Amoxicillin-Clavulanic Acid Resistance in the Genus Bifidobacterium

Abstract

Amoxicillin-clavulanic acid (AMC) is one of the most frequently prescribed antibiotic formulations in the Western world. Extensive oral use of this antimicrobial combination influences the gut microbiota. One of the most abundant early colonizers of the human gut microbiota is represented by different taxa of the Bifidobacterium genus, which include many members that are considered to bestow beneficial effects upon their host. In the current study, we investigated the impact of AMC administration on the gut microbiota composition, comparing the gut microbiota of 23 children that had undergone AMC antibiotic therapy to that of 19 children that had not been treated with antibiotics during the preceding 6 months. Moreover, we evaluated AMC sensitivity by MIC test of 261 bifidobacterial strains, including reference strains for the currently recognized 64 bifidobacterial (sub)species, as well as 197 bifidobacterial isolates of human origin. These assessments allowed the identification of four bifidobacterial strains that exhibit a high level of AMC insensitivity, which were subjected to genomic and transcriptomic analyses to identify the putative genetic determinants responsible for this AMC insensitivity. Furthermore, we investigated the ecological role of AMC-resistant bifidobacterial strains by in vitro batch cultures.IMPORTANCE Based on our results, we observed a drastic reduction in gut microbiota diversity of children treated with antibiotics, which also affected the abundance of Bifidobacterium, a bacterial genus commonly found in the infant gut. MIC experiments revealed that more than 98% of bifidobacterial strains tested were shown to be inhibited by the AMC antibiotic. Isolation of four insensitive strains and sequencing of their genomes revealed the identity of possible genes involved in AMC resistance mechanisms. Moreover, gut-simulating in vitro experiments revealed that one strain, i.e., Bifidobacterium breve PRL2020, is able to persist in the presence of a complex microbiota combined with AMC antibiotic.

Keywords: antibiotics; bifidobacteria; comparative genomics.

FIG 1

Evaluation of the microbiota composition of amoxicillin-clavulanic acid (AMC) and control (CTRL) samples. (a) Whisker plot based on observed OTUs identified from AMC and CTRL samples. The x axis represents the different groups, while the y axis indicates the number of observed operational taxonomic units (OTUs). The boxes represent 50% of the data set, distributed between the first and the third quartiles. The median divides the boxes into the interquartile range, while “X” represents the mean. (b) Microbiota composition of AMC and CTRL samples based on 16S rRNA profiling normalized with a quantitative microbiome profiling approach employing flow cytometric enumeration of microbial cells for each sample. The x axis represents the different analyzed samples, while the y axis indicates normalized OTUs for each sample. (c) Beta diversity in AMC and CTRL samples. The predicted PCoA results encompassing all 43 analyzed samples are reported and the different clusters are represented by different colors.

FIG 2

Comparative genomic and resistome analyses of AMC-resistant Bifidobacterium strains. (a) Venn diagrams. The numbers in the central circles represent the numbers of genes in the core genomes of the B. breve and B. longum subsp. longum strains analyzed. The numbers in the overlapping sections represent the numbers of shared genes between AMC-resistant strains, while the numbers in the ovals depict the number of TUGs for each strain. (b) Predicted resistomes of the AMC-resistant Bifidobacterium strains.

FIG 3

Transcriptional modulation of B. breve PRL2020 genes in the presence of AMC. The heatmap displays the subset of significantly upregulated encoding genes. Red boxes highlight the ORFs PRL2020_1181, PRL2020_1167, and PRL2020_1282, which are putatively involved in the AMC resistance mechanism. The color scale (green) at the top of the figure indicates increased transcription levels compared to those of the reference samples. The EggNOG letter for each significant upregulated gene is reported. Each letter stands for a function, as follows: S, function unknown; L, replication, recombination, and repair; R, general function prediction only; G, carbohydrate transport and metabolism; J, translation, ribosomal structure, and biogenesis; E, amino acid transport and metabolism; K, transcription; P, inorganic ion transport and metabolism; C, energy production and conversion; V, defense mechanisms; T, signal transduction mechanisms; H, coenzyme transport and metabolism; and I, lipid transport and metabolism.

FIG 4

Growth experiments in fecal medium. (a) Comparison of the absolute abundances of the coculture experiments in MRS_AMC and the reference condition. (b) Relative percentage difference between AMC-treated and untreated samples based on the absolute abundance of the bacterial species corresponding to the strain analyzed (B. breve for PRL2020 and M1D strains and B. longum for 39B and 1898B strains).