Frederiek-Maarten Kerckhof

Butyrate-producing Clostridium cluster XIVa species specifically colonize mucins in an in vitro gut model

The human gut is colonized by a complex microbiota with multiple benefits. Although the surface-attached, mucosal microbiota has a unique composition and potential to influence human health, it remains difficult to study in vivo. Therefore, we performed an in-depth microbial characterization (human intestinal tract chip (HITChip)) of a recently developed dynamic in vitro gut model, which simulates both luminal and mucosal gut microbes (mucosal-simulator of human intestinal microbial ecosystem (M-SHIME)). Inter-individual differences among human subjects were confirmed and microbial patterns unique for each individual were preserved in vitro. Furthermore, in correspondence with in vivo studies, Bacteroidetes and Proteobacteria were enriched in the luminal content while Firmicutes rather colonized the mucin layer, with Clostridium cluster XIVa accounting for almost 60% of the mucin-adhered microbiota. Of the many acetate and/or lactate-converting butyrate producers within this cluster, Roseburia intestinalis and Eubacterium rectale most specifically colonized mucins. These 16S rRNA gene-based results were confirmed at a functional level as butyryl-CoA:acetate-CoA transferase gene sequences belonged to different species in the luminal as opposed to the mucin-adhered microbiota, with Roseburia species governing the mucosal butyrate production. Correspondingly, the simulated mucosal environment induced a shift from acetate towards butyrate. As not only inter-individual differences were preserved but also because compared with conventional models, washout of relevant mucin-adhered microbes was avoided, simulating the mucosal gut microbiota represents a breakthrough in modeling and mechanistically studying the human intestinal microbiome in health and disease. Finally, as mucosal butyrate producers produce butyrate close to the epithelium, they may enhance butyrate bioavailability, which could be useful in treating diseases, such as inflammatory bowel disease.

Conceptualizing functional traits and ecological characteristics of methane-oxidizing bacteria as life strategies

Methane-oxidizing bacteria (MOB) possess the ability to use methane for energy generation and growth, thereby, providing a key ecosystem service that is highly relevant to the regulation of the global climate. MOB subgroups have different responses to key environmental controls, reflecting on their functional traits. Their unique features (C1-metabolism, unique lipids and congruence between the 16S rRNA and pmoA gene phylogeny) have facilitated numerous environmental studies, which in combination with the availability of cultured representatives, yield the most comprehensive ecological picture of any known microbial functional guild. Here, we focus on the broad MOB subgroups (type I and type II MOB), and aim to conceptualize MOB functional traits and observational characteristics derived primarily from these environmental studies to be interpreted as microbial life strategies. We focus on the functional traits, and the conditions under which these traits will render different MOB subgroups a selective advantage. We hypothesize that type I and type II MOB generally have distinct life strategies, enabling them to predominate under different conditions and maintain functionality. The ecological characteristics implicated in their adopted life strategies are discussed, and incorporated into the Competitor-Stress tolerator-Ruderal functional classification framework as put forward for plant communities. In this context, type I MOB can broadly be classified as competitor-ruderal while type II MOB fit more within the stress tolerator categories. Finally, we provide an outlook on MOB applications by exemplifying two approaches where their inferred life strategies could be exploited thereby, putting MOB into the context of microbial resource management.

Absolute quantification of microbial taxon abundances

High-throughput amplicon sequencing has become a well-established approach for microbial community profiling. Correlating shifts in the relative abundances of bacterial taxa with environmental gradients is the goal of many microbiome surveys. As the abundances generated by this technology are semi-quantitative by definition, the observed dynamics may not accurately reflect those of the actual taxon densities. We combined the sequencing approach (16S rRNA gene) with robust single-cell enumeration technologies (flow cytometry) to quantify the absolute taxon abundances. A detailed longitudinal analysis of the absolute abundances resulted in distinct abundance profiles that were less ambiguous and expressed in units that can be directly compared across studies. We further provide evidence that the enrichment of taxa (increase in relative abundance) does not necessarily relate to the outgrowth of taxa (increase in absolute abundance). Our results highlight that both relative and absolute abundances should be considered for a comprehensive biological interpretation of microbiome surveys.

Chronic cigarette smoke exposure induces microbial and inflammatory shifts and mucin changes in the murine gut

Inflammatory bowel diseases (IBD) are complex multifactorial diseases characterized by an inappropriate host response to an altered commensal microbiome and dysfunctional mucus barrier. Cigarette smoking is the best known environmental risk factor in IBD. Here, we studied the influence of chronic smoke exposure on the gut microbiome, mucus layer composition and immune factors in conventional mice. We compared smoke-exposed with air-exposed mice (n = 12) after a smoke exposure of 24 weeks. Both Illumina sequencing (n = 6) and denaturing gradient gel electrophoresis (n = 12) showed that bacterial activity and community structure were significantly altered in the colon due to smoke exposure. Interestingly, an increase of Lachnospiraceae sp. activity in the colon was observed. Also, the mRNA expression of Muc2 and Muc3 increased in the ileum, whereas Muc4 increased in the distal colon of smoke-exposed mice (n = 6). Furthermore, we observed increased Cxcl2 and decreased Ifn-γ in the ileum, and increased Il-6 and decreased Tgf-β in the proximal colon. Tight junction gene expression remained unchanged. We infer that the modulating role of chronic smoke exposure as a latently present risk factor in the gut may be driven by the altered epithelial mucus profiles and changes in microbiome composition and immune factors.

Optimized Cryopreservation of Mixed Microbial Communities for Conserved Functionality and Diversity

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at −80°C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

Characterization of Staphylococcus and Corynebacterium Clusters in the Human Axillary Region

The skin microbial community is regarded as essential for human health and well-being, but likewise plays an important role in the formation of body odor in, for instance, the axillae. Few molecular-based research was done on the axillary microbiome. This study typified the axillary microbiome of a group of 53 healthy subjects. A profound view was obtained of the interpersonal, intrapersonal and temporal diversity of the human axillary microbiota. Denaturing gradient gel electrophoresis (DGGE) and next generation sequencing on 16S rRNA gene region were combined and used as extent to each other. Two important clusters were characterized, where Staphylococcus and Corynebacterium species were the abundant species. Females predominantly clustered within the Staphylococcus cluster (87%, n = 17), whereas males clustered more in the Corynebacterium cluster (39%, n = 36). The axillary microbiota was unique to each individual. Left-right asymmetry occurred in about half of the human population. For the first time, an elaborate study was performed on the dynamics of the axillary microbiome. A relatively stable axillary microbiome was noticed, although a few subjects evolved towards another stable community. The deodorant usage had a proportional linear influence on the species diversity of the axillary microbiome.

Conversion of biogas to bioproducts by algae and methane oxidizing bacteria.

Biogas produced by anaerobic digestion is typically converted into electricity and low value heat. In this study, biogas is microbially transformed into valuable bioproducts. As proof of principle, the production of feed additives, i.e. lipids and polyhydroxybutyrate, out of biogas was evaluated. In a first stage, the CO₂ in a synthetic biogas was photosynthetically fixed by an algae Scenedesmus sp. culture at an average rate of 192 ± 9 mg CO₂ L⁻¹ liquid d⁻¹, resulting in concomitant O₂ production. After N-depletion, more than 30% of the 220 ± 7 mg lipids g⁻¹ total organic carbon were unsaturated. In a second stage, the theoretical resulting gas mixture of 60% CH₄ and 40% O₂ was treated by a methane oxidizing Methylocystis parvus culture, with oxidation rates up to 452 ± 7 mg⁻¹ CH₄-C L⁻¹ liquid d⁻¹. By repeated N-limitation, concentrations of 295 ± 50 mg intracellular polyhydroxybutyrate g⁻¹ cell dry weight were achieved. Finally, a one-stage approach with controlled coculturing of both microbial groups resulted in harvestable bioflocs. This is the first time that a total microbial conversion of both greenhouse gases into biomass was achieved without external O₂ provision. Based on these results, a biotechnological approach is discussed whereby all kinds of biogas can be transformed into valuable bioproducts.

Biotic Interactions in Microbial Communities as Modulators of Biogeochemical Processes: Methanotrophy as a Model System

Microbial interaction is an integral component of microbial ecology studies, yet the role, extent, and relevance of microbial interaction in community functioning remains unclear, particularly in the context of global biogeochemical cycles. While many studies have shed light on the physico-chemical cues affecting specific processes, (micro)biotic controls and interactions potentially steering microbial communities leading to altered functioning are less known. Yet, recent accumulating evidence suggests that the concerted actions of a community can be significantly different from the combined effects of individual microorganisms, giving rise to emergent properties. Here, we exemplify the importance of microbial interaction for ecosystem processes by analysis of a reasonably well-understood microbial guild, namely, aerobic methane-oxidizing bacteria (MOB). We reviewed the literature which provided compelling evidence for the relevance of microbial interaction in modulating methane oxidation. Support for microbial associations within methane-fed communities is sought by a re-analysis of literature data derived from stable isotope probing studies of various complex environmental settings. Putative positive interactions between active MOB and other microbes were assessed by a correlation network-based analysis with datasets covering diverse environments where closely interacting members of a consortium can potentially alter the methane oxidation activity. Although, methanotrophy is used as a model system, the fundamentals of our postulations may be applicable to other microbial guilds mediating other biogeochemical processes.

Exploration and prediction of interactions between methanotrophs and heterotrophs

Methanotrophs can form the basis of a methane-driven food web on which heterotrophic microorganisms can feed. In return, these heterotrophs can stimulate growth of methanotrophs in co-culture by providing growth additives. However, only a few specific interactions are currently known. We incubated nine methanotrophs with 25 heterotrophic strains in a pairwise miniaturized co-cultivation setup. Through principal component analysis and k-means clustering, methanotrophs and heterotrophs could be grouped according to their interaction behaviour, suggesting strain-dependent methanotroph-heterotroph complementarity. Co-cultivation significantly enhanced the growth parameters of three methanotrophs. This was most pronounced for Methylomonas sp. M5, with a threefold increase in maximum density and a fourfold increase in maximum increase in density in co-culture with Cupriavidus taiwanensis LMG 19424. In contrast, co-cultivation with Methylobacterium radiotolerans LMG 2269 and Pseudomonas aeruginosa LMG 12228 inhibited growth of most methanotrophs. Functional genomic analysis suggested the importance of vitamin metabolism for co-cultivation success. The generated data set was then successfully exploited as a proof-of-principle for predictive modelling of co-culture responses based on other interactions of the same heterotrophs and methanotrophs, yielding values of the area under the receiver operating characteristic curve of 0.73 upon 50% missing values for the maximum increase in density parameter. As such, these modelling-based tools were shown to hold great promise in reducing the amount of data that needs to be generated when conducting large co-cultivation studies.

Strain-Specific Transfer of Antibiotic Resistance from an Environmental Plasmid to Foodborne Pathogens

Pathogens resistant to multiple antibiotics are rapidly emerging, entailing important consequences for human health. This study investigated if the broad-host-range multiresistance plasmid pB10, isolated from a wastewater treatment plant, harbouring amoxicillin, streptomycin, sulfonamide, and tetracycline resistance genes, was transferable to the foodborne pathogens Salmonella spp. or E. coli O157:H7 and how this transfer alters the phenotype of the recipients. The transfer ratio was determined by both plating and flow cytometry. Antibiotic resistance profiles were determined for both recipients and transconjugants using the disk diffusion method. For 14 of the 15 recipient strains, transconjugants were detected. Based on plating, transfer ratios were between 6.8 × 10−9 and 3.0 × 10−2 while using flow cytometry, transfer ratios were between <1.0 × 10−5 and 1.9 × 10−2. With a few exceptions, the transconjugants showed phenotypically increased resistance, indicating that most of the transferred resistance genes were expressed. In summary, we showed that an environmental plasmid can be transferred into foodborne pathogenic bacteria at high transfer ratios. However, the transfer ratio seemed to be recipient strain dependent. Moreover, the newly acquired resistance genes could turn antibiotic susceptible strains into resistant ones, paving the way to compromise human health.