Modelling upper respiratory tract diseases: getting grips on host-microbe interactions in chronic rhinosinusitis using in vitro technologies

Chronic rhinosinusitis or CRS is a widespread disease that occurs in about 11 % of the adult population. However, the exact causes and mechanisms behind the development of this disease are not yet fully understood. Possible causes are an inadequate or excessive reaction of the immune system, or a loss of the balance within the microbial community in the human nose and sinuses and the balance between this sinonasal microbial community and the host’s epithelium. Investigation of these hypotheses, with the final goal to improve treatment methods, requires more realistic laboratory models of the sinonasal epithelium, as well as of the microbial community that is present on this epithelium, and last but not least, the interaction between both.

In this article, published in the journal Microbiome, we provided an overview of the currently available laboratory models to mimic and investigate the human nose and sinus epithelium, as well as the interactions among a number of microbes present in this niche, and between these microorganisms and their human host. Remarkably, most researchers investigate interactions with one or a limited number of microorganisms, whereas in reality a complex microbial community is present in the nose and sinuses. For this reason, we propose strategies to develop improved model systems which take into account the healthy or diseased state of the human host epithelium, being the environment in which the microbes are present, as well as the rich microbial community itself. It is indeed in this complex environment that disease environment occurs and in which it will be treated.

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Comparing the healthy nose and nasopharynx microbiota reveals continuity as well as niche- specificity

How can bacteria in the respiratory tract maintain our health and prevent infections from occurring? To answer this question, a better characterization of the collection of bacteria that are present in this human body niche is necessary. A good starting point is the identification of all bacteria that are present under healthy conditions. Therefore, we set up a large-scale citizen-science study, where we collected samples from 100 enthusiastic healthy volunteers. These volunteers were willing to donate a swab sample of their nose and nasopharynx. The bacterial DNA from all these samples was collected.

After processing of all these samples, we got a better idea about the bacteria that are present in the nose of healthy people. Our results, published in Frontiers in Microbiology, show that overall, the healthy nose and nasopharynx are mostly dominated by only a few bacterial species. Furthermore, these bacterial profiles in the nasopharynx could be grouped into at least four bacterial types (you can compare this to blood types) dependent on the bacterium that is most present: a type dominated by Moraxella, by Streptococcus or by Fusobacterium, and finally a mixed type of Staphylococcus, Dolosigranulum and Corynebacterium. Almost all individuals could be grouped into one of these four bacterial types. Interestingly, in the nose, only the Moraxella and the mixed type were found.

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Enhancing the viability of Lactobacillus rhamnosus GG after spray drying and during storage.

Increasing knowledge about the human microbiome has led to a growing awareness of the potential of applying probiotics to improve our health. The pharmaceutical industry shows an emerging interest in pharmaceutical formulations containing these beneficial microbes, the so-called pharmabiotics. An important manufacturing step is the drying of the probiotics, as this can increase the stability and shelf life of the finished pharmabiotic product. Unfortunately, drying also puts stress on microbial cells, thus causing a decrease in viability.

In this research article, published in the International Journal of Pharmaceutics, we aimed to examine the effect of different drying media and protective excipients on the viability of the prototype probiotic strain Lactobacillus rhamnosus GG after spray drying and during subsequent storage for 28 weeks. The presence of phosphates in the drying medium showed to have a superior protective effect, especially during long-term storage at room temperature. Addition of lactose or trehalose resulted in significantly improved survival rates after drying as well as during long-term storage for the tested excipients. Both disaccharides are characterized by a high glass transition temperature. Maltodextrin showed less protective capacities compared to lactose and trehalose in all tested conditions. The usage of mannitol or dextran resulted in sticky powders and low yields, so further testing was not possible. In addition to optimizing the viability, future research will also explore the functionality of cellular probiotic components after spray drying in order to safeguard the probiotic activity of the formulated pharmabiotics.

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Multifactorial inhibition of lactobacilli against the respiratory tract pathogen Moraxella catarrhalis

Probiotics, mainly lactic acid bacteria (LAB), are widely focused on gastrointestinal applications. However, recent microbiome studies indicate that LAB can be endogenous members of other human body sites such as the upper respiratory tract (URT). Interestingly, DNA-based microbiome research suggests an inverse correlation between the presence of LAB and the occurrence of important URT pathogens such as Moraxella catarrhalis which linked to otitis media, sinusitis and chronic obstructive pulmonary disease. However, a direct interaction between these microbes has not been explored in detail. Our results, now published in ‘Beneficial Microbes’ demonstrate that many of the Lactobacillus strains tested, exhibit antipathogenic activities against M. catarrhalis using agar-based assays, time course analysis, biofilm assays and MIC testing. Lactic acid was shown to be a key metabolite in these activities. In addition, cell line assays for adhesion competition and immunomodulation were used to substantiate the inhibitory effect of lactobacilli against M. catarrhalis. The well-documented strain Lactobacillus rhamnosus GG was shown to decrease the adhesion of M. catarrhalis to Calu-3 nasopharyngeal cells and to inhibit inflammation markers which were activated by M. catarrhalis.

This study suggests that several lactobacilli and their key metabolite lactic acid are possible candidates for probiotic therapeutic interventions against URT infections.

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Large-scale genome analysis of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus reveals taxonomic inconsistencies

The closely related species of the Lactobacillus casei group (L. casei, L. paracasei and L. rhamnosus) are extensively studied because of their applications in food fermentations and as probiotics. Our results, now published in mSystems, show that many strains in this group are incorrectly classified. Surprisingly many bacteria classified as L. casei are misclassified and should be relabeled as L. paracasei instead. We found that reclassifying them to their most closely related type strain improves the functional predictive power of their taxonomy.

In addition, our findings may spark increased interest in the L. casei species. We found that after reclassification, only 10 genomes remain classified as L. casei. Moreover, these strains show very interesting properties. First, they all appear to be catalase positive. This suggests that they have an increased oxidative stress resistance. Second, we isolated a L. casei strain, AMBR2, from the human upper respiratory tract and discovered that it harbors one or even two large, glycosylated putative surface adhesins. This might inspire further exploration of this strain as a potential probiotic organism.

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Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics.

The increasing knowledge about the human microbiome leads to the awareness of how important probiotics can be for our health. Although further substantiation is required, it appears that several pathologies could be treated or prevented by the administration of pharmaceutical formulations containing such live health-beneficial bacteria. These pharmabiotics need to provide their effects until the end of shelf life, which can be optimally achieved by drying them before further formulation. However, drying processes, including spray-, freeze-, vacuum- and fluidized bed drying, induce stress on probiotics, thus decreasing their viability. Several protection strategies can be envisaged to enhance their viability, including addition of protective agents, controlling the process parameters and prestressing the probiotics prior to drying. Moreover, probiotic viability needs to be maintained during long-term storage. Overall, lower storage temperature and low moisture content result in good survival rates. Attention should also be given to the rehydration conditions of the dried probiotics, as this can exert an important effect on their revival. By describing not only the characteristics, but also the viability results obtained by the most relevant drying techniques in the probiotic industry, we hope to facilitate the deliberate choice of drying process and protection strategy for specific probiotic and pharmabiotic applications.

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