A long-awaited taxogenomic investigation of the family Halomonadaceae.

The family Halomonadaceae is the largest family composed of halophilic bacteria, with more than 160 species with validly published names as of July 2023. Several classifications to circumscribe this family are available in major resources, such as those provided by the List of Prokaryotic names with Standing in Nomenclature (LPSN), NCBI Taxonomy, Genome Taxonomy Database (GTDB), and Bergey's Manual of Systematics of Archaea and Bacteria (BMSAB), with some degree of disagreement between them. Moreover, regardless of the classification adopted, the genus Halomonas is not phylogenetically consistent, likely because it has been used as a catch-all for newly described species within the family Halomonadaceae that could not be clearly accommodated in other Halomonadaceae genera. In the past decade, some taxonomic rearrangements have been conducted on the Halomonadaceae based on ribosomal and alternative single-copy housekeeping gene sequence analysis. High-throughput technologies have enabled access to the genome sequences of many type strains belonging to the family Halomonadaceae; however, genome-based studies specifically addressing its taxonomic status have not been performed to date. In this study, we accomplished the genome sequencing of 17 missing type strains of Halomonadaceae species that, together with other publicly available genome sequences, allowed us to re-evaluate the genetic relationship, phylogeny, and taxonomy of the species and genera within this family. The approach followed included the estimate of the Overall Genome Relatedness Indexes (OGRIs) such as the average amino acid identity (AAI), phylogenomic reconstructions using amino acid substitution matrices customized for the family Halomonadaceae, and the analysis of clade-specific signature genes. Based on our results, we conclude that the genus Halovibrio is obviously out of place within the family Halomonadaceae, and, on the other hand, we propose a division of the genus Halomonas into seven separate genera and the transfer of seven species from Halomonas to the genus Modicisalibacter, together with the emendation of the latter. Additionally, data from this study demonstrate the existence of various synonym species names in this family.

A citizen-science-enabled catalogue of the vaginal microbiome and associated factors.

Understanding the composition and function of the vaginal microbiome is crucial for reproductive and overall health. Here we established the Isala citizen-science project to analyse the vaginal microbiomes of 3,345 women in Belgium (18-98 years) through self-sampling, 16S amplicon sequencing and extensive questionnaires. The overall vaginal microbiome composition was strongly tied to age, childbirth and menstrual cycle phase. Lactobacillus species dominated 78% of the vaginal samples. Specific bacterial taxa also showed to co-occur in modules based on network correlation analysis. Notably, the module containing Lactobacillus crispatus, Lactobacillus jensenii and Limosilactobacillus taxa was positively linked to oestrogen levels and contraceptive use and negatively linked to childbirth and breastfeeding. Other modules, named after abundant taxa (Gardnerella, Prevotella and Bacteroides), correlated with multiple partners, menopause, menstrual hygiene and contraceptive use. With this resource-rich vaginal microbiome map and associated health, life-course, lifestyle and dietary factors, we provide unique data and insights for follow-up clinical and mechanistic research.

Lactobacillus isalae sp. nov., isolated from the female reproductive tract.

A novel strain of the genus Lactobacillus, named AMBV1719T, was isolated from the vagina of a healthy participant in our large-scale citizen science project on the female microbiome, named Isala. Phylogenetic analysis showed that the 16S rRNA gene of AMBV1719T is most similar to that of Lactobacillus taiwanensis with a sequence similarity of 99.873 %. However, a genome-wide comparison using average nucleotide identity (ANI) revealed that isolate AMBV1719T showed the highest ANI with Lactobacillus paragasseri JCM 5343T, with a value of only 88.17 %. This low ANI value with the most closely related strains known to date indicated that AMBV1719T represents a distinct species. This strain has a limited ability to degrade carbon sources compared to Lactobacillus gasseri, indicating its adaptation to the host. Its genome has a length of 2.12 Mb with a G+C content of 34.8 mol%. We thus propose the name Lactobacillus isalae sp. nov. for this novel species, with AMBV1719T (=LMG 32886T=CECT 30756T) as the type strain.

Salivary microbiome of healthy women of reproductive age.

IMPORTANCE: The salivary microbiome has been proven to play a crucial role in local and systemic diseases. Moreover, the effects of biological and lifestyle factors such as oral hygiene and smoking on this microbial community have already been explored. However, what was not yet well understood was the natural variation of the saliva microbiome in healthy women and how this is associated with specific use of hormonal contraception and with the number of different sexual partners with whom microbiome exchange is expected regularly. In this paper, we characterized the salivary microbiome of 255 healthy women of reproductive age using an in-depth questionnaire and self-sampling kits. Using the large metadata set, we were able to investigate the associations of several host-related and lifestyle variables with the salivary microbiome profiles. Our study shows a high preservation between individuals.

Lactiplantibacillus carotarum AMBF275T sp. nov. isolated from carrot juice fermentation.

A novel strain of the genus Lactiplantibacillus, named AMBF275T, was isolated from fermented carrot juice, a salted fermented beverage dominated by lactic acid bacteria. The results of phylogenetic analysis indicated that the 16S rRNA gene of AMBF275T is most similar to the 16S rRNA gene of Lactiplantibacillus garii FI11369T with a sequence similarity of 99.4 %. However, a genome-wide comparison using average nucleotide identity (ANI) revealed that AMBF275T and L. garii FI11369T have an ANI of only 82.35 %. ANI values between AMBF275T and other representative strains of species of the genus Lactiplantibacillus from the Genome Taxonomy Database (GTDB) were even lower than this 82.35 %, indicating that AMBF275T represents a distinct species. We thus propose the name Lactiplantibacillus carotarum sp. nov. for this novel species, with AMBF275T (=LMG 32885T, =CECT 30757T) as the type strain.

After the storm-Perspectives on the taxonomy of Lactobacillaceae.

In 2020, a taxonomic reorganization of the lactic acid bacteria reclassified over 300 species in 7 genera and 2 families into one family, the Lactobacillaceae, with 31 genera including 23 new genera to include organisms formerly classified as Lactobacillus species. This communication aims to provide a debrief on the taxonomic reorganization of lactobacilli to identify shortcomings in the proposed taxonomic framework, and to outline perspectives and opportunities provided by the current taxonomy of the Lactobacillaceae. The current taxonomy of lactobacilli not only necessitates becoming familiar with 23 new genus names but also provides substantial new opportunities in scientific discovery and regulatory approval of these organisms. First, description of new species in the Lactobacillaceae is facilitated and a solid framework for description of novel genera is provided. Second, the current taxonomy greatly enhances the resolution of genus-level sequencing approaches (e.g., 16S rRNA-based metagenomics) when identifying the composition and function of microbial communities. Third, the current taxonomy greatly facilitates the formulation of hypotheses linking phylogeny to metabolism and ecology of lactobacilli.

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.

Spontaneous Riboflavin-Overproducing Limosilactobacillus reuteri for Biofortification of Fermented Foods.

Riboflavin-producing lactic acid bacteria represent a promising and cost-effective strategy for food biofortification, but production levels are typically insufficient to support daily human requirements. In this study, we describe the novel human isolate Limosilactobacillus reuteri AMBV339 as a strong food biofortification candidate. This strain shows a high natural riboflavin (vitamin B2) overproduction of 18.36 µg/ml, biomass production up to 6 × 1010 colony-forming units/ml (in the typical range of model lactobacilli), and pH-lowering capacities to a pH as low as 4.03 in common plant-based (coconut, soy, and oat) and cow milk beverages when cultured up to 72 h at 37°C. These properties were especially pronounced in coconut beverage and butter milk fermentations, and were sustained in co-culture with the model starter Streptococcus thermophilus. Furthermore, L. reuteri AMBV339 grown in laboratory media or in a coconut beverage survived in gastric juice and in a simulated gastrointestinal dialysis model with colon phase (GIDM-colon system) inoculated with fecal material from a healthy volunteer. Passive transport of L. reuteri AMBV339-produced riboflavin occurred in the small intestinal and colon stage of the GIDM system, and active transport via intestinal epithelial Caco-2 monolayers was also demonstrated. L. reuteri AMBV339 did not cause fecal microbiome perturbations in the GIDM-colon system and inhibited enteric bacterial pathogens in vitro. Taken together, our data suggests that L. reuteri AMBV339 represents a promising candidate to provide riboflavin fortification of plant-based and dairy foods, and has a high application potential in the human gastrointestinal tract.

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.

Latilactobacillus fragifolii sp. nov., isolated from leaves of a strawberry plant (Fragaria x ananassa).

Thirteen Gram-positive, catalase-positive, rod-shaped single colonies were obtained after culturing a strawberry leaf on de Man-Rogosa-Sharpe agar. Based on 16S rRNA gene and rpoA gene sequence similarities, ranging between 99.0-100% and 96.5-100%, respectively, the 13 isolates were found to be closely related to each other. Two of the independent isolates, AMBP162T and AMBP252, were whole-genome sequenced, and showed to be undistinguishable with an average nucleotide identity (ANI) value of 100 %. Compared to the reference genomes for all species in the family Lactobacillaceae, the AMBP162T genome was most similar to the reference strain of Latilactobacillus curvatus with ANI of only 89.5 %, indicating they were a different species. Based on genotypic and phenotypic data, a novel Latilactobacillus species, Latilactobacillus fragifolii sp. nov., with the type strain AMBP162T (=LMG 32285T=CECT 30357T) is proposed.

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.

The nasal mutualist Dolosigranulum pigrum AMBR11 supports homeostasis via multiple mechanisms.

Comparing the nasal microbiome of healthy individuals and chronic rhinosinusitis (CRS) patients revealed Dolosigranulum pigrum as a species clearly associated with nasal health, although isolates obtained from healthy individuals are scarce. In this study, we explored the properties of this understudied lactic acid bacterium by integrating comparative genomics, habitat mining, cultivation, and functional characterization of interaction capacities. Mining 10.000 samples from the Earth Microbiome Project of 17 habitat types revealed that Dolosigranulum is mainly associated with the human nasal cavity. D. pigrum AMBR11 isolated from the nose of a healthy individual exerted antimicrobial activity against Staphylococcus aureus, decreased proinflammatory cytokine production in airway epithelial cells, and Galleria mellonella larvae mortality induced by this important nasal pathobiont. Furthermore, the strain protected the nasal barrier function in a mouse model using interleukin-4 as disruptive cytokine. Hence, D. pigrum AMBR11 is a mutualist with high potential as topical live biotherapeutic product.

Case-Control Microbiome Study of Chronic Otitis Media with Effusion in Children Points at Streptococcus salivarius as a Pathobiont-Inhibiting Species.

Chronic otitis media with effusion (OME) has been associated with a shift in microbiome composition and microbial interaction in the upper respiratory tract (URT). While most studies have focused on potential pathogens, this study aimed to find bacteria that could be protective against OME through a case-control microbiome study and characterization of isolates from healthy subjects. The URT and ear microbiome profiles of 70 chronic OME patients and 53 controls were compared by 16S rRNA amplicon sequencing. Haemophilus influenzae was the most frequent classic middle ear pathobiont. However, other taxa, especially Alloiococcus otitis, were also frequently detected in the ear canal of OME patients. Streptococci of the salivarius group and Acinetobacter lwoffii were more abundant in the nasopharynx of healthy controls than in OME patients. In addition to the microbiome analysis, 142 taxa were isolated from healthy individuals, and 79 isolates of 13 different Streptococcus species were tested for their pathobiont-inhibiting potential. Of these, Streptococcus salivarius isolates showed a superior capacity to inhibit the growth of H. influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, A. otitis, and Corynebacterium otitidis S. salivarius strains thus show potential as a probiotic for prevention or treatment of OME based on their overrepresentation in the healthy nasopharynx and their ability to inhibit the growth of respiratory pathobionts. (This study has been registered at ClinicalTrials.gov under registration no. NCT03109496.)IMPORTANCE The majority of probiotics marketed today target gastrointestinal health. This study searched for bacteria native to the human upper respiratory tract, with a beneficial potential for respiratory and middle ear health. Comparison of the microbiomes of children with chronic otitis media with effusion (OME) and of healthy controls identified Streptococcus salivarius as a health-associated and prevalent inhabitant of the human nasopharynx. However, beneficial potential should be assessed at strain level. Here, we also isolated specific S. salivarius strains from the healthy individuals in our study. These isolates showed a beneficial safety profile and efficacy potential to inhibit OME pathogens in vitro These properties will now have to be evaluated and confirmed in human clinical studies.

The Dwindling Microbiota of Aerobic Vaginitis, an Inflammatory State Enriched in Pathobionts with Limited TLR Stimulation.

While bacterial vaginosis (BV) is a well-known type of vaginal dysbiosis, aerobic vaginitis (AV) is an inflammatory condition that remains understudied and under-recognised. It predisposes women to serious complications including urogenital infections and pregnancy problems. Here, we investigated the bacterial community in AV to explore its possible role in AV pathogenesis. We collected vaginal lavage fluid samples of women (n = 58) classified by wet-mount microscopy as suffering from AV or BV and included an asymptomatic reference group without signs of AV or BV. AV samples showed reduced absolute abundances of bacteria in general and specifically of lactobacilli by qPCR, but 16S rRNA gene sequencing and amplicon sequence variant analysis revealed that Lactobacillus remained the dominant taxon in 25% of the AV samples studied. The other AV samples showed high relative abundances of Streptococcus agalactiae and, unexpectedly, the anaerobes Gardnerella vaginalis and Prevotella bivia in more than half of the AV samples studied. Yet, despite increased relative abundance of these potential pathogens or pathobionts in the AV bacterial communities, the AV samples only slightly stimulated Toll-like receptor 4 and showed reduced activation of Toll-like receptor 2/6, receptors of two pathways central to mucosal immunity. Our findings indicate that the reduced total bacterial abundance with associated enrichment in certain pathobionts in AV might be mainly a consequence of the inflammatory conditions and/or altered hormonal regulation rather than bacteria being a major cause of the inflammation.

Lactobacilli Have a Niche in the Human Nose.

Although an increasing number of beneficial microbiome members are characterized for the human gut and vagina, beneficial microbes are underexplored for the human upper respiratory tract (URT). In this study, we demonstrate that taxa from the beneficial Lactobacillus genus complex are more prevalent in the healthy URT than in patients with chronic rhinosinusitis (CRS). Several URT-specific isolates are cultured, characterized, and further explored for their genetic and functional properties related to adaptation to the URT. Catalase genes are found in the identified lactobacilli, which is a unique feature within this mostly facultative anaerobic genus. Moreover, one of our isolated strains, Lactobacillus casei AMBR2, contains fimbriae that enable strong adherence to URT epithelium, inhibit the growth and virulence of several URT pathogens, and successfully colonize nasal epithelium of healthy volunteers. This study thus demonstrates that specific lactobacilli are adapted to the URT and could have a beneficial keystone function in this habitat.

Impact of a lactobacilli-containing gel on vulvovaginal candidosis and the vaginal microbiome.

Vulvovaginal candidosis (VVC) is a common condition with severe symptoms and high recurrence rates. Probiotic lactobacilli are explored as alternatives to azole treatments. Although the vaginal microbiota is generally not depleted in lactobacilli during VVC, studies indicate that the functionality and antimicrobial activity of the lactobacilli is impaired. We selected three strains from the Lactobacillus genus complex (L. rhamnosus GG, L. pentosus KCA1 and L. plantarum WCFS1) based on in vitro evaluation and formulated them in a gel for vaginal application. This gel was evaluated in 20 patients suffering from acute VVC, who were followed for four weeks including a 10-day treatment period. The microbiome was assessed through 16S rRNA (bacteria) and internal transcribed spacer (ITS; fungi) amplicon sequencing, supplemented with quantitative PCR, culture and microscopy for Candida evaluation. 45% of women did not require rescue medication (3×200 mg fluconazole), implying an improvement of their symptoms. These women showed similar end concentrations of fungi as women treated with fluconazole. Moreover, fluconazole appeared to reduce numbers of endogenous lactobacilli. Our study points towards important aspects for future selection of lactobacilli for probiotic use in VVC and the need to investigate possible negative influences of azoles on the vaginal bacterial community.

A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae.

The genus Lactobacillus comprises 261 species (at March 2020) that are extremely diverse at phenotypic, ecological and genotypic levels. This study evaluated the taxonomy of Lactobacillaceae and Leuconostocaceae on the basis of whole genome sequences. Parameters that were evaluated included core genome phylogeny, (conserved) pairwise average amino acid identity, clade-specific signature genes, physiological criteria and the ecology of the organisms. Based on this polyphasic approach, we propose reclassification of the genus Lactobacillus into 25 genera including the emended genus Lactobacillus, which includes host-adapted organisms that have been referred to as the Lactobacillus delbrueckii group, Paralactobacillus and 23 novel genera for which the names Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus are proposed. We also propose to emend the description of the family Lactobacillaceae to include all genera that were previously included in families Lactobacillaceae and Leuconostocaceae. The generic term 'lactobacilli' will remain useful to designate all organisms that were classified as Lactobacillaceae until 2020. This reclassification reflects the phylogenetic position of the micro-organisms, and groups lactobacilli into robust clades with shared ecological and metabolic properties, as exemplified for the emended genus Lactobacillus encompassing species adapted to vertebrates (such as Lactobacillus delbrueckii, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensensii, Lactobacillus johnsonii and Lactobacillus acidophilus) or invertebrates (such as Lactobacillus apis and Lactobacillus bombicola).

Anterior Nares Diversity and Pathobionts Represent Sinus Microbiome in Chronic Rhinosinusitis.

It is generally believed that the microbiome plays a role in the pathophysiology of chronic rhinosinusitis (CRS), though its exact contribution to disease development and severity remains unclear. Here, samples were collected from the anterior nares, nasopharynx, and maxillary and ethmoid sinuses of 190 CRS patients and from the anterior nares and nasopharynx of 100 controls. Microbial communities were analyzed by Illumina sequencing of the V4 region of 16S rRNA. The phenotype and patient characteristics were documented, and several serum inflammatory markers were measured. Our data indicate a rather strong continuity for the microbiome in the different upper respiratory tract (URT) niches in CRS patients, with the microbiome in the anterior nares being most similar to the sinus microbiome. Bacterial diversity was reduced in CRS patients without nasal polyps compared to that in the controls but not in CRS patients with nasal polyps. Statistically significant differences in the presence/absence or relative abundance of several taxa were found between the CRS patients and the healthy controls. Of these, Dolosigranulum pigrum was clearly more associated with URT samples from healthy subjects, while the Corynebacterium tuberculostearicum, Haemophilus influenzae/H. aegyptius, and Staphylococcus taxa were found to be potential pathobionts in CRS patients. However, CRS versus health as a predictor explained only 1 to 2% of the variance in the microbiome profiles in an adonis model. A history of functional endoscopic sinus surgery, age, and sex also showed a minor association. This study thus indicates that functional studies on the potential beneficial versus pathogenic activity of the different indicator taxa found here are needed to further understand the pathology of CRS and its different phenotypes. (This study has been registered at ClinicalTrials.gov under identifier NCT02933983.)IMPORTANCE There is a clear need to better understand the pathology and specific microbiome features in chronic rhinosinusitis patients, but little is known about the bacterial topography and continuity between the different niches of the upper respiratory tract. Our work showed that the anterior nares could be an important reservoir for potential sinus pathobionts. This has implications for the diagnosis, prevention, and treatment of CRS. In addition, we found a potential pathogenic role for the Corynebacterium tuberculostearicum, Haemophilus influenzae/H. aegyptius, and Staphylococcus taxa and a potential beneficial role for Dolosigranulum Finally, a decreased microbiome diversity was observed in patients with chronic rhinosinusitis without nasal polyps compared to that in healthy controls but not in chronic rhinosinusitis patients with nasal polyps. This suggests a potential role for the microbiome in disease development or progression of mainly this phenotype.

A Genome-Based Species Taxonomy of the Lactobacillus Genus Complex.

There are more than 200 published species within the Lactobacillus genus complex (LGC), the majority of which have sequenced type strain genomes available. Although genome-based species delimitation cutoffs are accepted as the gold standard by the community, these are seldom actually checked for new or already published species. In addition, the availability of genome data is revealing inconsistencies in the species-level classification of many strains. We constructed a de novo species taxonomy for the LGC based on 2,459 publicly available genomes, using a 94% core nucleotide identity cutoff. We reconciled these de novo species with published species and subspecies names by (i) identifying genomes of type strains and (ii) comparing 16S rRNA genes of the genomes with 16S rRNA genes of type strains. We found that genomes within the LGC could be divided into 239 de novo species that were discontinuous and exclusive. Comparison of these de novo species to published species led to the identification of nine sets of published species that can be merged and one species that can be split. Further, we found at least eight de novo species that constitute new, unpublished species. Finally, we reclassified 74 genomes on the species level and identified for the first time the species of 98 genomes. Overall, the current state of LGC species taxonomy is largely consistent with genome-based species delimitation cutoffs. There are, however, exceptions that should be resolved to evolve toward a taxonomy where species share a consistent diversity in terms of sequence divergence.IMPORTANCE The Lactobacillus genus complex is a group of bacteria that constitutes an important source of strains with medical and food applications. The number of bacterial whole-genome sequences available for this taxon has been increasing rapidly in recent years. Despite this wealth of information, the species within this group are still largely defined by older techniques. Here, we constructed a completely new species-level taxonomy for the Lactobacillus genus complex based on ∼2,500 whole-genome sequences. As a result of this effort, we found that many genomes are not classified to their correct species, and we were able to correct these. In addition, we found that some published species are abnormally large, while others are too small. Finally, we discovered at least eight completely novel species that have not been published before. Our work will help the field to evolve toward a more meaningful and complete taxonomy, based on whole-genome sequences.

Comparative genome analysis of Lactobacillus mudanjiangensis, an understudied member of the Lactobacillus plantarum group.

The genus Lactobacillus is known to be extremely diverse and consists of different phylogenetic groups that show a diversity that is roughly equal to the expected diversity of a typical bacterial genus. One of the most prominent phylogenetic groups within this genus is the Lactobacillus plantarum group, which contains the understudied Lactobacillus mudanjiangensis species. Before this study, only one L. mudanjiangensis strain, DSM 28402T, had been described, but without whole-genome analysis. In this study, three strains classified as L. mudanjiangensis were isolated from three different carrot juice fermentations and their whole-genome sequence was determined, together with the genome sequence of the type strain. The genomes of all four strains were compared with publicly available L. plantarum group genome sequences. This analysis showed that L. mudanjiangensis harboured the second largest genome size and gene count of the whole L. plantarum group. In addition, all members of this species showed the presence of a gene coding for a cellulose-degrading enzyme. Finally, three of the four L. mudanjiangensis strains studied showed the presence of pili on scanning electron microscopy (SEM) images, which were linked to conjugative gene regions, coded on a plasmid in at least two of the strains studied.