High-level colonisation of the human gut by Verrucomicrobia following broad-spectrum antibiotic treatment
Introduction
The repertoire of the human microbiome, its mutualism with the host and its consequences for health are essential challenges for microbiologists [1]. Different tools have been used to study the ecosystem of the human microbiome, yielding increasingly complex results. The composition of this ecosystem is influenced by diverse factors such as age, geographic provenance, environment and dietary habits [2] as well as the use of probiotics or antibiotics [2], [3]. In addition, alterations in the human gut have been suggested to play a role in several diseases [2]. Because 80% of bacteria in the human gut remain uncultured, metagenomic tools have been used to elucidate gut diversity [1]. Whilst molecular approaches are associated with bias, metagenomics is considered the gold standard for human gut studies [1].
In the 1960s, the effects of antibiotics on the human gut microbiota were largely studied with culture-dependent methods [3]. However, these studies focused on short courses of antibiotics and their impact on the few cultivable bacteria from the most studied phyla, such as Bacteroidetes, Proteobacteria and Firmicutes. The impact of an antibiotic on the gut microbiota depends on the spectrum of the agent, the dose, the route of administration, its pharmacokinetic and pharmacodynamic properties, and in vivo inactivation of the antibiotic. Microbial disturbances that occurred during antibiotherapy were reversible after treatment, although some patients needed months to recover [3], [4], [5]. Molecular tools, such as metagenomics and pyrosequencing, have yielded a broader view of the impact of antibiotics on the gut microflora. In all studies, a reduction in bacterial diversity was observed; therefore, modification of richness appears to be independent of the antimicrobial agent [3]. Thus, antibiotics such as vancomycin [6] and ciprofloxacin [7] were responsible for a critical reduction in the gut biomass. In other culture-dependent studies, no changes in richness were observed upon the use of antibiotics such as metronidazole [3].
The Verrucomicrobia phylum, which is phylogenetically closely related to Planctomycetes and Chlamydiae, is primarily comprised of environmental microorganisms [8]. In this phylum, Akkermansia muciniphila is a Gram-negative mucin-degrading bacterium that was first isolated from human faeces [9]. The genome of this bacterium was recently sequenced [10]. TaqMan® PCR, 454 sequencing and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting have been used to detect this species in stools from patients who received chemotherapy with or without concomitant antibiotics who were previously negative for the bacterium. Moreover, A. muciniphila, which co-colonises with Ruminococcus, enterotype 3 cluster, is suspected to contribute to the route used to generate energy from fermentable substrates [11]. To the best of our knowledge, the antibiotic susceptibility of A. muciniphila has not been studied to date.
As part of a larger study on gut microbiota from patients of various geographic origins and with various dietary habits [12], we report that the Verrucomicrobia phylotype was found unexpectedly in >40% of the total gut microbiota from two patients treated with broad-spectrum and extended antibiotherapy based on microbiota analyses, including pyrosequencing and electron microscopy.
Section snippets
Stool samples
Patient A was a 38-year-old male who was observed by one of our research team members (DR) for a Coxiella burnetii vascular infection complicated by an oesophageal fistula that required an antibiotic regimen combining doxycycline (400 mg/day), hydroxychloroquine (600 mg/day), piperacillin/tazobactam (12 g/day) and teicoplanin (600 mg/day) at the time of stool collection. Previous treatments for Patient A included imipenem, metronidazole and ciprofloxacin. Patient B was a 62-year-old man admitted
Pyrosequencing
For Patient A, who suffered a C. burnetii CB109 strain vascular infection (GenBank accession no. AY575667.1), pyrosequencing generated 106 043 reads. The abundance of reads classified in the phylum range was 42 592 reads from Firmicutes (53.46%), 36 463 from Verrucomicrobia (44.91%), 910 from Actinobacteria (1.12%), 381 from Proteobacteria (0.47%) and 36 from Bacteroidetes (0.044%). Phylotype analysis identified 47 different phylotypes corresponding to known species from six phyla and 33 genera.
Discussion
We serendipitously identified high-level colonisation by the Verrucomicrobia phylum in stools from patients receiving a broad-spectrum antibiotic regimen. Akkermansia muciniphila was the only species from this phylum that was present in both stools and was associated with up to 44.9% of the reads abundance in the first stool sample and up to 84.6% of the reads abundance in the second stool sample. Pyrosequencing of 16S rRNA amplicons was used to explore the gut microbiota and the V6 region was
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