Carbapenem- and cefiderocol-resistant Enterobacterales in surface water in Kumasi, Ashanti Region, Ghana

Abstract Background MDR pathogens including ESBL- and/or carbapenemase-producing Enterobacterales (ESBL-PE and CPE) increasingly occur worldwide in the One Health context. Objectives This proof-of-principle study investigated the occurrence of ESBL-PE in surface water in the Ashanti Region in Ghana, sub-Saharan Africa (SSA), and investigated their additional genotypic and phenotypic antimicrobial resistance features as part of the Surveillance Outbreak Response Management and Analysis System (SORMAS). Methods From 75 water samples overall, from nine small to medium-sized river streams and one pond spatially connected to a channelled water stream in the greater area of Kumasi (capital of the Ashanti Region in Ghana) in 2021, we isolated 121 putative ESBL-PE that were subsequently subjected to in-depth genotypic and phenotypic analysis. Results Of all 121 isolates, Escherichia coli (70.25%) and Klebsiella pneumoniae (23.14%) were the most prevalent bacterial species. In addition to ESBL enzyme-production of mostly the CTX-M-15 type, one-fifth of the isolates carried carbapenemase genes including blaNDM-5. More importantly, susceptibility testing not only confirmed phenotypic carbapenem resistance, but also revealed two isolates resistant to the just recently approved last-resort antibiotic cefiderocol. In addition, we detected several genes associated with heavy metal resistance. Conclusions ESBL-PE and CPE occur in surface water sources in and around Kumasi in Ghana. Further surveillance and research are needed to not only improve our understanding of their exact prevalence and the reservoir function of water sources in SSA but should include the investigation of cefiderocol-resistant isolates.


Introduction
Antimicrobial resistance (AMR) has emerged as an urgent global health crisis, severely limiting available therapeutic options including last-resort antibiotics such as carbapenems.Sub-Saharan Africa (SSA) bears a high burden of AMR, with a significant number of deaths attributed to ESBL-and carbapenemase-producing Enterobacterales (ESBL-PE and CPE) such as Escherichia coli and Klebsiella pneumoniae. 1 In Ghana, multiple studies have revealed an increasing number of hospital-and community-associated infections with ESBL producers. 2Like many other low-and middle-income countries (LMICs), Ghana faces challenges regarding unclean water due to open drains, limited sanitation and healthcare, and a lack of public education and environmental consciousness.While there is evidence suggesting that environmental sources might serve as potential transmission points for AMR bacteria in SSA, the extent of environmental contamination including surface water with ESBL-PE and CPE remains largely unknown.One of the few available studies revealed overall high rates of ESBL-producing E. coli in rivers in two cities in Ghana between 2018 and 2020. 3In a broader African context, data on the presence of ESBL-PE and particularly CPE in environmental settings across the continent are exceptionally scarce.In addition, nothing is known about the occurrence of Enterobacterales resistant to the recently approved antibiotic cefiderocol in these niches.
This proof-of-principle study's objective was to complement the Surveillance Outbreak Response Management and Analysis System (SORMAS) project 4 with data on the occurrence of ESBL-PE and CPE in surface water from Kumasi, the capital city of the Ashanti Region in Ghana.

Sampling strategy and bacterial isolation
We collected 75 surface water samples from 10 randomly chosen periurban sites in the Kumasi area in Ghana (Figure 1).This included nine small-to medium-sized river streams and one pond spatially connected to a channelled water stream.Note that the individual sampling spots are representative of the variety of water sources found in the Kumasi area, which are used by animals and humans alike.Observed activities around the water bodies included irrigation and collection of water for households, laundry and livestock.Sampling of 1 L water volume per site was performed once a week over 7-8 weeks between July and September 2021.Water samples were collected directly by filling a sterile plastic container with surface water.Samples were refrigerated at +4°C and further processed at the laboratory of the Kumasi Centre for Collaborative Research, Ghana, within 4 h of collection.Samples were then prefiltered using a sterile gauze (PZN: 04046708, FESMED Verbandmittel GmbH, Frankenberg/Sa., Germany) to retain macroscopic particles.This was followed by filtration through a sterile 1.20 µm, and ultimately 0.45 µm, pore-sized membrane filter (Millipore, Merck, Darmstadt, Germany).Filtration was supported by the 'All-Glass Filter Holder Kit' (XX5514700) combined with a pump (WP6122050, 220 V/50 Hz, both from Millipore).Similar to a previous study, 5 a 6 mm-sized piece of the 0.45 µm filter was transferred to 10 LB broths (Lennox, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) containing 2 µg/mL cefotaxime (VWR International, Darmstadt, Germany) and cultured at 37°C and 200 rpm overnight.On the following day, 1.8 mL of the culture was pelleted at 20 000×g for 1 min and resuspended in 1 mL of the same culture medium supplemented with glycerol (anhydrous; Merck, Darmstadt, Germany) at a final concentration of 20%.Samples were then stored at −80°C and transported on dry ice to the Friedrich-Loeffler-Institut, Germany.One hundred microlitres of an overnight culture was then plated on CHROMagar Orientation (MastDiagnostica GmbH, Reinfeld, Germany) supplemented with 2 µg/mL cefotaxime, and incubated overnight at 37°C.Cefotaxime-resistant colonies of E. coli and other Enterobacterales were subcultured until pure cultures were obtained and selected for further characterization.All isolates were stored at −80°C in LB broth and glycerol at a final concentration of 20%.

WGS
Total DNA was extracted using the MasterPure DNA Purification Kit for Blood, v. 2 (Lucigen, Middleton, WI, USA), according to the manufacturer's instructions.DNA was then quantified fluorometrically using the Qubit 4 Fluorometer and the corresponding dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).DNA was shipped to SeqCenter in Pittsburgh, PA, USA, and sequenced on an Illumina NextSeq 2000 after library preparation using the Illumina DNA Prep kit and IDT 10 bp UDI indices (Illumina, San Diego, CA, USA), resulting in 2 × 151 bp reads.Demultiplexing, quality control and adapter trimming were performed using bcl-convert v. 3.9.3(https://support-docs.illumina.com/SW/BCL_Convert/Content/SW/FrontPages/BCL_Convert.htm).

Phenotypic antimicrobial susceptibility testing
MICs were determined with the automated VITEK 2 system (AST-N428 and AST-XN24; bioMérieux, Marcy l'Étoile, France), according to the manufacturer's instructions.Susceptibility to cefiderocol was assessed by disc diffusion tests using cefiderocol 30 μg discs (Mast Diagnostics, Merseyside, UK).Isolates assigned to technical uncertainty (Enterobacterales, 18-22 mm 15 ) were re-analysed using a commercial broth microdilution kit (ComASP, Liofilchem, Waltham, MA, USA) according to the manufacturer's instructions.All results were interpreted according to the published breakpoints and guidelines of EUCAST. 15
We performed antimicrobial susceptibility testing for these isolates to verify carbapenem resistance phenotypes.In addition, we evaluated susceptibility to the recently approved and important last-resort siderophore cephalosporin antibiotic cefiderocol (Table 1).
The bla OXA-181 -positive isolates were only resistant to ertapenem, highlighting the weak hydrolytic activity of this OXA-48-like carbapenemase against carbapenems. 16In contrast, Eger et al.  isolates carrying bla NDM-5 or bla OXA-48 showed higher MICs of imipenem and/or meropenem.Notably, the CPE were predominantly associated with internationally recognized high-risk clonal lineages, such as E. cloacae ST171, E. coli ST410 and ST1588, and K. pneumoniae ST25.Many studies have consistently demonstrated that successful clonal lineages carry multiple AMR determinants, can be rapidly transmitted among and persist in different host species and ecosystems, may cause severe disease in animals and humans, and are globally distributed (e.g. the study by Eger et al. 17 ).Environmental implications of AMR emergence predominantly originate from high-resource settings, leaving a significant knowledge gap in LMICs.The facilitation of AMR occurs through the discharge of sewage and antimicrobial residues into the environment and the inadequate treatment of human and animal waste.In Ghana, like in other countries in SSA, the drainage systems comprise open drains and street gutters fed from various sources including households, hospitals and industries, subsequently contaminating the environment.In fact, we frequently observed private household sewage and excrement from livestock and feral domestic animals near the sampling spots.Reducing environmental contamination with AMR bacteria primarily includes public education programmes to not only raise awareness about the risks associated with untreated effluent discharge into the environment but also regarding the usage of lake and river water for domestic purposes.In addition, improving basic sanitation and increasing the number and efficacy of sewage treatment plants is crucial.
To the best of our knowledge, this is the first study identifying cefiderocol-resistant Enterobacterales in surface water from SSA.Both resistant isolates, PBIO3888 and PBIO3903, were collected shortly after the international approval and clinical use of cefiderocol in 2019.Interestingly, the corresponding sampling location is at the Kumasi Zoo compound, which, although accessrestricted, receives wastewater from the zoo enclosures, visitors and the nearby central marketplace.Recent studies have suggested several mechanisms contributing to cefiderocol resistance, including gene alterations in the iron transport pathway and nutrient uptake (e.g.cirA and ompC). 18However, a BLAST analysis of the amino acid sequences of CirA (UniProt accession P17315), OmpF (UniProt accession P02931) and OmpC (UniProt accession P06996), using E. coli K-12 as a reference, did not reveal any potential resistance-mediating mutations in our isolates.This suggests that cefiderocol resistance may be attributed to overexpression of the NDM-5 carbapenemase.
Ghana faces the challenge of environmental contamination with heavy metals, particularly those associated with illegal gold mining activities. 19Heavy metals in the environment may co-select for AMR in bacteria, which is caused by either single mechanisms conferring cross-resistance to both antibiotics and heavy metals, and/or the occurrence of different resistance determinants located on the same genetic element. 20Except for seven of the E. coli isolates in this study, all CPE were positive for genes associated with multi-metal RND efflux pump activity (silABCEFPRS), which typically confer resistance to various heavy metals, including silver.Genes involved in copper resistance (pcoABCDRS; 17/24), followed by arsenic resistance (arsD; 8/24), mercury resistance (merRT; 7/24) and tellurium resistance (terD; 4/24) also occurred.Consequently, additional assessment of not only heavy metal resistance determinants but also heavy metal residues should be an elementary component of surface water surveillance approaches. 5ven though this is a proof-of-concept study, it could have been improved by considering more sampling sites, a longer sampling period, and by obtaining relevant metadata including weather conditions and physicochemical water parameters during sampling.These issues will be addressed in a follow-up, large-scale study.

Conclusions
In conclusion, we not only found ESBL-PE and CPE but also, more importantly, two isolates resistant to the recently approved antibiotic cefiderocol in surface water samples in Ghana, also indicating the potential for transmission to humans and animals.Our For isolates with zone diameters within the range of technical uncertainty (Enterobacterales, 18-22 mm 15 ), the MIC of cefiderocol was determined by broth microdilution.
Eger et al.
study suggests that further exploration of transmission pathways related to environmental contamination is warranted.

Figure 1 .
Figure 1.Map of the Kumasi area (Ghana).The selected water sources are labelled from (A) to (J).Sample sizes were (A) n = 8, (B) n = 8, (C) n = 8, (D) n = 8, (E) n = 7, (F) n = 7, (G) n = 7, (H) n = 7, (I) n = 7 and (J) n = 8.The CPE isolates are assigned to their sampling locations.The inset image outlined in red shows images of the Kumasi Zoo water bodies (location G) where the two cefiderocol-resistant isolates (highlighted in red) were isolated.The map was created in QGIS v. 3.20 using Bing Aerial (http://ecn.t3.tiles.virtualearth.net/tiles/a{q}.jpeg?g=1) as a map source with TM World Borders 0.3 overlay (https://thematicmapping.org/downloads/world_borders.php; last accessed on 23 January 2024).Microsoft product screenshots were reprinted with permission from Microsoft Corporation.Major surface waters are highlighted in pale blue based on the OpenStreetMap data for Ghana (CC BY-SA 2.0 DEED; https://download.geofabrik.de/africa/ghana.html).The Digital Elevation Model (DEM) layer uses a single band with values from 185.2 to 388.5.For reference, the overall location of Kumasi is indicated on the Ghana overview map with Regions (https://vemaps.com/ghana/gh-04by Vemaps.com).

Table 1 .
Overview of the CPE and their phenotypic and genotypic properties Predictions for carbapenemase genes are based on alignments of sequences from the AMRFinderPlus database. 14Default settings of identity (use a curated threshold if it exists, and ≥90.0%otherwise) and coverage (≥50.0%).