Draft genome sequences of three emerging beta-lactamase-producing Escherichia coli in the camel production system in Northern Kenya

Introduction

Antimicrobial resistance (AMR), especially on the readily available beta-lactam antibiotics continues to threaten effective healthcare management and global economic success in livestock farming (Fashae et al., 2021; Kiiru et al., 2012). Recently, concerted efforts have been employed to explore the AMR situation following a One Health Approach Previously, we and others have demonstrated an increased occurrence of broad-spectrum producing Enterobacteriaceae in livestock (Akunda et al., 2023; Nüesch-Inderbinen et al., 2020). Importantly, extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae have been implicated in the development of multi-drug resistance in both humans and animals. However, there still exists a gap in whole genome sequencing data on Enterobacteriaceae harboring AMR genes in camels (Camelus dromedaries), a key component of livestock farming in arid and semi-arid regions of Northern Kenya. We therefore aimed at sequencing the genomes of extended-spectrum beta lactamase Enterobacteriaceae using Escherichia coli (E. coli) as our model organism.

Methods

Here, we report three draft genome sequences of beta-lactamase-producing E. coli isolated from camel fecal samples in Laikipia county, Northern Kenya as previously described (Akunda et al., 2023). Fecal samples were collected from healthy camels reared under both ranching systems and pastoralism, transported in Cary Blair media (HiMedia Lab, Mumbai, India) and stored at 4°C awaiting processing.

The samples were cultured on MacConkey agar plate (HiMedia Lab, Mumbai, India) and incubated at 37°C for 18 hours. A single colony per MacConkey agar plate that displayed E. coli traits morphologically was identified through Gram staining and the IMViC (indol, methyl red, Voges Proskauer, and citrate) biochemical method and subjected to antimicrobial susceptibility testing (AST) against the beta-lactam antibiotic spectrum. Briefly, antimicrobial susceptibility testing (AST) was performed on Mueller Hinton agar (HiMedia Lab, Mumbai, India) using Kirby Bauer disk diffusion method. Antibiotics tested included, Ampicillin-10 μg (AMP), Chloramphenicol-30 μg (CHL), Tetracycline-30 μg (TCY), Gentamycin-10 μg (GEN), Streptomycin-10 μg (STR1), Trimethoprim-sulfamethoxazole-25 μg (SXT), Norfloxacin-10 μg (NOR), Ciprofloxacin-5 μg (CIP), Cefaclor-30 μg (CEC), Ceftriaxone-30 μg (CRO), Cefotaxime-30 μg (CTX), Cefuroxime-30 μg (CXM), Cefepime-30 μg (FEP), Amoxicillin–Clavulanate-20/10 μg (AMC), and Ceftazidime-30 μg (CAZ). The antibiotic susceptibility profile of the sequenced isolates is as shown in Table 1.

Production of beta-lactamase genes was assessed by conducting PCR to confirm genes encoding for CTX-M, TEM, CMY, SHV and OXA for the resistant isolates (Livermore et al., 2007), results are as shown in Table 1. Briefly, a 20 ul PCR reaction was prepared by adding 10 ul of the Platinum II Hot-Start PCR Master Mix (Invitrogen, Carlsbad, CA, U.S), 0.4 ul of 10 nmol of forward and reverse primers, 4 ul of Platinum GC enhancer, 0.2 ul nuclease free water and 5 ul DNA. Cycling was performed on Veriti™ Dx 96-well Thermal Cycler (Thermofisher, Waltham, Massachusetts, U.S) using primers and PCR conditions as in Table 2. The respective gene fragment sizes were confirmed in 1% agarose gel (Invitrogen, Carlsbad, CA, U.S). ESBL producing E. coli isolates were phenotypically confirmed using double-disc synergy test (DDST) (Drieux et al., 2008).

Genomic DNA of the confirmed ESBL producing E. coli isolates was extracted using Isolate II Genomic DNA Kit (Bioline, UK) and sequencing performed using Oxford Nanopore Technologies (ONT, Oxford, UK). Briefly, the manufacturer’s sample barcodes (Native Barcoding Expansion 1-12) (EXP-NBD104) and sequencing adapters (Ligation Sequencing kit) (SQK-LSK109)) were added following kit instructions. Sequencing was performed on MinION device using R9.4.1 flow cell to generate 186, 38153, and 31152 raw reads for the E. coli strains IPR_LC17, IPR_LC19 and IPR_LC20, respectively. Basecalling and demultiplexing of raw reads were performed using Guppy v3.6.1 (https://nanoporetech.com). Adaptors were trimmed using PORECHOP v0.2.4 (Wick et al., 2017) and poor quality reads, less than 500 bp and with an average quality score of below 10 were removed using NANOFLIT v2.8.0 (De Coster et al., 2018).

E. coli PR_LC17 genome was assembled using Canu v2.2 (Koren et al., 2017) while E. coli (IPR_LC19) and E. coli (IPR_LC20) were assembled using Flye v2.9.1 (Kolmogorov et al., 2019). The taxonomy of the organism was confirmed using public databases for molecular typing and microbial genome diversity (PubMLST) (Jolley et al., 2018). Quality of the assembled genomes was assessed using QUAST v5.1.0 (Gurevich et al., 2013) and annotated using NCBI’s PGAP (Tatusova et al., 2016). The assembly metrics and annotation summaries are as shown in Table 3.