A glimpse of antimicrobial resistance gene diversity in kefir and yoghurt

Abstract

Antimicrobial resistance (AMR) is a global threat gaining more and more practical significance every year. The main determinants of AMR are the antimicrobial resistance genes (ARGs). Since bacteria can share genetic components via horizontal gene transfer, even non-pathogenic bacteria may provide ARG to any pathogens which they become physically close to (e.g. in the human gut). In addition, fermented food naturally contains bacteria in high amounts. In this study, we examined the diversity of ARG content in various kefir and yoghurt samples (products, grains, bacterial strains) using a unified metagenomic approach. We found numerous ARGs of commonly used fermenting bacteria. Even with the strictest filter restrictions, we identified ARGs undermining the efficacy of aminocoumarins, aminoglycosides, carbapenems, cephalosporins, cephamycins, diaminopyrimidines, elfamycins, fluoroquinolones, fosfomycins, glycylcyclines, lincosamides, macrolides, monobactams, nitrofurans, nitroimidazoles, penams, penems, peptides, phenicols, rifamycins, tetracyclines and triclosan. In the case of gene lmrD, we detected genetic environment providing mobility of this ARG. Our findings support the theory that during the fermentation process, the ARG content of foods can grow due to bacterial multiplication. The results presented suggest that the starting culture strains of fermented foods should be monitored and selected in order to decrease the intake of ARGs via foods.

Figure 1

Bacterial content of the samples. (a) The number of reads classified bacterial by Kraken2 on the NCBI NT database. Metagenome includes the samples deriving from grains, milk or products. (b) Relative abundances of the most common bacterial species in the grain and product samples.

Figure 2

Antimicrobial resistance (AMR) abundance of the samples. (a) Violin plot representing the distribution of the total AMR fragments per kilobase per million fragments (FPKM) per sample, grouped by type and source. The horizontally jittered dots represent the FPKM of the samples. (b) The AMR abundance diversity ($\beta$-diversity) of the samples. It is plotted on the first two axes of principal coordinate analysis (PCoA) performed on Bray-Curtis distance which was calculated using the relative abundances of contigs harbouring ARGs. The symbols show the type, the colours the source, while the numbers correspond to the sequence number in the Sample ID. Some samples (k_s_01, k_s_04, k_s_05, k_s_06, k_p_08) are not shown as their ORFs did not meet filtering criteria.

Figure 3

Antimicrobial resistance (AMR) abundance in kefir and yoghurt samples. (a) Relative abundance of AMR genes. ORFs having at least 60% length and 90% base sequence identity with the reference ARG sequence are shown. Some samples (k_s_01, k_s_04, k_s_05, k_s_06, k_p_08) are not shown as their ORFs did not meet filtering criteria. (b) Relative abundance of drug classes related to the ARGs identified in the samples.

Figure 4

Identified ARGs and their most probable bacteria of origin. The gene names that are too long have been abbreviated (w1: Bifidobacterium adolescentis rpoB mutants conferring resistance to rifampicin; w3: Escherichia coli marR mutant conferring antibiotic resistance; w4: E. coli soxS with mutation conferring antibiotic resistance; w5: E. coli UhpT with mutation conferring resistance to fosfomycin).

Figure 5

Changes during kefir fermentation. (a) Antimicrobial resistance gene (ARG) abundance expressed as fragments per kilobase per million fragments (FPKM) based on the alignment of bacterial reads to the ARG harbouring contigs. (b) ARGs and their most likely origins. (c) Relative abundances of bacteria with a probable ARG content.

Figure 6

Identified ARGs excluding nudged findings. The coverage and identity of detected open reading frames (ORFs) by antimicrobial resistance genes (ARGs). The ORF covered proportion of the reference ARG sequence and the identity % of predicted protein (colour). The gene names which are too long have been abbreviated (w1: Bifidobacterium adolescentis rpoB mutants conferring resistance to rifampicin; w2: Escherichia coli EF-Tu mutants conferring resistance to Pulvomycin; w3: E. coli marR mutant conferring antibiotic resistance; w4: E. coli soxS with mutation conferring antibiotic resistance; w5: E. coli UhpT with mutation conferring resistance to fosfomycin).