Acquisition and carriage of genetically diverse multi-drug resistant gram-negative bacilli in hospitalised newborns in The Gambia

Abstract

Background: This detailed genomic study characterised multi-drug resistant-Gram negative bacilli (MDR-GNB) carriage in neonates < 2 kg and paired mothers at a low-resource African hospital.

Methods: This cross-sectional cohort study was conducted at the neonatal referral unit in The Gambia with weekly neonatal skin and peri-anal sampling and paired maternal recto-vaginal swabs. Prospective bacteriological culture used MacConkey agar with species identification by API20E and API20NE. All GNB isolates underwent whole genome sequencing on Illumina Miseq platform. Multi-Locus Sequence Typing and SNP-distance analysis identified strain type and relatedness.

Results: 135 swabs from 34 neonates and 21 paired mothers, yielded 137 GNB isolates, of which 112 are high quality de novo assemblies. Neonatal MDR-GNB carriage prevalence is 41% (14/34) at admission with 85% (11/13) new acquisition by 7d. Multiple MDR and ESBL-GNB species are carried at different timepoints, most frequently K. pneumoniae and E. coli, with heterogeneous strain diversity and no evidence of clonality. 111 distinct antibiotic resistance genes are mostly beta lactamases (Bla-AMPH, Bla-PBP, CTX-M-15, Bla-TEM-105). 76% (16/21) and 62% (13/21) of mothers have recto-vaginal carriage of ≥1 MDR-GNB and ESBL-GNB respectively, mostly MDR-E. coli (76%, 16/21) and MDR-K. pneumoniae (24%, 5/21). Of 21 newborn-mother dyads, only one have genetically identical isolates (E. coli ST131 and K. pneumoniae ST3476).

Conclusions: Gambian hospitalised neonates exhibit high MDR and ESBL-GNB carriage prevalence with acquisition between birth and 7d with limited evidence supporting mother to neonate transmission. Genomic studies in similar settings are required to further understand transmission and inform targeted surveillance and infection prevention policies.

Fig. 1. Overview of screening, enrolment, carriage sampling and isolate sequencing for neonates and mothers.

^aOther reasons for non-recruitment included weight >2 kg on study scales (n = 2) and no staff available to perform study procedures (n = 2). ^bTwo mothers were sampled in absence of neonatal paired swabs and two sets of twins were included. ^c36 neonates were enrolled but 2 were not sampled, due to rapid deterioration and death (n = 1) and lack of consent for neonatal sampling (n = 1). ^dOne participant did not meet eligibility for peri-anal samples due to imperforate anus. ^eTwo neonates did not have skin swabs taken: One met exclusion criteria; One had consent withdrawn for repeat sampling. ^fOne neonate did not have peri-anal swabs taken as consent was withdrawn for repeat sampling. ^gOne newborn did not have skin or peri-anal samples taken due to error. GNB Gram-Negative Bacilli, WGS Whole Genome Sequencing.

Fig. 2. Multi-locus sequencing types and genetic relatedness of K. pneumoniae and E. coli identified from paired newborn-mother carriage samples.

a Klebsiella pneumoniae. b Escherichia coli. SNP-distance was used to determine genetic relatedness of isolates using the core-genome alignment obtained from Roary. Sequence type (ST) were determine using the mlst package https://github.com/tseemann/mlst. SNP distances were imported into R to generate the circus plots using the circlize package. In the top half of the circus plot, the inner segments indicate whether the isolates were collected from a neonate (brown) or mother (red), mothers’ study ID (MXX) and day of sampling labelled on the outside separated by underscore (_). Sample IDs highlighted with the blue font are neonate-mother pairs that have the ST. The bottom half indicates the ST types of K. pneumoniae or E. coli. Connecting lines joining the upper and lower halves of the circus plot indicates to which ST a particular isolate belongs.

Fig. 3. Phylogenetic trees of K. pneumoniae and E. coli isolates identified from paired newborn-mother carriage samples.

a Klebsiella pneumoniae. b Escherichia coli. A maximum likelihood phylogenetic tree was constructed from core-genome SNPs using RAxML with 100 bootstraps. For both species isolates are clustered by STs. Also shown are the presence (black-filled square) or absence (white empty-square) of antimicrobial resistance genes (AMR).