Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

Is the persistence of Listeria monocytogenes in food processing facilities and its resistance to pathogen intervention linked to its phylogeny?

The foodborne pathogen Listeria monocytogenes is differentiated into four distinct lineages which differ in their virulence. It remains unknown, however, whether the four lineages also differ with respect to their ability to persist in food processing facilities, their resistance to high pressure, a preservation method that is used commercially for Listeria control on ready-to-eat meats, and their ability to form biofilms. This study aimed to determine differences in the pressure resistance and biofilm formation of 59 isolates of L. monocytogenes representing lineages I and II. Furthermore, the genetic similarity of 9 isolates of L. monocytogenes that were obtained from a meat processing facility over a period of 1 year and of 20 isolates of L. monocytogenes from food processing facilities was analyzed to assess whether the ability of the lineages of L. monocytogenes to persist in these facilities differs. Analysis of 386 genomes with respect to the source of isolation revealed that genomes of lineage II are over-represented in meat isolates when compared with clinical isolates. Of the 38 strains of Lm. monocytogenes that persisted in food processing facilities (this study or published studies), 31 were assigned to lineage II. Isolates of lineage I were more resistant to treatments at 400 to 600 MPa. The thickness of biofilms did not differ between lineages. In conclusion, strains of lineage II are more likely to persist in food processing facilities while strains of lineage I are more resistant to high pressure.IMPORTANCEListeria monocytogenes substantially contributes to the mortality of foodborne disease in developed countries. The virulence of strains of four lineages of L. monocytogenes differs, indicating that risks associated with the presence of L. monocytogenes are lineage specific. Our study extends the current knowledge by documentation that the lineage-level phylogeny of L. monocytogenes plays a role in the source of isolation, in the persistence in food processing facilities, and in the resistance to pathogen intervention technologies. In short, the control of risks associated with the presence of L. monocytogenes in food is also lineage specific. Understanding the route of contamination L. monocytogenes is an important factor to consider when designing improved control measures.

Competitiveness of reutericyclin producing and nonproducing Limosilactobacillus reuteri in food and intestinal ecosystems: a game of rock, paper, and scissors?

The ecological relationships among antimicrobial producing, resistant, and sensitive strains have been proposed to follow rock-paper-scissors dynamics, but evidence is mainly based on Gram-negative bacteriocins in vitro. The ecological relevance of antimicrobials in vivo or in situ has not been systematically studied. This study therefore aimed to analyze binary and ternary competitions among reutericyclin-producing strain Limosilactobacillus reuteri TMW1.656, its reutericyclin-resistant, nonproducing isogenic derivative L. reuteri TMW1.656∆rtcN, and the reutericyclin-sensitive, nonproducing L. reuteri TMW1.656∆rtcN∆rtcT in vitro (liquid culture and static plate), in situ (sourdough fermentation), and in vivo (gut of germ-free mice). In liquid culture, L. reuteri TMW1.656 had a higher fitness than TMW1.656∆rtcN and TMW1.656∆rtcN∆rtcT. Limosilactobacillus reuteri TMW1.656∆rtcN∆rtcT had a higher fitness than TMW1.656∆rtcN. On agar plates, L. reuteri TMW1.656 had a higher fitness than TMW1.656∆rtcN∆rtcT. In situ, reutericyclin production and resistance had no influence on the fitness of the strains. In vivo, TMW1.656 had an advantage over TMW1.656∆rtcN and TMW1.656∆rtcN∆rtcT. Ternary competitions showed reutericyclin production was ecologically beneficial in all ecosystems. The findings support the ecological importance of reutericyclin in a variety of environments/niches, providing an explanation for the acquisition of the reutericyclin gene cluster in L. reuteri and its contribution to the ecological fitness of Streptococcus mutans.

Selection of adjunct cultures for the ripening of plant cheese analogues.

Fermentation contributes to the taste and odor of plant cheeses. The selection of functional cultures for the fermentation of plant cheeses, however, is in its infancy. This study aimed to select lactic acid bacteria for ripening of soy and lupin cheese analogues. Bacillus velezensis and B. amyloliquefaciens were used for germination of seeds to produce proteolytic enzymes; Lactococcus lactis and Lactiplantibacillus plantarum served as primary acidifying cultures. Levilactobacillus hammesii, Furfurilactobacillus milii, or Lentilactobacillus buchneri were assessed as adjunct cultures for the ripening of plant cheese. Growth of bacilli was inhibited at low pH. Both Lc. lactis and Lp. plantarum were inactived during plant cheese ripening. Cell counts of Lv. hammesii remained stable over 45 d of ripening while Ff. milii and Lt. buchneri grew slowly. Sequencing of full length 16S rRNA genes confirmed that the inocula the plant cheeses accounted for more than 98% of the bacterial communities. HPLC analysis revealed that Lt. buchneri metabolized lactate to acetate and 1,2-propanediol during ripening. Bacilli enhanced proteolysis as measured by quantification of free amino nitrogen, and the release of glutamate. LC-MS/MS analysis quantified kokumi-active dipeptides. The concentrations of γ-Glu-Leu, γ-Glu-Ile, and γ-Glu-Ala, γ-Glu-Cys in unripened cheeses were increased by seed germination but γ-Glu-Phe was degraded. Lt. buchneri but not Lv. hammesii or Ff. milii accumulated γ-Glu-Val, γ-Glu-Ile or γ-Glu-Leu during ripening, indicating strain-specific differences. In conclusion, a consortium of bacilli, acidification cultures and adjunct cultures accumulates taste- and kokumi-active compounds during ripening of plant cheeses.

A phylogenomic analysis of Limosilactobacillus reuteri reveals ancient and stable evolutionary relationships with rodents and birds and zoonotic transmission to humans.

BACKGROUND: Gut microbes play crucial roles in the development and health of their animal hosts. However, the evolutionary relationships of gut microbes with vertebrate hosts, and the consequences that arise for the ecology and lifestyle of the microbes are still insufficiently understood. Specifically, the mechanisms by which strain-level diversity evolved, the degree by which lineages remain stably associated with hosts, and how their evolutionary history influences their ecological performance remain a critical gap in our understanding of vertebrate-microbe symbiosis. RESULTS: This study presents the characterization of an extended collection of strains of Limosilactobacillus reuteri and closely related species from a wide variety of hosts by phylogenomic and comparative genomic analyses combined with colonization experiments in mice to gain insight into the long-term evolutionary relationship of a bacterial symbiont with vertebrates. The phylogenetic analysis of L. reuteri revealed early-branching lineages that primarily consist of isolates from rodents (four lineages) and birds (one lineage), while lineages dominated by strains from herbivores, humans, pigs, and primates arose more recently and were less host specific. Strains from rodent lineages, despite their phylogenetic divergence, showed tight clustering in gene-content-based analyses. These L. reuteri strains but not those ones from non-rodent lineages efficiently colonize the forestomach epithelium of germ-free mice. The findings support a long-term evolutionary relationships of L. reuteri lineages with rodents and a stable host switch to birds. Associations of L. reuteri with other host species are likely more dynamic and transient. Interestingly, human isolates of L. reuteri cluster phylogenetically closely with strains from domesticated animals, such as chickens and herbivores, suggesting zoonotic transmissions. CONCLUSIONS: Overall, this study demonstrates that the evolutionary relationship of a vertebrate gut symbiont can be stable in particular hosts over time scales that allow major adaptations and specialization, but also emphasizes the diversity of symbiont lifestyles even within a single bacterial species. For L. reuteri, symbiont lifestyles ranged from autochthonous, likely based on vertical transmission and stably aligned to rodents and birds over evolutionary time, to allochthonous possibly reliant on zoonotic transmission in humans. Such information contributes to our ability to use these microbes in microbial-based therapeutics.

Socializing at the Air-Liquid Interface: a Functional Genomic Analysis on Biofilm-Related Genes during Pellicle Formation by Escherichia coli and Its Interaction with Aeromonas australiensis.

Pellicles are biofilms that form at the air-liquid interface. We demonstrated that specific strains of Escherichia coli formed pellicles in single cultures when cocultured with Carnobacterium maltaromaticum and E. coli O157:H7 but not with Aeromonas australiensis. Therefore, a combination of comparative genomic, mutational, and transcriptome analyses were applied to identify the unique genes in pellicle formation and investigate gene regulation under different growth phases. Here, we report that pellicle-forming strains do not harbor unique genes relative to non-pellicle-forming strains; however, the expression level of biofilm-related genes differed, especially for the genes encoding curli. Further, the regulatory region of curli biosynthesis is phylogenetically different among pellicle- and non-pellicle-forming strains. The disruption on modified cellulose and regulatory region of curli biosynthesis abolished pellicle formation in strains of E. coli. Besides, the addition of quorum sensing molecules (C4-homoserine lactones [C4-HSL]), synthesized by Aeromonas species, to pellicle formers abolished pellicle formation and implied a role of quorum sensing on pellicle formation. The deletion of autoinducer receptor sdiA in E. coli did not restore pellicle formation when cocultured with A. australiensis but modulated expression level of genes for curli and cellulose biosynthesis, resulting in a thinner layer of pellicle. Taken together, this study identified genetic determinants for pellicle formation and characterized the switching between pellicle to surface-associated biofilm in a dual-species environment, facilitating better understanding of the mechanisms for pellicle formation in E. coli and related organisms. IMPORTANCE To date, most attention has focused on biofilm formation on solid surfaces. By comparison, the knowledge on pellicle formation at the air-liquid interface is more limited and few studies document how bacteria decide on whether to form biofilms on solid surfaces or pellicles at the air-liquid interface to the surface-associated biofilms at the bottom. In this report, we characterized the regulation of biofilm-related genes during pellicle formation and document that interspecies communication via quorum sensing contributes to regulating the switch from pellicle to surface-associated biofilm. The discoveries expand the current view of regulatory cascades associated with pellicle formation.

Comparative genomic analysis of Periweissella and the characterization of novel motile species.

The genus Periweissella was proposed as a novel genus in the Lactobacillaceae in 2022. However, the phylogenetic relationship between Periweissella and other heterofermentative lactobacilli, and the genetic and physiological properties of this genus remain unclear. This study aimed to determine the phylogenetic relationship between Periweissella and the two closest genera, Weissella and Furfurilactobacillus, by the phylogenetic analysis and calculation of (core gene) pairwise average amino acid identity. Targeted genomic analysis showed that fructose bisphosphate aldolase was only present in the genome of Pw. cryptocerci. Mannitol dehydrogenase was found in genomes of Pw. beninensis, Pw. fabaria, and Pw. fabalis. Untargeted genomic analysis identified the presence of flagellar genes in Periweissella but not in other closely related genera. Phenotypes related to carbohydrate fermentation and motility matched the genotypes. Motility genes were organized in a single operon and the proteins shared a high amino acid similarity in the genus Periweissella. The relatively low similarity of motility operons between Periweissella and other motile lactobacilli indicated the acquisition of motility by the ancestral species. Our findings facilitate the phylogenetic, genetic, and phenotypic understanding of the genus Periweissella.ImportanceThe genus Periweissella is a heterofermentative genus in the Lactobacillaceae which includes predominantly isolates from cocoa fermentations in tropical climates. Despite the relevance of the genus in food fermentations, genetic and physiological properties of the genus are poorly characterized and genome sequences became available only after 2020. This study characterized strains of the genus by functional genomic analysis, and by determination of metabolic and physiological traits. Phylogenetic analysis revealed that Periweissella is the evolutionary link between rod-shaped heterofermentative lactobacilli and the coccoid Leuconostoc clade with the genera Weissella and Furfurilactobacillus as closest relatives. Periweissella is the only heterofermentative genus in the Lactobacillaceae which comprises predominantly motile strains. The genomic, physiological, and metabolic characterization of Periweissella may facilitate the potential use of strains of the genus as starter culture in traditional or novel food fermentations.

Physiological and genomic characterization of Lactiplantibacillus plantarum isolated from Indri indri in Madagascar.

AIMS: Indri indri is a lemur of Madagascar which is critically endangered. The analysis of the microbial ecology of the intestine offers tools to improve conservation efforts. This study aimed to achieve a functional genomic analysis of three Lactiplantibacillus plantarum isolates from indris. METHODS AND RESULTS: Samples were obtained from 18 indri; 3 isolates of Lp. plantarum were obtained from two individuals. The three isolates were closely related to each other, with <10 single nucleotide polymorphisms, suggesting that the two individuals shared diet-associated microbes. The genomes of the three isolates were compared to 96 reference strains of Lp. plantarum. The three isolates of Lp. plantarum were not phenotypically resistant to antibiotics but shared all 17 genes related to antimicrobial resistance that are part of the core genome of Lp. plantarum. The genomes of the three indri isolates of Lp. plantarum also encoded for the 6 core genome genes coding for enzymes related to metabolism of hydroxybenzoic and hydroxycinnamic acids. The phenotype for metabolism of hydroxycinnamic acids by indri isolates of Lp. plantarum matched the genotype. CONCLUSIONS: Multiple antimicrobial resistance genes and gene coding for metabolism of phenolic compounds were identified in the genomes of the indri isolates, suggesting that Lp. plantarum maintains antimicrobial resistance in defense of antimicrobial plant secondary pathogens and that their metabolism by intestinal bacteria aids digestion of plant material by primate hosts.

Role of thiols and ascladiol production in patulin degradation by lactobacilli.

Patulin is a mycotoxin contaminant in various foods with apple products being its major dietary source. Yeast can reduce patulin levels during fermentation via biotransformation and thiol-adduct formation, with the ability of patulin to react with thiols being well known. Conversion of patulin to ascladiol by lactobacilli has been sparsely reported, while the contribution of thiols in reduction of patulin levels by lactobacilli remains undocumented. In this study, 11 strains of lactobacilli were screened for ascladiol formation in apple juice fermentation. Highest bioconversion was obtained for Lactiplantibacillus plantarum strains followed by Levilactobacillus brevis TMW1.465. Ascladiol production was also detected in several other lactobacilli species albeit in trace amounts. Reduction in patulin levels by Fructilactobacillus sanfranciscensis DMS 20451 and its glutathione reductase (ΔgshR) negative mutant was also assayed to determine the contribution of thiols. The hydrocinnamic acid reductase of Furfurilactobacillus milii did not contribute to reduction of patulin levels. In conclusion, this study demonstrated the potential of various lactobacilli in reduction of patulin levels via biotransformation of patulin to ascladiol, while also providing evidence for the role of thiol formation by lactobacilli and its presence in reducing patulin levels during fermentation.

Composition and activity of antifungal lipopeptides produced by Bacillus spp. in daqu fermentation.

Daqu is a solid-state fermentation and saccharification starter for the Chinese liquor baijou. During the daqu stage, amylolytic and proteolytic enzymes are produced by Bacillus and fungi. Bacillus spp. also produce lipopeptides with a broad spectrum of antimicrobial activities but direct evidence for their impact on community assembly in daqu is lacking. This study aimed to study the interaction between Bacillus spp. and fungi in daqu models. The antifungal activity of surfactin, fengycin, and iturin A was initially assessed in vitro. Iturin A displayed the strongest antifungal activity (MIC = 10-50 mg/L). In situ antifungal activity of B. amyloliquefaciens and B. velezensis against molds was observed in a simple daqu model inoculated with single strains of Bacillus species. Formation of lipopeptides in situ was supported by quantification of mRNA encoding for enzymes for surfactin, fengycin, and iturin A biosynthesis. In situ antifungal activity of Bacillus species was also observed in a complex daqu model that was inoculated with 8 bacterial or fungal strains plus one of the three strains of Bacillus. A relationship of lipopeptides to in situ antifungal activity was further supported by detection of the lipopeptides by liquid chromatography coupled to mass spectrometry. Both results indicated that B velezensis FUA2155 had higher antifungal activity in the daqu model, and was the only strain that produced multiple iturin A congeners in situ. Taken together, this study provides evidence that production of lipopeptides by Bacillus species in daqu may impact community assembly and hence product quality.

Conversion of hydroxycinnamic acids by Furfurilactobacillus milii in sorghum fermentations: Impact on profile of phenolic compounds in sorghum and on ecological fitness of Ff. milii.

The conversion of phenolic compounds by lactobacilli in food fermentations contributes to food quality. The metabolism of phenolics by lactobacilli has been elucidated in the past years but information on the contribution of specific enzymes in food fermentations remains scarce. This study aimed to address this gap by disruption of genes coding for the hydroxycimmanic acid reductase Par1, the hydroxycinnamic acid decarboxylase Pad, the hydrocinnamic esterase EstR, and strains with disruption of all three genes in Furfurilactobacillus milii FUA3583. The conversion of phenolics by Ff. milii and its isogenic mutants in sorghum fermentations was studied by LC-UV and LC-UV-MS/MS analyses. Ff. milii FUA3583 converted hydroxycinnamic acids predominantly with Par1. Vinylphenols were detected only in mutants lacking par1. A phenotype for the estR defective mutant was not identified. The formation of pyrano-3-deoxyanthocyanidins was observed only after fermentation with strains expressing Pad. Specifically, formation of these compounds was low with Ff. milii FUA3583, substantially increased in the Par1 mutant and abolished in all mutants with disrupted pad. Competition experiments with Ff. milii FUA3583 and its isogenic mutants demonstrated that expression of one of the two metabolic pathways for hydroxycinnamic acids increases the ecological fitness of the strain. Disruption of EstR in a Δpar1Δpar2Δpad background improved ecological fitness, indirectly demonstrating a phenotype of the esterase in Ff. milii. The documentation of the functionality of genes coding for conversion of hydroxycinnamic acids may support the selection of starter cultures for improved quality of fermented cereal products.

Characterization of the Glucan-Branching Enzyme GlgB Gene from Swine Intestinal Bacteria.

Starch hydrolysis by gut microbiota involves a diverse range of different enzymatic activities. Glucan-branching enzyme GlgB was identified as the most abundant glycosidase in Firmicutes in the swine intestine. GlgB converts α-(1→4)-linked amylose to form α-(1→4,6) branching points. This study aimed to characterize GlgB cloned from a swine intestinal metagenome and to investigate its potential role in formation of α-(1→4,6)-branched α-glucans from starch. The branching activity of purified GlgB was determined with six different starches and pure amylose by quantification of amylose after treatment. GlgB reduced the amylose content of all 6 starches and amylose by more than 85% and displayed a higher preference towards amylose. The observed activity on raw starch indicated a potential role in the primary starch degradation in the large intestine as an enzyme that solubilizes amylose. The oligosaccharide profile showed an increased concentration of oligosaccharide introduced by GlgB that is not hydrolyzed by intestinal enzymes. This corresponded to a reduced in vitro starch digestibility when compared to untreated starch. The study improves our understanding of colonic starch fermentation and may allow starch conversion to produce food products with reduced digestibility and improved quality.

Supercharged MPNs? Automated Determination of High-Throughput Most Probable Number (htMPN) Using Chip-Based 3D Digital PCR.

Surface plating on agar and most probable number (MPN) are the standard methods for determining bacterial viability but both have limitations. Here we present a novel cell count method, high-throughput MPN (htMPN), that uses a chip-based digital PCR instrument to accelerate and to improve the quantification of viable or sublethally injured cells. This method tracks growth of up to 20,000 individual bacterial cells on a single chip. Single cells were grown in the individual wells of the chip at their optimal temperature until the cell density was high enough to detect the fluorescent signal with cell-permeant or cell-impermeant DNA-intercalating fluorescent dyes. This method based on microfluidic devices implemented in digital PCR equipment was equivalent to surface plating in determining cell counts of Escherichia coli, Salmonella enterica serovar Typhimurium, Fructilactobacillus sanfranciscensis, Pseudomonas putida, and vegetative cells but not spores of Bacillus subtilis. Viable E. coli could be enumerated within 7 h. Culture of strict aerobes was restricted to strains that are capable of nitrate respiration; organisms requiring complex media that also contain double-stranded DNA were detected after treatment of growth media with DNase before inoculation. Our approach not only monitors the frequency distribution of bacterial growth and determines cell counts with high reliability but also detected heat-injured cells of S. Typhimurium that escaped detection by the surface plating. Overall, the method accelerates detection of viable bacterial cells, facilitates automation, and offers new possibilities for the analysis of individual bacterial cells. IMPORTANCE htMPN uses chip-based fluorescence acquisition and is a simple and compact tool for automatic viable cell enumeration with applications in microbiological research. This method applies to a wide range of anaerobic or facultative anaerobic species and improves accuracy by reducing the number of pipetting steps. In addition, the method offers an additional tool for single-cell microbiology. The single cell time-to-detection times have been used as an important criterion for the physiological state of bacterial cells after sublethal stress, and htMPNs support the acquisition of such data with an unprecedented number of cells. In particular, htMPN provides an anaerobic environment and enables a long incubation time to increase the recovery rate of sublethally injured cells. Given its reproducibility and reliability, our approach can potentially be applied to quantify viable cells in samples from environmental, clinical, or food samples to reduce the risk of underestimation of the number of viable bacterial cells.

Furfurilactobacillus milii sp. nov., isolated from fermented cereal foods.

Genomic characterization of Furfurilactobacillus rossiae revealed that strains which were previously identified as F. rossiae are genetically heterogeneous. The 16S rRNA gene sequences of strains FUA3430, FUA3583, C5, FUA3115 and FUA3119, were 99.6 % identical to F. rossiae but the core genome analysis revealed that these strains share less than 93 % average nucleotide identity (ANI) with the F. rossiae type strain DSM 15814T. Because the ANI value is below the threshold for delineation of bacterial species, we propose the novel species Furfurilactobacillus milii sp. nov. with the type strain FUA3430T (=DSM 113338T=LMG 32478T). Strains of F. milii have smaller genomes than F. rossiae, lack the pdu-cbi-cob-hem cluster which is responsible for 1,2-propanediol utilization in F. rossiae, and lack genes involved in ethanolamine utilization. Two strains of the novel species (FUA3430T and FUA3583) were compared to F. rossiae FUA3214. Analysis of the cellular fatty acid composition and metabolite analysis did not reveal significant differences between F. milii sp. nov. and F. rossiae FUA3124. Although the growth requirements with respect to temperature and pH were very similar, only the strain of F. rossiae utilized melibiose and d-xylose. Morphological differences were also seen in the colony and cell size of the novel compared to F. rossiae.

Genetic Determinants of Stress Resistance in Desiccated Salmonella enterica.

Enteric pathogens, including Salmonella, are capable of long-term survival after desiccation and resist heat treatments that are lethal to hydrated cells. The mechanisms of dry-heat resistance differ from those of wet-heat resistance. To elucidate the mechanisms of dry-heat resistance in Salmonella, screening of the dry-heat resistance of 108 Salmonella strains, representing 39 serotypes, identified the 22 most resistant and the 8 most sensitive strains for comparative genome analysis. A total of 289 genes of the accessory genome were differently distributed between resistant and sensitive strains. Among these genes, 28 proteins with a putative relationship to stress resistance were selected for to quantify relative gene expression before and after desiccation and expression by solid-state cultures on agar plates relative to cultures growing in liquid culture media. Of these 28 genes, 15 genes were upregulated (P < 0.05) after desiccation or by solid-state cultures on agar plates. These 15 genes were cloned into the low-copy-number vector pRK767 under the control of the lacZ promoter. The expression of 6 of these 15 genes increased (P < 0.05) resistance to dry heat and to treatment with pressure of 500 MPa. Our finding extends the knowledge of mechanisms of stress resistance in desiccated Salmonella to improve control of this bacterium in dry food. IMPORTANCE This study directly targeted an increasing threat to food safety and developed knowledge and targeted strategies that can be used by the food industry to help reduce the risk of foodborne illness in their dry products and thereby reduce the overall burden of foodborne illness. Genomic and physiological analyses have elucidated mechanisms of bacterial resistance to many food preservation technologies, including heat, pressure, disinfection chemicals, and UV light; however, information on bacterial mechanisms of resistance to dry heat is scarce. Mechanisms of tolerance to desiccation likely also contribute to resistance to dry heat, but this assumption has not been verified experimentally. It remains unclear how mechanisms of resistance to wet heat relate to dry-heat resistance. Thus, this study will fill a knowledge gap to improve the safety of dry foods.

Limosilactobacillus balticus sp. nov., Limosilactobacillus agrestis sp. nov., Limosilactobacillus albertensis sp. nov., Limosilactobacillus rudii sp. nov. and Limosilactobacillus fastidiosus sp. nov., five novel Limosilactobacillus species isolated from the vertebrate gastrointestinal tract, and proposal of six subspecies of Limosilactobacillus reuteri adapted to the gastrointestinal tract of specific vertebrate hosts.

Ten strains, BG-AF3-AT, pH52_RY, WF-MT5-AT, BG-MG3-A, Lr3000T, RRLNB_1_1, STM3_1T, STM2_1, WF-MO7-1T and WF-MA3-C, were isolated from intestinal or faecal samples of rodents, pheasant and primate. 16S rRNA gene analysis identified them as Limosilactobacillus reuteri. However, average nucleotide identity and digital DNA-DNA hybridization values based on whole genomes were below 95 and 70 %, respectively, and thus below the threshold levels for bacterial species delineation. Based on genomic, chemotaxonomic and morphological analyses, we propose five novel species with the names Limosilactobacillus balticus sp. nov. (type strain BG-AF3-AT=DSM 110574T=LMG 31633T), Limosilactobacillus agrestis sp. nov. (type strain WF-MT5-AT=DSM 110569T=LMG 31629T), Limosilactobacillus albertensis sp. nov. (type strain Lr3000T=DSM 110573T=LMG 31632T), Limosilactobacillus rudii sp. nov. (type strain STM3_1T=DSM 110572T=LMG 31631T) and Limosilactobacillus fastidiosus sp. nov. (type strain WF-MO7-1T=DSM 110576T=LMG 31630T). Core genome phylogeny and experimental evidence of host adaptation of strains of L. reuteri further provide a strong rationale to consider a number of distinct lineages within this species as subspecies. Here we propose six subspecies of L. reuteri: L. reuteri subsp. kinnaridis subsp. nov. (type strain AP3T=DSM 110703T=LMG 31724T), L. reuteri subsp. porcinus subsp. nov. (type strain 3c6T=DSM 110571T=LMG 31635T), L. reuteri subsp. murium subsp. nov. (type strain lpuph1T=DSM 110570T=LMG 31634T), L. reuteri subsp. reuteri subsp. nov. (type strain F 275T=DSM 20016T=ATCC 23272T), L. reuteri subsp. suis subsp. nov. (type strain 1063T=ATCC 53608T=LMG 31752T) and L. reuteri subsp. rodentium subsp. nov. (type strain 100-23T=DSM 17509T=CIP 109821T).

Characterization of γ-glutamyl cysteine ligases from Limosilactobacillus reuteri producing kokumi-active γ-glutamyl dipeptides.

γ-Glutamyl cysteine ligases (Gcls) catalyze the first step of glutathione synthesis in prokaryotes and many eukaryotes. This study aimed to determine the biochemical properties of three different Gcls from strains of Limosilactobacillus reuteri that accumulate γ-glutamyl dipeptides. Gcl1, Gcl2, and Gcl3 were heterologously expressed in Escherichia coli and purified by affinity chromatography. Gcl1, Gcl2, and Gcl2 exhibited biochemical with respect to the requirement for metal ions, the optimum pH and temperature of activity, and the kinetic constants for the substrates cysteine and glutamate. The substrate specificities of the three Gcls to 14 amino acids were assessed by liquid chromatography-mass spectrometry. All three Gcls converted ala, met, glu, and gln into the corresponding γ-glutamyl dipeptides. None of the three were active with val, asp, and his. Gcl1 and Gcl3 but not Gcl2 formed γ-glu-leu, γ-glu-ile, and γ-glu-phe; Gcl3 exhibited stronger activity with gly, pro, and asp when compared to Gcl2. Phylogenetic analysis of Gcl and the Gcl-domain of GshAB in lactobacilli demonstrated that most of Gcls were present in heterofermentative lactobacilli, while GshAB was identified predominantly in homofermentative lactobacilli. This distribution suggests a different ecological role of the enzyme in homofermentative and heterofermentative lactobacilli. In conclusion, three Gcls exhibited similar biochemical properties but differed with respect to their substrate specificity and thus the synthesis of kokumi-active γ-glutamyl dipeptides. KEY POINTS: • Strains of Limosilactobacillus reuteri encode for up to 3 glutamyl cysteine ligases. • Gcl1, Gcl2, and Gcl3 of Lm. reuteri differ in their substrate specificity. • Gcl1 and Gcl3 produce kokumi-active dipeptides.

Genetic Determinants of Hydroxycinnamic Acid Metabolism in Heterofermentative Lactobacilli.

Phenolic acids are among the most abundant phenolic compounds in edible parts of plants. Lactic acid bacteria (LAB) metabolize phenolic acids, but the enzyme responsible for reducing hydroxycinnamic acids to phenylpropionic acids (HcrB) was only recently characterized in Lactobacillus plantarum In this study, heterofermentative LAB species were screened for their hydroxycinnamic acid metabolism. Data on strain-specific metabolism in combination with comparative genomic analyses identified homologs of HcrB as putative phenolic acid reductases. Par1 and HcrF both encode putative multidomain proteins with 25% and 63% amino acid identity to HcrB, respectively. Of these genes, par1 in L. rossiae and hcrF in L. fermentum were overexpressed in response to hydroxycinnamic acids. The deletion of par1 in L. rossiae led to the loss of phenolic acid metabolism. The strain-specific metabolism of phenolic acids was congruent with the genotype of lactobacilli; however, phenolic acid reductases were not identified in strains of Weissella cibaria that reduced hydroxycinnamic acids to phenylpropionic acids. Phylogenetic analysis of major genes involved in hydroxycinnamic acid metabolism in strains of the genus Lactobacillus revealed that Par1 was found to be the most widely distributed phenolic acid reductase, while HcrB was the least abundant, present in less than 9% of Lactobacillus spp. In conclusion, this study increased the knowledge on the genetic determinants of hydroxycinnamic acid metabolism, explaining the species- and strain-specific metabolic variations in lactobacilli and providing evidence of additional enzymes involved in hydroxycinnamic acid metabolism of lactobacilli.IMPORTANCE The metabolism of secondary plant metabolites, including phenolic compounds, by food-fermenting lactobacilli is a significant contributor to the safety, quality, and nutritional quality of fermented foods. The enzymes mediating hydrolysis, reduction, and decarboxylation of phenolic acid esters and phenolic acids in lactobacilli, however, are not fully characterized. The genomic analyses presented here provide evidence for three novel putative phenolic acid reductases. Matching comparative genomic analyses with phenotypic analysis and quantification of gene expression indicates that two of the three putative phenolic acid reductases, Par1 and HcrF, are involved in reduction of hydroxycinnamic acids to phenylpropionic acids; however, the activity of Par2 may be unrelated to phenolic acids and recognizes other secondary plant metabolites. These findings expand our knowledge on the metabolic potential of lactobacilli and facilitate future studies on activity and substrate specificity of enzymes involved in metabolism of phenolic compounds.

The Locus of Heat Resistance Confers Resistance to Chlorine and Other Oxidizing Chemicals in Escherichia coli.

Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 CFU/ml higher than that for LHR-positive strains of E. coli The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY581/591, respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx2::gfp::amp Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx2::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island.IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.

Ecological Importance of Cross-Feeding of the Intermediate Metabolite 1,2-Propanediol between Bacterial Gut Symbionts.

Cross-feeding based on the metabolite 1,2-propanediol has been proposed to have an important role in the establishment of trophic interactions among gut symbionts, but its ecological importance has not been empirically established. Here, we show that in vitro growth of Lactobacillus reuteri (syn. Limosilactobacillus reuteri) ATCC PTA 6475 is enhanced through 1,2-propanediol produced by Bifidobacterium breve UCC2003 and Escherichia coli MG1655 from the metabolization of fucose and rhamnose, respectively. Work with isogenic mutants showed that the trophic interaction is dependent on the pduCDE operon in L. reuteri, which encodes the ability to use 1,2-propanediol, and the l-fucose permease (fucP) gene in B. breve, which is required for 1,2-propanediol formation from fucose. Experiments in gnotobiotic mice revealed that, although the pduCDE operon bestows a fitness burden on L. reuteri ATCC PTA 6475 in the mouse digestive tract, the ecological performance of the strain was enhanced in the presence of B. breve UCC2003 and the mucus-degrading species Bifidobacterium bifidum The use of the respective pduCDE and fucP mutants of L. reuteri and B. breve in the mouse experiments indicated that the trophic interaction was specifically based on 1,2-propanediol. Overall, our work established the ecological importance of cross-feeding relationships based on 1,2-propanediol for the fitness of a bacterial symbiont in the vertebrate gut.IMPORTANCE Through experiments in gnotobiotic mice that employed isogenic mutants of bacterial strains that produce (Bifidobacterium breve) and utilize (Lactobacillus reuteri) 1,2-propanediol, this study provides mechanistic insight into the ecological ramifications of a trophic interaction between gut symbionts. The findings improve our understanding on how cross-feeding influences the competitive fitness of L. reuteri in the vertebrate gut and revealed a putative selective force that shaped the evolution of the species. The findings are relevant since they provide a basis to design rational microbial-based strategies to modulate gut ecosystems, which could employ mixtures of bacterial strains that establish trophic interactions or a personalized approach based on the ability of a resident microbiota to provide resources for the incoming microbe.