Probiotics are defined as live microorganisms that, when ingested in appropriate numbers, exert a beneficial effect on the host health, including the prevention and treatment of some pathologies.1 It is generally accepted that a strain must satisfy a number of requirements to qualify as probiotic, such as the ability to survive to the gastrointestinal transit, adhere to mucus, and exhibit antimicrobial and immunostimulatory properties.2,3 According to the Official FEEDAP Scientific Opinion4 any bacterial strain carrying an acquired resistance to antimicrobial(s) that is shown to be due to chromosomal mutation(s) presents a low potential for horizontal spread and generally may be used for human nutrition.
Bifidobacterium longum is a microorganism present in the human gastrointestinal tract and is one of the 32 species that belong to the genus Bifidobacterium.5 It is considered an early colonizer of the gastrointestinal tract of infants5 and one of the major constituents of the infant’s intestinal microbiota, where it is predominant especially between 0 and 6 months. Among B. longum biotypes, B. longum W11 (LMG P-21586) has proven to be of particular interest. This biotype was investigated both in vitro and in vivo. B. longum W11 was characterized in vitro for its ability to drive the immune system response toward a pro-Th1 profile and thus potentially counteract the Th2-immune responses.6
Adhesion to human intestinal cells is considered an important property of probiotic bacteria, which has been selected for commercial use in food and in therapeutics. Adhesion enables probiotic strains to persist longer in the intestinal tract and, then, to stabilize the intestinal mucosal barrier, to provide competitive exclusion of pathogenic bacteria, and to better develop a metabolic and immune-modulatory activity. Several studies show that some strains of Bifidobacterium spp. can produce cell-bound or released exopolysaccharides and that exopolysaccharides production is a beneficial trait mediating for a proper anchoring to the intestinal epithelial cells7–9 and for commensal-host interaction through immune modulation.10,11
B. longum W11 was investigated for its production of exopolysaccharides, and the results obtained with SEM observation indicated that B. longum W11 is able to adhere to the HT-29 cell line and that the production of the exocellular polymers could be one of the factors contributing to its adhesion properties.12
In the last decade a rifamycin derivative, rifaximin, registered in many countries in Europe and the United States, has gained interest for its pharmacological, toxicological, and clinical characteristics. It has an excellent safety profile due to negligible intestinal absorption after oral administration.13 Its wide antimicrobial spectrum covers gram-positive and gram-negative bacteria, including aerobes and anaerobes.14
Rifaximin has been used successfully in the treatment of several intestinal disorders, including traveler’s diarrhea,15 diverticular disease,16 and small intestinal bacterial overgrowth.17 Recently, several publications have pointed out that rifaximin induces clinical remission of active Crohn’s disease.18–20
The efficacy of rifaximin is supported by alternative mechanisms of action that do not involve a direct bactericidal activity: alteration of virulence factors of enteric bacteria21; reduction of pathogen adhesion and internalization to the intestinal epithelium22; and reduction of inflammatory cytokine release.23,24
Moreover, such studies demonstrate that rifaximin does not disrupt the overall biostructure of the human microbiota, nor does it exert any cytotoxic or genotoxic activities, but it provokes changes in bacterial metabolism and bifidobacterial numbers that support a functional advantage to the host.25 Therefore, rifaximin could be a novel strategy for the treatment of intestinal diseases like irritable bowel syndrome and Crohn’s disease.
It is well known, however, that most antibiotic treatments can induce unfavorable side effects largely attributable to the disruption of the intestinal microbiota. The most widely discussed effect is antibiotic-associated diarrhea, a common and unintended consequence of antibiotic use.26 One strategy to diminish the negative side effects of antibiotic treatments is the parallel intake of probiotic bacteria. Anyway, even if some hours are let to pass between the intake of the antibiotic and the supplementation with probiotics, the positive impact of the latter is minimal as it is unable to affect the gut milieu and to integrate into the microbiota. In fact, the high sensitivity of the bacterial cells to the antibiotic molecule completely prevents a stable colonization of the intestine, thus ensuring only nonsignificant and transient effects.
In this way, the use of antibiotic-resistant beneficial bacteria could represent a good strategy, even if in this case a thorough documentation regarding the nontransmissibility of the resistance absolutely needs to be provided by the supplier.
In the light of this evidence and the suggested resistance to rifaximin carried by B. longum W11, the main aim of this study was to unequivocally demonstrate the nontransmissibility of the genes mediating the resistance to the antibiotic, therefore opening the possibility of a combined therapy with rifaximin and B. longum W11 that could be of particular relevance.
MATERIALS AND METHODS
EUCAST Disk Diffusion Method
An analysis was performed to verify whether the W11, B. longum, sample was resistant to rifaximin, an antibiotic of the group of rifamycins. As rifaximin is largely unabsorbed and remains localized in the gut, there is no formal resistance breakpoint for it. Most studies use a breakpoint of 32 mg/L between sensitive (32 mg/L) and resistant (32 mg/L) strains, although the manufacturer Alfa Wasserman recommends a resistance breakpoint of 64 mg/L.27 In this study, we used 5 different concentrations of antibiotics—32, 64, 128, 256, and 512 μg/mL—and tested rifaximin (Sigma, reconstituted in ethanol) in comparison with rifampicin, rifapentine (Sigma, reconstituted in H2O), and rifabutin (Sigma, reconstituted in DMSO), all rifamycin derivatives. The susceptibility of the B. longum W11 strain to antimicrobial agents was measured in comparison with the B. longum BL03 strain following the EUCAST guidelines.28 Bacteria were grown to the mid-log phase in MRS broth containing 0.05% L-cysteine-HCl. For inoculum preparation, test organisms were cultured for 24/48 hours anaerobically on Iso-sensitest agar (Oxoid) containing 0.05% L-cysteine-HCl at 37°C; bacteria were suspended in 0.9% saline solution to yield McFarland 0.5 and diluted 1:10 into the medium, so that the final test concentration of bacteria was approximately 1×106 CFU/mL. Susceptibility tests were performed by the disc diffusion method of Kirby-Bauer,28 and zones of inhibition were measured after 48 hours of incubation at 37°C.
Genomic DNA was isolated from the liquid cell culture of the W11 strain using the GenElute Bacterial Genomic DNA Kit according to the manufacturer’s instructions (Sigma-Aldrich, Milano, Italy).
All the tests and analyses pertaining to the W11 genome sequencing, the identification of the genes involved in the resistance to rifaximin, and the evaluation of their possible transmissibility were carried out by the Genomics Platform of PTP Science Park (Lodi, Italy).
The B. longum W11 genome was sequenced on Illumina MiSeq with a 250 PE reads module, generating a total of 4,568,832 paired-end reads, equal to a 800× coverage of the bacterial genome and 20,001,157 mate pair reads with an average insert size of 3 Kb. The raw sequencing reads, before the data processing, were trimmed to remove the sequencing primers, adapters, and low-quality bases using Trimmomatic software with the following parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 ILLUMINACLIP:Adapters.fa:2:30:10. Filtered sequencing reads were aligned with the reference B. longum genome (G CA_000730205.1) available from the latest version of the Ensemble Database (Ensembl Genomes v25), using BWA software. After mapping the reads with the reference bacterial genome, the alignment data were processed using FreeBayes software to perform the variation calling and identify the occurrence of mutations within the rpoB gene region. FreeBayes was run with the following parameters: ploidy 1; no-population-priors; no-mnps; no-indels; min-base-quality 30; min-mapping-quality 40; min-alternate-count 5; and min-coverage 10. The resulting VCF file was processed using SnpEff software to isolate the SNPs that could introduce amino acid changes in the protein sequence encoded by the rpoB gene.29 The search for transposable elements was carried out using TransposonePSI software (http://transposonpsi.sourceforge.net/) with default settings.
Antimicrobial Susceptibility Testing
In Figure 1, it is possible to see that the BL03 strain was inhibited by rifampicin, rifapentine, rifabutin, and rifaximin, at concentrations ranging from 32 to 512 μg/mL (100% susceptible). On the other side, the W11 strain was resistant to all drugs at concentrations ranging from 32 to 256 μg/mL and was inhibited by rifaximin, rifabutin, and rifaximin at a concentration of 512 μg/mL.
Mutations in rpoB
The mutations related to this resistance involve the rpoB gene (DNA-mediated RNA polymerase subunit beta) and are described in the literature.30,31 The analysis showed a mutation at the chromosomal DNA level, which causes a change in the triple of a specific amino acid (P564L) of the protein that leads to the resistance to rifamycin.
Figure 2 shows the protein sequence alignments of the rpoB genes for Escherichia coli (used as reference for the alignments), the reference B. longum (G CA_000730205.1), and the B. longum W11. Highlighted in red is Cluster II of the protein sequence, where rifamycin resistance mutations were described in the literature, whereas the positions of the amino acid changes on the 2 B. longum genomes are marked in blue. The arrow indicates the position of the mutation in the W11 rpoB gene (P to L), which is compatible with the rifamycin resistance.
Once the annotation and gene predictions were completed, the exact position of the rpoB gene was identified and a targeted search was conducted for transposable elements 200-Kbp upstream and downstream to the rpoB gene, using TransposonePSI software (http://transposonpsi.sourceforge.net/). No transposable elements were identified, confirming that the rpoB gene is not flanked by mobile genetic elements.
B. longum W11 is industrially manufactured according to a specific industrial fermentation process, which makes possible to englobe the bacterial cells in a protein matrix, thus rendering this probiotic more resistant to both gastroduodenal transit and stress that occurs during conservation of the finished product.
In the present paper, the bacterial genome of the B. longum W11 strain was fully sequenced using the NGS Illumina technology. Bioinformatics analysis of the assembled genome and its annotation highlighted the existence of a mutated rpoB gene in agreement with the observed resistance to rifamycin. Given the absence of transposable elements in the genomic area surrounding the rpoB gene, the resistance phenotype is to be considered stable.
According to this, B. longum W11 could be safely used in combined therapy with rifaximin, thus opening new focused frontiers in the probiotic era while preserving the necessary safety of use for consumers.
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