries. As part of the BARNARDS study, we examined the rectal microbiota of 2,931 neonates (term used up to 60 d) with clinical signs of sepsis and of 15,217 mothers screening for bla_CTX-M-15, bla_NDM, bla_KPC and bla_OXA-48-like genes, which were detected in 56.1%, 18.5%, 0% and 4.1% of neonates' rectal swabs and 47.1%, 4.6%, 0% and 1.6% of mothers' rectal swabs, respectively. Carbapenemase-positive bacteria were identified by MALDI-TOF MS and showed a high diversity of bacterial species (57 distinct species/genera) which exhibited resistance to most of the antibiotics tested. Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae/E. cloacae complex, the most commonly found isolates, were subjected to whole-genome sequencing analysis and revealed close relationships between isolates from different samples, suggesting transmission of bacteria between neonates, and between neonates and mothers. Associations between the carriage of antimicrobial resistance genes (ARGs) and healthcare/environmental factors were identified, and the presence of ARGs was a predictor of neonatal sepsis and adverse birth outcomes.

Fig. 1. Total number of rectal samples collected from mothers and neonates and characterized.

Diagram detailing the total number of mother and neonate rectal samples collected and screened for the presence of bla_CTX-M-15, bla_NDM, bla_KPC and bla_OXA-48-like genes, the number of Gram-negative isolates carrying carbapenemase genes, the number of isolates tested for antibiotic susceptibility and the number of EC (E. coli), ENT (E. cloacae complex) and KP (K. pneumoniae) isolates characterized by WGS and bioinformatics analysis. Isolates for WGS were chosen after culture on VE (vancomycin, ertapenem) agar. Recoverable isolates after −80 °C preservation were selected for gDNA extraction and WGS. Source data

Fig. 2. Prevalence of bla_CTX-M-15, bla_NDM and bla_OXA-48-like genes among the rectal swabs of neonates and mothers.

a–c, Prevalence of bla_CTX-M-15, bla_NDM and bla_OXA-48-like genes among the rectal swabs of neonates. The prevalence of all genes was higher in South-Asian countries compared to African countries, except for bla_KPC, which was not found among neonates. d–f, Prevalence of bla_CTX-M-15, bla_NDM and bla_OXA-48-like genes among the rectal swabs of mothers. A higher prevalence of genes was seen in MR from South-Asian countries compared to African countries. bla_KPC genes were found in three Indian and four Pakistani rectal samples from mothers. The BARNARDS network included the following hospitals: Bangladesh: BC and BK; Ethiopia: ES; India: IN; Nigeria: NN, NW and NK; Pakistan: PP and PC; Rwanda: RU and RK; and South Africa: ZAT. Coloured maps were created using MapChart (https://www.mapchart.net). g,h, Carriage of bla_CTX-M-15, bla_NDM and bla_OXA-48-like genes among neonates’ rectal swabs against age of neonates at rectal swab collection per continent: Asia (g) and Africa (h). The prevalence of each ARG is plotted. The total number of samples collected per day is shown in the circles below the graphs. From day 0, ARGs were detected in the neonatal rectal microbiota. There was a tendency to a decrease in prevalence of bla_NDM (53.7% to 27.7%) and bla_OXA-48-like (35.4% to 0%) genes among the Asian samples through the first 14 d of life. Source data

Fig. 3. Core genome characterization of E. coli isolates.

The phylogenetic tree of 253 E. coli genomes, including 150 from BARNARDS and 103 from other studies^–, is shown, using Roary (v.3.12.0) and FastTree (v.2.1.11). Isolates are coloured at the endpoint according to country and the outer ring abbreviation is labelled according to the sample source. STs for all isolates are shown in the text after the sample source. The additional two outer rings denote the presence of bla_NDM and bla_OXA-48-like genes. Clades containing isolates from the present study are highlighted in teal, green clades indicate E. coli neonatal sepsis isolates from other studies and pink clades relate to E. coli rectal carriage from different studies. Major STs are labelled around the phylogeny and isolates that belong to a mother–neonate pair are denoted by an orange triangle. For site acronyms, see Methods. Source data

Fig. 4. Core genome characterization of Enterobacter spp. isolates.

The phylogenetic tree of 209 Enterobacter spp. genomes including 111 from BARNARDS and 98 from other studies^,, is shown, using Roary (v.3.12.0) and FastTree (v.2.1.11). Isolates are coloured at the endpoint according to country and the outer ring abbreviation is labelled according to the sample source. STs for all isolates are shown in the text after the sample source. The additional two outer rings denote the presence of bla_NDM and bla_OXA-48-like genes. Clades containing isolates from the present study are highlighted in teal, green clades indicate Enterobacter spp. neonatal sepsis isolates from other studies and pink Enterobacter spp. rectal carriage from different studies. Major STs are labelled around the phylogeny. For site acronyms, see Methods. Source data

Fig. 5. Core genome characterization of K. pneumoniae isolates.

The phylogenetic tree of 268 K. pneumoniae genomes, including 161 from BARNARDS and 107 from other studies^–, is shown, using Roary (v.3.12.0) and FastTree (v.2.1.11). Isolates are coloured at the endpoint according to country and the outer ring abbreviation is labelled according to the sample source. STs for all isolates are shown in the text after the sample source. The additional two outer rings denote the presence of bla_NDM and bla_OXA-48-like genes. Clades containing isolates from the present study are highlighted in teal, green clades indicate K. pneumoniae neonatal sepsis isolates from other studies and pink K. pneumoniae rectal carriage from different studies. Major STs are labelled around the phylogeny and isolates that belong to a mother–neonate pair are denoted by an orange triangle. Any carriage isolates sequenced in the present study that are genetically similar to the isolate recovered from the corresponding neonatal blood culture are denoted by a yellow star. For site acronyms, see Methods. Source data

Extended Data Fig. 1. Carriage of bla_CTX-M-15, bla_NDM and bla_OXA-48-like genes among neonates’ rectal swabs by age of neonates at sample collection.

Carriage of bla_CTX-M-15, bla_NDM and bla_OXA-48-like genes among neonates’ rectal swabs against age of neonates at sample collection per biological sepsis status in a) Asia and b) Africa and per delivery type in c) Asia and d) Africa. Prevalences of each antibiotic resistance gene (ARG) are plotted. The total number of samples collected per day for each type of delivery is shown in the circles below the graphs. From day 0, ARGs were detected in the neonates’ faecal microbiota independently of biological sepsis status or delivery type. Plots were done in R studio using packages tidyr (v1.2.0), ggpubr (v0.4.3), gridExtra (v2.3), and egg (v0.4.3). Source data

Extended Data Fig. 2. Species diversity of the isolates recovered from rectal samples.

Diversity of carbapenemase positive Gram-negative bacterial isolates collected from a) neonates’ and b) mothers’ rectal swabs. The number of isolates collected per species is shown. Source data

Extended Data Fig. 3. Antimicrobial resistance profiles of the isolates recovered from rectal samples.

Antimicrobial resistance (AMR) profiles of the carbapenemase positive Gram-negative bacterial isolates collected from a) neonates’ and b) mothers’ rectal swabs per species. AMR profiles distributed by site for c) neonates’ and d) mothers’ isolates distributed by site are also shown. The number of isolates tested for antibiotic susceptibility per species (a, b) and per site (c, d) is shown in the legends. The overall percentage of resistant isolates to each antimicrobial tested is shown at the top of the corresponding bar. For antibiotics’ acronyms see methods. Source data

Extended Data Fig. 4. Whole genome single-nucleotide polymorphisms analysis of ST405 E. coli isolates.

Whole genome SNP analysis of ST405 E. coli (n = 24 isolates) using snippy v4.6.0 on paired end fastq, gubbins v2.3.4 to remove recombination and IQ-tree v2.0 to construct the phylogeny. iTOL v4 was used to visualise the phylogenetic tree. BCBR/BCMR, neonate (BR)/mother (MR) rectal isolates from Bangladesh, Chittagong Maa-O-Shishu Hospital, Chattogram (BC). BKBR/BKMR, neonate (BR)/mother (MR) rectal isolates from Bangladesh, Kumudini Women’s Medical College, Mirzapur (BK). PPBR, neonate (BR) rectal isolates from Pakistan Institute of Medical Sciences (PP), Islamabad. Reference, BC-MR1421-3. Dates of rectal swab collection are shown. Source data

Extended Data Fig. 5. Whole genome single-nucleotide polymorphisms analysis of ST11 and ST15 K. pneumoniae isolates.

a) SNP analysis of ST11 Klebsiella pneumoniae (n = 12 isolates) collected during this study and K. pneumoniae GCF_001462965, GCF_006351115 and GCF_900607955 (see SourceData for Enterobacter sp. trees) using snippy v4.6.0 on paired end fastq, gubbins v2.3.4 to remove recombination and IQ-tree v2.0 to construct the phylogeny. iTOL v4 was used to visualise the phylogenetic tree. BCBR/BCMR, neonate (BR)/mother (MR) rectal isolates from Bangladesh, Chittagong Maa-O-Shishu Hospital, Chattogram (BC). BKBR/BKMR, neonate (BR)/mother (MR) rectal isolates from Bangladesh, Kumudini Women’s Medical College, Mirzapur (BK). PPBR/PPMR, neonate (BR)/mother (MR) rectal isolates from Pakistan Institute of Medical Sciences (PP), Islamabad. Reference, PP-BR254-2. Dates of rectal swab collection are shown. b) SNP analysis of ST15 K. pneumoniae (n = 25 isolates; BARN) collected during this study and other K. pneumoniae isolates’ genome assemblies available in GeneBank (see SourceData for Klebsiella pneumoniae trees) using snippy v4.6.0 on paired end fastq, gubbins v2.3.4 to remove recombination and IQ-tree v2.0 to construct the phylogeny. iTOL v4 was used to visualise the phylogenetic tree. PPBR/PPMR, neonate (BR)/mother (MR) rectal isolates from Pakistan Institute of Medical Sciences (PP), Islamabad. Reference, PP-BR737-1. Dates of rectal swab collection are shown. Source data

Extended Data Fig. 6. Whole genome single-nucleotide polymorphisms analysis of ST418 E. hormaechei isolates.

SNP analysis of ST418 E. hormaechei (n = 16) collected during this study and Enterobacter hormaechei subsp. xiangfangensis GCF_1526085 using snippy v4.6.0 on paired end fastq, gubbins v2.3.4 to remove recombination and IQ-tree v2.0 to construct the phylogeny. iTOL v4 was used to visualise the phylogenetic tree. BCBR/BCMR, neonate (BR)/mother (MR) rectal isolates from Bangladesh, Chittagong Maa-O-Shishu Hospital, Chattogram (BC). BKBR/BKMR, neonate (BR)/mother (MR) rectal isolates from Bangladesh, Kumudini Women’s Medical College, Mirzapur (BK). Reference, BC-MR78-3. Dates of rectal swab collection are shown. Source data

Extended Data Fig. 7. Exploratory multivariable statistical analysis to identify associations between socio-demographic and clinical data and maternal and neonatal carriage of ARGs.

Forest plots representing exploratory multivariable statistical analysis to identify associations between socio-demographic and clinical data and maternal and neonatal carriage of ARGs. Bars represent ranges of odds ratio. Multiplicity-adjusted p-values that remained statistically significant at the 5% level are coloured in red. Z tests were used from multivariable logistic regression models and statistical tests were two-sided. a) Two multivariable models (MV) were performed to understand the association between WASH (water, sanitation and hygiene) related variables and maternal carriage of ARGs using the explanatory variables shown. MV2 results are displayed; In MV1, type of toilet in home did not gave an association with carriage of any of the ARGs. b) Two MV were performed to understand the association between birth healthcare environment features and carriage of ARGs among neonates from the birth cohort using the explanatory variables shown. MV1 results are shown. Perinatal asphyxia is not plotted given the wide confidence intervals. Source data

Extended Data Fig. 8. BARNARDS workflow for collection of samples and data in local sites and samples and isolates characterisation in Cardiff University.

Indian samples characterisation was performed locally.