Identification of potential novel combination antibiotic regimens based on drug-susceptibility and genetic diversity of Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries

Objectives Several recent studies highlight the high prevalence of resistance to multiple antibiotic classes used in current treatment regimens for neonatal sepsis and new treatment options are urgently needed. We aimed to identify potential new combination antibiotic treatment regimens by investigating the drug-resistance and genetic profiles of the most frequently isolated Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries (LMICs) in the NeoOBS study. Material and methods Gram-negative bacteria isolated from neonates with culture-confirmed sepsis from 13 clinical sites in nine countries, mainly LMICs, were analyzed. Culture-based identification was followed by whole-genome sequencing (WGS). Minimal inhibitory concentrations (MICs) for 8 antibiotics were determined for a representative subset of 108 isolates. Results Five bacterial species, Klebsiella pneumoniae (n=135), Acinetobacter baumannii (n=80), Escherichia coli (n=34), Serratia marcescens (n=33) and Enterobacter cloacae complex (ECC) (n=27) accounted for most Gram-negative bacterial isolates received (309/420, 74%). Extended-spectrum β-lactamases (ESBL) genes mostly belonging to CTX-M-15 were found in 107 (79%) K. pneumoniae isolates and 13 (38%) E. coli , as well as in 6 (18%) and 10 (37%) S. marcescens and ECC isolates, respectively. Carbapenem resistance genes were present in 41 (30%) K. pneumoniae, while 73 (91%) of A. baumannii isolates were predicted to be MDR based on carbapenem resistance genes. Apart from A. baumannii, in which two major pandemic lineages predominated, a wide genetic diversity occurred at the intraspecies level with different MDR clones occurring at the different sites. Phenotypic testing showed resistance to the WHO first- and second- line recommended treatment regimens: 74% of K. pneumoniae isolates were resistant to gentamicin and 85% to cefotaxime; E. coli isolates showed resistance to ampicillin, gentamicin and cefotaxime in 90%, 38% and 47%, respectively. For the novel antibiotic regimens involving different combinations of flomoxef, fosfomycin and amikacin, the overall predicted MIC-determined susceptibility for Enterobacterales isolates was 71% (n=77) to flomoxef-amikacin, 76% (n=82) to flomoxef-fosfomycin and 79% (n=85) to fosfomycin-amikacin combinations, compared to 31% and 22% isolates susceptible to ampicillin-gentamicin and cefotaxime, respectively. ESBL-producing Enterobacterales isolates were 100% susceptible both to flomoxef-fosfomycin and flomoxef-amikacin and 92% to fosfomycin-amikacin. Conclusion Enterobacterales carried multiple resistance genes to cephalosporins, carbapenems and aminoglycosides. ESBL-producing K. pneumoniae and E. coli isolates were highly susceptible to the three new antibiotic combination regimens planned to be evaluated in the currently recruiting GARDP-sponsored NeoSep1 trial. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was made possible with support from Bill & Melinda Gates Foundation; German Federal Ministry of Education and Research; German Federal Ministry of Health; Government of the Principality of Monaco; the Indian Council for Medical Research; Japanese Ministry of Health, Labour and Welfare; Netherlands Ministry of Health, Welfare and Sport; South African Medical Research Council; UK Department of Health and Social Care (UK National Institute of Health Research and the Global Antimicrobial Resistance Innovation Fund, GAMRIF); and Wellcome Trust. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Health Research Ethics Committee (HREC) of Stellenbosch University gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors