Bifidobacterium breve PRL2020: Antibiotic-Resistant Profile and Genomic Detection of Antibiotic Resistance Determinants

Abstract

Antibiotics are one of the greatest scientific achievements of modern medicine, but excessive use is creating challenges for the future of medicine. Antibiotic resistance (AR) is thought to cause changes in bowel habits and an increased risk of gastroenteritis, but it may also increase the risk of overweight, obesity, autoimmune and atopic diseases, and a low response to vaccines and cancer, likely mediated by antibiotic-induced gut dysbiosis. Probiotic add-on therapy could partially prevent antibiotic-induced gut dysbiosis, but their antibiotic sensitivity features likely limits this potential. The EFSA (European Food Safety Authority) guidelines consider the use of probiotics whose antibiotic-resistant profile could be transferable an important hazard. Recently, a strain of B. breve (PRL2020) has shown to be resistant to amoxicillin and amoxicillin-clavulanate (AC) by applying the microdilution protocol according EFSA guidelines. After verifying that horizontal gene transfer is unlikely to take place, this feature suggests its concomitant use with these specific antibiotics. The results of our tests demonstrated that the strain PRL2020 is indeed endowed with amoxicillin- and AC-resistant properties and that it is also insensitive to ampicillin. In-depth analysis of the annotated genome sequence of B. breve PRL2020 was employed to query the Comprehensive Antibiotic Resistance Database (CARD) using Resistance Gene Identifier (RGI) software (version 5.2.1). The similarity among the AR determinants found was studied through nucleotide sequence alignment, and it was possible to verify not only the absence of genes explaining these features in the flanking regions but also the presence of genetic sequences (rpoB and erm(X)) putatively responsible for rifampicin and erythromycin resistance. Both features are not phenotypically expressed, and for these antibiotics, the strain is within the EFSA limits. Analysis of the flanking regions of these genes revealed possible mobile elements upstream and downstream only in the case of the erm(X) gene, but the features of the Insertion Sequences (IS) are described as not to cause horizontal transfer. Our findings on strain PRL2020 demonstrate that its AR profile is compatible with antibiotics when taken with the aim of reducing the risk of dysbiosis.

Keywords: LMG S-32458; amoxicillin; ampicillin; clavulanic acid; erythromycin; microbiota; probiotics; rifaximin.

Figure 1

Molecular (BoxAR1) typing results obtained to confirm the singular pattern of different colonies of B. breve PRL2020. In lines 1 and 2: PRL2020 grown on TOS-MUP agar plates; in line 3: PRL2020 grown on agar plates with clindamycin at 8 µg/mL; in line 4: PRL2020 grown on agar plates with erythromycin at 0.25 µg/mL; in line 5: PRL2020 grown on agar plates with clindamycin at 4 µg/mL and erythromycin at 0.25 µg/mL; in line 6: PRL2020 grown from titres with erythromycin at 0.25 µg/mL; in line 7: PRL2020 grown from titres with clindamycin at 8 µg/mL; in line 8: PRL2020 grown on TOS-MUP agar plates with amoxicillin at 8 µg/mL; in line 9: PRL2020 grown on TOS-MUP agar plates with amoxicillin at 16 µg/mL. M: marker for 200 bp; TOS: transgalactosylated oligosaccharides; MUP: mupirocin.

Figure 2

Alignment of the locus IHV18_09970 of B. breve PRL2020 with wild-type B. adolescentis available in CARD (GenBank Acc. No: WP_041777404.1). The mutation sites conferring rifampicin resistance are highlighted in green, while the mutated residues potentially involved in resistance to rifampicin found in B. breve PRL2020 compared to the sequence of B. adolescentis are reported in red. Cluster 1, 2 and 3 are highlighted by blue, orange, and grey rectangles, respectively.

Figure 3

Flanking regions (±30 Kb) of the rpoB gene in B. breve PRL2020. The flanking regions (in blue, genes coding for functional proteins) of the locus IHV18_09970 (rpoB) (in red), identified by CARD analysis as putative rifampicin resistance determinant, do not reveal the presence of any genes linked to mobile genetic elements within the 60 Kb analysed. In grey, genes coding for hypothetical proteins.

Figure 4

Flanking regions (±30 Kb) of the erm(X) gene in B. breve PRL2020 (contig 5). Flanking regions of the loci IHV18_06605 and IHV18_06625 [erm(X), in red], which were identified by CARD analysis as putative erythromycin resistance determinants. In blue, genes coding for functional proteins; in yellow, genes coding for transposases; and in grey, genes encoding for hypothetical proteins.

Figure 5

Flanking regions (±30 Kb) of the erm(X) gene in B. breve PRL2020 (contig 4). Flanking regions of the locus IHV18_03440 [erm(X), in red], which were identified by CARD analysis as putative erythromycin resistance determinants. In blue, genes coding for functional proteins; in yellow, genes coding for transposases; and in grey, genes encoding for hypothetical proteins.

Figure 6

Protein sequence alignment of the locus IHV18_08715 of B. breve PRL2020 (MB196), with the bcrA sequence retrieved from the genome sequence of strain B. breve JCM 7017.