Colonisation of hospital surfaces from low- and middle-income countries by extended spectrum β-lactamase- and carbapenemase-producing bacteria

Abstract

Hospital surfaces can harbour bacterial pathogens, which may disseminate and cause nosocomial infections, contributing towards mortality in low- and middle-income countries (LMICs). During the BARNARDS study, hospital surfaces from neonatal wards were sampled to assess the degree of environmental surface and patient care equipment colonisation by Gram-negative bacteria (GNB) carrying antibiotic resistance genes (ARGs). Here, we perform PCR screening for extended-spectrum β-lactamases (bla_CTX-M-15) and carbapenemases (bla_NDM, bla_OXA-48-like and bla_KPC), MALDI-TOF MS identification of GNB carrying ARGs, and further analysis by whole genome sequencing of bacterial isolates. We determine presence of consistently dominant clones and their relatedness to strains causing neonatal sepsis. Higher prevalence of carbapenemases is observed in Pakistan, Bangladesh, and Ethiopia, compared to other countries, and are mostly found in surfaces near the sink drain. Klebsiella pneumoniae, Enterobacter hormaechei, Acinetobacter baumannii, Serratia marcescens and Leclercia adecarboxylata are dominant; ST15 K. pneumoniae is identified from the same ward on multiple occasions suggesting clonal persistence within the same environment, and is found to be identical to isolates causing neonatal sepsis in Pakistan over similar time periods. Our data suggests persistence of dominant clones across multiple time points, highlighting the need for assessment of Infection Prevention and Control guidelines.

Fig. 1. Maps indicating prevalence of ARG per site and country.

Prevalence of bla_CTX-M-15 (a), bla_NDM (b), and bla_OXA-48-like (c) genes per country and per hospital site. The total number of samples collected per country and per hospital site were used as denominators to calculate the prevalence of each antibiotic resistance gene (ARG) per country and per hospital site, respectively. Abbreviations for BARNARDS hospitals are detailed in the “Methods” section, map pins showing latitude and longitude, and coloured according to the ARG prevalence in that site. Source data are provided in Supplementary Table 2.

Fig. 2. Bacterial species diversity carrying carbapenemase genes.

Bacterial species carrying bla_NDM (a) and bla_OXA-48-like (b) among countries and sites, according to PCR screening and MALDI-TOF MS identification (a total of 175 bacterial isolates from 27 bacterial species; and 3 “unidentified” isolates). Source data are provided in Supplementary Data 4.

Fig. 3. A Sankey diagram linking bacterial species, plasmids and carbapenemase ARGs.

There were n = 95 isolates with an identifiable Inc plasmid type (due to assembly fragmentation, it was not always possible to assemble and analyse whole plasmids). The carbapenemase variant NDM and OXA-48-like group are divided into variants, and the Inc type detected per ARG-variant is shown. Source data are provided in Supplementary Data 5 and 6 and as Source Data file.

Fig. 4. Timeline showing nine potential clusters of bacterial isolates recovered from hospital surface swabs (HSS) and whether the same strain of bacteria was found in sepsis isolates during BARNARDS.

Sequence types (STs) and antimicrobial resistance genes (ARGs) are shown, and the highlighted background around isolates indicated these were within 10 pairwise SNPs. The different pink tone for ST20 K. pneumoniae indicates distinct sub-clusters identified among these isolates in Pakistan (over 1000 SNPs). Abbreviations for BARNARDS hospitals are detailed in the “Methods” section. The figure was created using Adobe Illustrator v26.5. Source data are provided in Supplementary Data 5 and 6.

Fig. 5. Workflow of sample collection and processing.

Diagram detailing the total number of hospital surface swabs (HSS) collected, showing Gram-negative bacteria (GNB) growth, and screened for the presence of bla_CTX-M-15, bla_NDM, bla_KPC and bla_OXA-48-like antimicrobial resistance genes (ARGs), the number of GNB isolates recovered carrying carbapenemase genes, the number of isolates characterised by whole genome sequencing (WGS) and, where WGS data was sufficient, bioinformatic analysis was performed. 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. Data cleaning is also detailed; where data regarding hospital surfaces and collection dates was available, statistical analysis and data analysis were performed.