Characterization of antimicrobial-resistant Gram-negative bacteria that cause neonatal sepsis in seven low- and middle-income countries

Abstract

Antimicrobial resistance in neonatal sepsis is rising, yet mechanisms of resistance that often spread between species via mobile genetic elements, ultimately limiting treatments in low- and middle-income countries (LMICs), are poorly characterized. The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) network was initiated to characterize the cause and burden of antimicrobial resistance in neonatal sepsis for seven LMICs in Africa and South Asia. A total of 36,285 neonates were enrolled in the BARNARDS study between November 2015 and December 2017, of whom 2,483 were diagnosed with culture-confirmed sepsis. Klebsiella pneumoniae (n = 258) was the main cause of neonatal sepsis, with Serratia marcescens (n = 151), Klebsiella michiganensis (n = 117), Escherichia coli (n = 75) and Enterobacter cloacae complex (n = 57) also detected. We present whole-genome sequencing, antimicrobial susceptibility and clinical data for 916 out of 1,038 neonatal sepsis isolates (97 isolates were not recovered from initial isolation at local sites). Enterobacterales (K. pneumoniae, E. coli and E. cloacae) harboured multiple cephalosporin and carbapenem resistance genes. All isolated pathogens were resistant to multiple antibiotic classes, including those used to treat neonatal sepsis. Intraspecies diversity of K. pneumoniae and E. coli indicated that multiple antibiotic-resistant lineages cause neonatal sepsis. Our results will underpin research towards better treatments for neonatal sepsis in LMICs.

Fig. 1. Flow chart detailing the enrolment of neonates and incidence of sepsis (both clinically diagnosed and confirmed by culture) per site.

The numbers of neonates with missing clinical data for the onset and outcome of sepsis are shown per site. The numbers of isolates collected from neonatal blood cultures are shown per site, with a breakdown by preliminary characterization (as determined by Gram stain) in the flow chart below. The final numbers of isolates included for analysis are highlighted in orange. BB, baby blood; ID, identification (of species); ND, not determined.

Fig. 2. World map showing the BARNARDS study clinical site locations.

The study sites are indicated by coloured squares. The African sites were located in Ethiopia (ES (green)), Nigeria (NK (cyan), NN (light blue) and NW (dark blue)), Rwanda (RK (dark purple) and RU (light purple)) and South Africa (ZAT; olive). The Asian sites were located in Bangladesh (BC (dark pink) and BK (light pink)), India (IN (orange)) and Pakistan (PC (peach) and PP (burgundy)). The numbers next to each clinical site location represent the total number of GNB identified. Inset: the stacked bar graph shows the distribution of the top ten GNB species recovered from blood cultures at the local sites. The onset of neonatal sepsis (EOS, LOS or ND) for GNB per clinical site is represented as a pie chart. The outcome of neonatal sepsis is shown for each continent.

Fig. 3. AMR of neonatal sepsis-causing pathogens.

a, Percentages of antimicrobial-resistant aetiological agents of neonatal sepsis, coloured according to bacterial species/group (n = 885 isolates of GNB). The MICs of the antibiotics were determined by agar dilution and the results were interpreted according to EUCAST guidelines and documents^,. AMC, amoxicillin/clavulanate; AMK, amikacin; AMP, ampicillin; ATM, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; CRO, ceftriaxone; CST, colistin; CTX, cefotaxime; ETP, ertapenem; FEP, cefepime; FOF, fosfomycin; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; MEM, meropenem; TGC, tigecycline; TOB, tobramycin; TZP, piperacillin/tazobactam. b, Sunburst diagram detailing the class A (red), B (yellow) and D (green) carbapenemase resistance genes detected. The second ring from the centre shows the carbapenemase genes identified. The distributions across species and clinical sites are shown in the outer rings. ABU, Acinetobacter baumannii; CFI, Citrobacter freundii; ECO, Escherichia coli; ENT, Enterobacter cloacae complex; KMI, Klebsiella michiganensis; KPN, Klebsiella pneumoniae; KQI, Klebsiella quasipneumoniae; PRO, Providencia rettgeri; SER, Serratia marcescens.

Fig. 4. Sankey diagram showing the distribution of the different plasmid types detected linking to the GNB in which they were found.

Plasmid types (left) found to carry carbapenemase AMR genes are colour coded and linked to the GNB species (right) in which the plasmid type was identified. Particular carbapenemase genes are shown on the far right.

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

a, Five-hundred-and-fifty-nine isolates incorporating a global collection. Blue shading indicates K. pneumoniae isolates from the BARNARDS collection. The branch labels are coloured according to country of origin. b, Detailed core genome characterization of 309 K. pneumoniae isolates (n = 258 BARNARDS). Yellow shading indicates isolates from other studies^, causing neonatal sepsis. The outermost rings represent infant outcome (orange) and onset of sepsis (green), followed by the ST, where asterisks represent previously unknown STs. The leaf labels are the code names (coloured according to the study site) of isolates. The branch symbols in the centre denote the carriage of carbapenemase ARGs (bla_NDM variants (circles) and bla_OXA-48 group variants (squares)). NA, not applicable. Source data

Fig. 6. Core genome characterization of E. coli isolates.

a, Three-hundred-and-sixty isolates incorporating a global collection. Blue shading indicates E. coli isolates from the BARNARDS collection (n = 75). The branch labels are coloured according to country of origin. b, Detailed core genome characterization of 87 E. coli isolates (n = 75 BARNARDS). Yellow shading represents isolates from other studies^, causing neonatal sepsis. The colours on the right represent infant outcome (orange) and onset of sepsis (green), followed by the ST. The numbers and code names (coloured according to study site) of isolates are also given. The branch symbols denote the carriage of carbapenemase ARGs (bla_NDM-5 (circles) and bla_OXA-181 (squares). NF, not found. Source data