Antimicrobial potential of known and novel probiotics on in vitro periodontitis biofilms.

Several oral diseases are characterized by a shift within the oral microbiome towards a pathogenic, dysbiotic composition. Broad-spectrum antimicrobials are often part of patient care. However, because of the rising antibiotic resistance, alternatives are increasingly desirable. Alternatively, supplying beneficial species through probiotics is increasingly showing favorable results. Unfortunately, these probiotics are rarely evaluated comparatively. In this study, the in vitro effects of three known and three novel Lactobacillus strains, together with four novel Streptococcus salivarius strains were comparatively evaluated for antagonistic effects on proximal agar growth, antimicrobial properties of probiotic supernatant and the probiotic's effects on in vitro periodontal biofilms. Strain-specific effects were observed as differences in efficacy between genera and differences within genera. While some of the Lactobacillus candidates were able to reduce the periodontal pathobiont A. actinomycetemcomitans, the S. salivarius strains were not. However, the S. salivarius strains were more effective against periodontal pathobionts P. intermedia, P. gingivalis, and F. nucleatum. Vexingly, most of the Lactobacillus strains also negatively affected the prevalence of commensal species within the biofilms, while this was lower for S. salivarius strains. Both within lactobacilli and streptococci, some strains showed significantly more inhibition of the pathobionts, indicating the importance of proper strain selection. Additionally, some species showed reductions in non-target species, which can result in unexpected and unexplored effects on the whole microbiome.

Development of a live biotherapeutic throat spray with lactobacilli targeting respiratory viral infections.

Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut-targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the formulation of a throat spray with live lactobacilli exhibiting several in vitro mechanisms of action against respiratory viral infections, including induction of interferon regulatory pathways and direct inhibition of respiratory viruses. Rational selection of Lactobacillaceae strains was based on previously documented beneficial properties, up-scaling and industrial production characteristics, clinical safety parameters, and potential antiviral and immunostimulatory efficacy in the URT demonstrated in this study. Using a three-step selection strategy, three strains were selected and further tested in vitro antiviral assays and in formulations: Lacticaseibacillus casei AMBR2 as a promising endogenous candidate URT probiotic with previously reported barrier-enhancing and anti-pathogenic properties and the two well-studied model strains Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 that display immunomodulatory capacities. The three strains and their combination significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV-229E coronavirus in co-culture models with bacteria, virus, and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway-inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form. Thus, the developed spray with live lactobacilli will be further explored in the clinic as a potential broad-acting live biotherapeutic strategy against respiratory viral diseases.

Randomized, Double-Blind, Placebo-Controlled Trial of a Throat Spray with Selected Lactobacilli in COVID-19 Outpatients.

Primary care urgently needs treatments for coronavirus disease 2019 (COVID-19) patients because current options are limited, while these patients who do not require hospitalization encompass more than 90% of the people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a throat spray containing three Lactobacillaceae strains with broad antiviral properties in a randomized, double-blind, placebo-controlled trial. Before the availability of vaccines, 78 eligible COVID-19 patients were randomized to verum (n = 41) and placebo (n = 37) within 96 h of a positive PCR-based SARS-CoV-2 diagnosis, and a per-protocol analysis was performed. Symptoms and severity were reported daily via an online diary. Combined nose-throat swabs and dried blood spots were collected at regular time points in the study for microbiome, viral load, and antibody analyses. The daily reported symptoms were highly variable, with no added benefit for symptom resolution in the verum group. However, based on 16S V4 amplicon sequencing, the acute symptom score (fever, diarrhea, chills, and muscle pain) was significantly negatively associated with the relative abundance of amplicon sequence variants (ASVs) that included the applied lactobacilli (P < 0.05). Furthermore, specific monitoring of these applied lactobacilli strains showed that they were detectable via quantitative PCR (qPCR) analysis in 82% of the patients in the verum group. At the end of the trial, a trend toward lower test positivity for SARS-CoV-2 was observed for the verum group (2/30; 6.7% positive) than for the placebo group (7/27; 26% positive) (P = 0.07). These data indicate that the throat spray with selected antiviral lactobacilli could have the potential to reduce nasopharyngeal viral loads and acute symptoms but should be applied earlier in the viral infection process and substantiated in larger trials. IMPORTANCE Viral respiratory tract infections result in significant health and economic burdens, as highlighted by the COVID-19 pandemic. Primary care patients represent 90% of those infected with SARS-CoV-2, yet their treatment options are limited to analgesics and antiphlogistics, and few broadly acting antiviral strategies are available. Microbiome or probiotic therapy is a promising emerging treatment option because it is based on the multifactorial action of beneficial bacteria against respiratory viral disease. In this study, an innovative topical throat spray with select beneficial lactobacilli was administered to primary COVID-19 patients. A remote study setup (reducing the burden on hospitals and general practitioners) was successfully implemented using online questionnaires and longitudinal self-sampling. Our results point toward the potential mechanisms of action associated with spray administration at the levels of viral loads and microbiome modulation in the upper respiratory tract and pave the way for future clinical applications of beneficial bacteria against viral diseases.

Selective targeting of skin pathobionts and inflammation with topically applied lactobacilli.

Tailored skin microbiome modulation approaches with probiotics are highly challenging. Here, we show that lactobacilli are underestimated members of the skin microbiota. We select specific strains of nomadic lactobacilli for their functional applicability on the skin and capacity to inhibit growth and inflammation by skin pathobionts. The strains are formulated as microcapsules for topical formulations and tested in patients with mild-to-moderate acne. The selected lactobacilli are able to reduce inflammatory lesions in a pilot and placebo-controlled study. Daily application for 8 weeks is associated with an in vivo temporary modulation of the microbiome, including a reduction in relative abundance of staphylococci and Cutibacterium acnes, and an increase in lactobacilli. The reduction in inflammatory lesions is still apparent 4 weeks after the topical application of the lactobacilli ended, indicating a possible additional immunomodulatory effect. This study shows that carefully selected and formulated lactobacilli are a viable therapeutic option for common acne lesions.

Microbial enrichment and storage for metagenomics of vaginal, skin, and saliva samples.

Few validated protocols are available for large-scale collection, storage, and analysis of microbiome samples from the vagina, skin, and mouth. To prepare for a large-scale study on the female microbiome by remote self-sampling, we investigated the impact of sample collection, storage, and host DNA depletion on microbiome profiling. Vaginal, skin, and saliva samples were analyzed using 16S rRNA gene amplicon and metagenomic shotgun sequencing, and qPCR. Of the two tested storage buffers, the eNAT buffer could keep the microbial composition stable during various conditions. All three tested host DNA-depletion approaches showed a bias against Gram-negative taxa. However, using the HostZERO Microbial DNA and QIAamp DNA Microbiome kits, samples still clustered according to body site and not by depletion approach. Therefore, our study showed the effectiveness of these methods in depleting host DNA. Yet, a suitable approach is recommended for each habitat studied based on microbial composition.

Cotton and Surgical Face Masks in Community Settings: Bacterial Contamination and Face Mask Hygiene.

During the current COVID-19 pandemic, the use of face masks has become increasingly recommended and even mandatory in community settings. To evaluate the risk of bacterial cross-contamination, this study analyzed the bacterial bioburden of disposable surgical masks and homemade cotton masks, and surveyed the habits and face mask preferences of the Flemish population. Using culture approaches and 16S rRNA gene amplicon sequencing, we analyzed the microbial community on surgical and/or cotton face masks of 13 healthy volunteers after 4 h of wearing. Cotton and surgical masks contained on average 1.46 × 105 CFU/mask and 1.32 × 104 CFU/mask, respectively. Bacillus, Staphylococcus, and Acinetobacter spp. were mostly cultured from the masks and 43% of these isolates were resistant to ampicillin or erythromycin. Microbial profiling demonstrated a consistent difference between mask types. Cotton masks mainly contained Roseomonas, Paracoccus, and Enhydrobacter taxa and surgical masks Streptococcus and Staphylococcus. After 4 h of mask wearing, the microbiome of the anterior nares and the cheek showed a trend toward an altered beta-diversity. According to dedicated questions in the large-scale Corona survey of the University of Antwerp with almost 25,000 participants, only 21% of responders reported to clean their cotton face mask daily. Laboratory results indicated that the best mask cleaning methods were boiling at 100°C, washing at 60°C with detergent or ironing with a steam iron. Taken together, this study suggests that a considerable number of bacteria, including pathobionts and antibiotic resistant bacteria, accumulate on surgical and even more on cotton face masks after use. Based on our results, face masks should be properly disposed of or sterilized after intensive use. Clear guidelines for the general population are crucial to reduce the bacteria-related biosafety risk of face masks, and measures such as physical distancing and increased ventilation should not be neglected when promoting face mask use.

The role of lactobacilli in inhibiting skin pathogens.

The human skin microbiota forms a key barrier against skin pathogens and is important in modulating immune responses. Recent studies identify lactobacilli as endogenous inhabitants of healthy skin, while inflammatory skin conditions are often associated with a disturbed skin microbiome. Consequently, lactobacilli-based probiotics are explored as a novel treatment of inflammatory skin conditions through their topical skin application. This review focuses on the potential beneficial role of lactobacilli (family Lactobacillaceae) in the skin habitat, where they can exert multifactorial local mechanisms of action against pathogens and inflammation. On one hand, lactobacilli have been shown to directly compete with skin pathogens through adhesion inhibition, production of antimicrobial metabolites, and by influencing pathogen metabolism. The competitive anti-pathogenic action of lactobacilli has already been described mechanistically for common different skin pathogens, such as Staphylococcus aureus, Cutibacterium acnes, and Candida albicans. On the other hand, lactobacilli also have an immunomodulatory capacity associated with a reduction in excessive skin inflammation. Their influence on the immune system is mediated by bacterial metabolites and cell wall-associated or excreted microbe-associated molecular patterns (MAMPs). In addition, lactobacilli can also enhance the skin barrier function, which is often disrupted as a result of infection or in inflammatory skin diseases. Some clinical trials have already translated these mechanistic insights into beneficial clinical outcomes, showing that topically applied lactobacilli can temporarily colonize the skin and promote skin health, but more and larger clinical trials are required to generate in vivo mechanistic insights and in-depth skin microbiome analysis.