Antibiotic Susceptibility, Resistance Gene Determinants and Corresponding Genomic Regions in Lactobacillus amylovorus Isolates Derived from Wild Boars and Domestic Pigs

Abstract

Restrictions on the use of antibiotics in pigs lead to the continuous search for new probiotics serving as an alternative to antibiotics. One of the key parameters for probiotic bacteria selection is the absence of horizontally transmissible resistance genes. The aim of our study was to determine antibiotic susceptibility profiles in 28 Lactobacillus amylovorus isolates derived from the digestive tract of wild boars and farm pigs by means of the broth microdilution method and whole genome sequencing (WGS). We revealed genetic resistance determinants and examined sequences flanking resistance genes in these strains. Our findings indicate that L. amylovorus strains from domestic pigs are predominantly resistant to tetracycline, erythromycin and ampicillin. WGS analysis of horizontally transmissible genes revealed only three genetic determinants (tetW, ermB and aadE) of which all tetW and ermB genes were present only in strains derived from domestic pigs. Sequence analysis of coding sequences (CDS) in the neighborhood of the tetW gene revealed the presence of site-specific recombinase (xerC/D), site-specific DNA recombinase (spoIVCA) or DNA-binding transcriptional regulator (xre), usually directly downstream of the tetW gene. In the case of ermB, CDS for omega transcriptional repressor or mobilization protein were detected upstream of the ermB gene.

Keywords: Lactobacillus amylovorus; antibiotic resistance; domestic pigs; ermB; tetW; wild boars.

Figure 1

The phylogenetic tree of a tetW gene, showing the relationship between the tetW genes from L. amylovorus (received from our study and NCBI) and the tetW sequences of selected bacterial species showing the highest similarity to L. amylovorus tetW genes (based on the blastn analysis). The evolutionary history was inferred using the Maximum Likelihood method and the Tamura 3-parameter model [36]. Bootstrap values (1000 replicates) were applied and the percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. There were a total of 1932 positions in the final dataset. The phylogenetic tree was rooted with the tetW from Bifidobacterium longum subsp. suillum JCM 19995 as an outgroup. Evolutionary analyses were conducted in MEGA X [34].

Figure 2

Organization of CDS in the contigs harboring tetW and xerC/D in L. amylovorus strains and comparison with plasmid pPMRA301 and plasmid p2 from L. amylovorus PMRA3 and GLR1118, respectively. Yellow arrow—CDS associated with mobility (e.g., IS—transposase, tr—putative transposase, int—putative integrase), red arrow—tetW gene, teal arrow—CDS with COG/PROKKA annotation, gray arrow—hypothetical protein, orange—XerC/D site-specific recombinase, green rectangle—tetW regulatory protein (trp), light blue—unknown misc. feature. The gray zones between sequences represent blastn sequence identity. *Plasmid p2 from GLR1118 shown only CDS identical to contigs bearing tetW resistance.

Figure 3

Organization of CDS in the contigs harboring tetW and site-specific recombinase spoIVCA or tetW and xre (DNA-binding transcriptional regulator) in L. amylovorus strains. Yellow arrow—CDS associated with mobility (e.g., IS—transposase), red arrow—tetW gene, teal arrow—CDS with functional annotation, gray arrow—hypothetical protein, orange—SpoIVCA site-specific recombinase, green rectangle—tetW regulatory protein (trp), blue arrow—CDS identified in other bacterial spp. (e.g., Treponema succinifaciens DSM 2489 or Victivallales bacterium CCUG 44730). The gray zones between sequences represent blastn sequence identity (generated by EasyFig).

Figure 4

Organization of CDS in the contigs harboring ermB gene. (A) contigs bearing omega transcriptional repressor (omtr) near ermB. (B) CDS pattern with mobilization protein (mp) near the ermB gene. (A) and (B): dark blue arrow—23S rRNA methyl transferase (rmt) and rRNA adenine methyltransferase gene (ramt), red arrow—ermB gene, teal arrow—CDS with functional annotation, gray arrow—hypothetical protein, pink arrow—omega transcriptional repressor (omtr), light blue rectangle—palindromatic sequences, yellow arrow—CDS associated with mobility (IS—transposase). The gray zones between sequences represent blastn sequence identity.