Profiling of vaginal Lactobacillus jensenii isolated from preterm and full-term pregnancies reveals strain-specific factors relating to host interaction.

Each year, 15 million infants are born preterm (<37 weeks gestation), representing the leading cause of mortality for children under the age of five. Whilst there is no single cause, factors such as maternal genetics, environmental interactions, and the vaginal microbiome have been associated with an increased risk of preterm birth. Previous studies show that a vaginal microbiota dominated by Lactobacillus is, in contrast to communities containing a mixture of genera, associated with full-term birth. However, this binary principle does not fully consider more nuanced interactions between bacterial strains and the host. Here, through a combination of analyses involving genome-sequenced isolates and strain-resolved metagenomics, we identify that L. jensenii strains from preterm pregnancies are phylogenetically distinct from strains from full-term pregnancies. Detailed analysis reveals several genetic signatures that distinguish preterm birth strains, including genes predicted to be involved in cell wall synthesis, and lactate and acetate metabolism. Notably, we identify a distinct gene cluster involved in cell surface protein synthesis in our preterm strains, and profiling the prevalence of this gene cluster in publicly available genomes revealed it to be predominantly present in the preterm-associated clade. This study contributes to the ongoing search for molecular biomarkers linked to preterm birth and opens up new avenues for exploring strain-level variations and mechanisms that may contribute to preterm birth.

Maternal breastfeeding is associated with offspring microbiome diversity; a secondary analysis of the MicrobeMom randomized control trial.

Background: Microbial dysbiosis in infancy can influence long-term health outcomes such as childhood obesity. The aim of this study is to explore relationships among maternal well-being during pregnancy, breastfeeding, and the infant gut microbiome. Methods: This is a secondary analysis of healthy pregnant women from the MicrobeMom study, a double-blind randomized control trial of maternal probiotic supplementation (Bifidobacterium breve 702258) versus placebo antenatally and up to 3 months postpartum. Maternal well-being was assessed using the WHO-5 well-being index at 16 weeks' and 34 weeks' gestation. Breastfeeding practices were recorded at discharge from hospital and at 1 month postpartum. Infant stool samples were obtained at 1 month of age. Next generation shotgun sequencing determined infant microbial diversity. Independent sample t-tests and Mann-Whitney U tests informed adjusted regression analysis, which was adjusted for delivery mode, antibiotics during delivery, maternal age and body mass index (BMI), and probiotic vs. control study group. Results: Women (n = 118) with at least one measure of well-being were on average 33 years (SD 3.93) of age and 25.09 kg/m2 (SD 3.28) BMI. Exclusive breastfeeding was initiated by 65% (n = 74). Any breastfeeding was continued by 69% (n = 81) after 1 month. In early and late pregnancy, 87% (n = 97/111) and 94% (n = 107/114) had high well-being scores. Well-being was not associated with infant microbial diversity at 1 month. In adjusted analysis, exclusive breastfeeding at discharge from hospital was associated with infant microbial beta diversity (PC2; 0.254, 95% CI 0.006, 0.038). At 1 month postpartum, any breastfeeding was associated with infant microbial alpha diversity (Shannon index; -0.241, 95% CI -0.498, -0.060) and observed species; (-0.325, 95% CI -0.307, -0.060), and infant microbial beta diversity (PC2; 0.319, 95% CI 0.013, 0.045). Exclusive breastfeeding at 1 month postpartum was associated with infant alpha diversity (Shannon index -0.364, 95% CI -0.573, -0.194; Simpson index 0.339, 95% CI 0.027, 0.091), and infant's number of observed microbial species (-0.271, 95% CI -0.172, -0.037). Conclusion: Breastfeeding practices at 1 month postpartum were associated with lower microbial diversity and observed species in infants at 1 month postpartum, which is potentially beneficial to allow greater abundance of Bifidobacterium. Clinical trial registration: ISRCTN53023014.

Detailed mapping of Bifidobacterium strain transmission from mother to infant via a dual culture-based and metagenomic approach.

A significant proportion of the infant gut microbiome is considered to be acquired from the mother during and after birth. Thus begins a lifelong and dynamic relationship with microbes that has an enduring impact on host health. Based on a cohort of 135 mother-infant (F = 72, M = 63) dyads (MicrobeMom: ISRCTN53023014), we investigated the phenomenon of microbial strain transfer, with a particular emphasis on the use of a combined metagenomic-culture-based approach to determine the frequency of strain transfer involving members of the genus Bifidobacterium, including species/strains present at low relative abundance. From the isolation and genome sequencing of over 449 bifidobacterial strains, we validate and augment metagenomics-based evidence to reveal strain transfer in almost 50% of dyads. Factors important in strain transfer include vaginal birth, spontaneous rupture of amniotic membranes, and avoidance of intrapartum antibiotics. Importantly, we reveal that several transfer events are uniquely detected employing either cultivation or metagenomic sequencing, highlighting the requirement for a dual approach to obtain an in-depth insight into this transfer process.

Ability of Bifidobacterium breve 702258 to transfer from mother to infant: the MicrobeMom randomized controlled trial.

BACKGROUND: The composition of the infant microbiome can have a variety of short- and long-term implications for health. It is unclear if maternal probiotic supplementation in pregnancy can affect the infant gut microbiome. OBJECTIVE: This study aimed to investigate if maternal supplementation of a formulation of Bifidobacterium breve 702258 from early pregnancy until 3 months postpartum could transfer to the infant gut. STUDY DESIGN: This was a double-blinded, placebo-controlled, randomized controlled trial of B breve 702258 (minimum 1 × 109 colony-forming units) or placebo taken orally from 16 weeks' gestation until 3 months postpartum in healthy pregnant women. The primary outcome was presence of the supplemented strain in infant stool up to 3 months of life, detected by at least 2 of 3 methods: strain-specific polymerase chain reaction, shotgun metagenomic sequencing, or genome sequencing of cultured B breve. A total of 120 individual infants' stool samples were required for 80% power to detect a difference in strain transfer between groups. Rates of detection were compared using the Fisher exact test. RESULTS: A total of 160 pregnant women with average age of 33.6 (3.9) years and mean body mass index of 24.3 (22.5-26.5) kg/m2, of whom 43% were nulliparous (n=58), were recruited from September 2016 to July 2019. Neonatal stool samples were obtained from 135 infants (65 in intervention and 70 in control group). The presence of the supplemented strain was detected through at least 2 methods (polymerase chain reaction and culture) in 2 infants in the intervention group (n=2/65; 3.1%) and none in the control group (n=0; 0%; P=.230). CONCLUSION: Direct mother-to-infant strain transfer of B breve 702258 occurred, albeit infrequently. This study highlights the potential for maternal supplementation to introduce microbial strains into the infant microbiome.

Effect of a bacteriocin-producing Streptococcus salivarius on the pathogen Fusobacterium nucleatum in a model of the human distal colon.

The gut microbiome is a vast reservoir of microbes, some of which produce antimicrobial peptides called bacteriocins that may inhibit specific bacteria associated with disease. Fusobacterium nucleatum is an emerging human bacterial pathogen associated with gastrointestinal diseases including colorectal cancer (CRC). In this study, fecal samples of healthy donors were screened for potential bacteriocin-producing probiotics with antimicrobial activity against F. nucleatum. A novel isolate, designated as Streptococcus salivarius DPC6993 demonstrated a narrow-spectrum of antimicrobial activity against F. nucleatum in vitro. In silico analysis of the S. salivarius DPC6993 genome revealed the presence of genes involved in the production of the bacteriocins salivaricin A5 and salivaricin B. After 6 h in a colon fermentation model, there was a significant drop in the number of F. nucleatum in samples that had been simultaneously inoculated with S. salivarius DPC6993 + F. nucleatum DSM15643 compared to those inoculated with F. nucleatum DSM15643 alone (mean ± SD: 9243.3 ± 3408.4 vs 29688.9 ± 4993.9 copies/µl). Furthermore, 16S rRNA amplicon analysis revealed a significant difference in the mean relative abundances of Fusobacterium between samples inoculated with both S. salivarius DPC6993 and F. nucleatum DSM15643 (0.05%) and F. nucleatum DSM15643 only (0.32%). Diversity analysis indicated minimal impact exerted by S. salivarius DPC6993 on the surrounding microbiota. Overall, this study highlights the ability of a natural gut bacterium to target a bacterial pathogen associated with CRC. The specific targeting of CRC-associated pathogens by biotherapeutics may ultimately reduce the risk of CRC development and positively impact CRC outcomes.

In Vitro and In Silico Based Approaches to Identify Potential Novel Bacteriocins from the Athlete Gut Microbiome of an Elite Athlete Cohort.

Exercise reduces inflammation, fatigue, and aids overall health. Additionally, physical fitness has been associated with desirable changes in the community composition of the athlete gut microbiome, with health-associated taxa being shown to be increased in active individuals. Here, using a combination of in silico and in vitro methods, we investigate the antimicrobial activity of the athlete gut microbiome. In vitro approaches resulted in the generation of 284 gut isolates with inhibitory activity against Clostridioides difficile and/or Fusobacterium nucleatum, and the most potent isolates were further characterized, and potential bacteriocins were predicted using both MALDI-TOF MS and whole-genome sequencing. Additionally, metagenomic reads from the faecal samples were used to recover 770 Metagenome Assembled Genomes (MAGs), of which 148 were assigned to be high-quality MAGs and screened for the presence of putative bacteriocin gene clusters using BAGEL4 software, with 339 gene clusters of interest being identified. Class I was the most abundant bacteriocin class predicted, accounting for 91.3% of predictions, Class III had a predicted abundance of 7.5%, and Class II was represented by just 1% of all predictions.

Shotgun sequencing of the vaginal microbiome reveals both a species and functional potential signature of preterm birth.

An association between the vaginal microbiota and preterm birth (PTB) has been reported in several research studies. Population shifts from high proportions of lactobacilli to mixed species communities, as seen with bacterial vaginosis, have been linked to a twofold increased risk of PTB. Despite the increasing number of studies using next-generation sequencing technologies, primarily involving 16S rRNA-based approaches, to investigate the vaginal microbiota during pregnancy, no distinct microbial signature has been associated with PTB. Shotgun metagenomic sequencing offers a powerful tool to reveal community structures and their gene functions at a far greater resolution than amplicon sequencing. In this study, we employ shotgun metagenomic sequencing to compare the vaginal microbiota of women at high risk of preterm birth (n = 35) vs. a low-risk control group (n = 14). Although microbial diversity and richness did not differ between groups, there were significant differences in terms of individual species. In particular, Lactobacillus crispatus was associated with samples from a full-term pregnancy, whereas one community state-type was associated with samples from preterm pregnancies. Furthermore, by predicting gene functions, the functional potential of the preterm microbiota was different from that of full-term equivalent. Taken together, we observed a discrete structural and functional difference in the microbial composition of the vagina in women who deliver preterm. Importance: with an estimated 15 million cases annually, spontaneous preterm birth (PTB) is the leading cause of death in infants under the age of five years. The ability to accurately identify pregnancies at risk of spontaneous PTB is therefore of utmost importance. However, no single cause is attributable. Microbial infection is a known risk factor, yet the role of vaginal microbes is poorly understood. Using high-resolution DNA-sequencing techniques, we investigate the microbial communities present in the vaginal tracts of women deemed high risk for PTB. We confirm that Lactobacillus crispatus is strongly linked to full-term pregnancies, whereas other microbial communities associate with PTB. Importantly, we show that the specific functions of the microbes present in PTB samples differs from FTB samples, highlighting the power of our sequencing approach. This information enables us to begin understanding the specific microbial traits that may be influencing PTB, beyond the presence or absence of microbial taxa.

Whey protein effects on energy balance link the intestinal mechanisms of energy absorption with adiposity and hypothalamic neuropeptide gene expression.

We tested the hypothesis that dietary whey protein isolate (WPI) affects the intestinal mechanisms related to energy absorption and that the resulting energy deficit is compensated by changes in energy balance to support growth. C57BL/6 mice were provided a diet enriched with WPI with varied sucrose content, and the impact on energy balance-related parameters was investigated. As part of a high-sucrose diet, WPI reduced the hypothalamic expression of pro-opiomelanocortin gene expression and increased energy intake. The energy expenditure was unaffected, but epididymal weight was reduced, indicating an energy loss. Notably, there was a reduction in the ileum gene expression for amino acid transporter SLC6a19, glucose transporter 2, and fatty acid transporter 4. The composition of the gut microbiota also changed, where Firmicutes were reduced. The above changes indicated reduced energy absorption through the intestine. We propose that this mobilized energy in the adipose tissue and caused hypothalamic changes that increased energy intake, acting to counteract the energy deficit arising in the intestine. Lowering the sucrose content in the WPI diet increased energy expenditure. This further reduced epididymal weight and plasma leptin, whereupon hypothalamic ghrelin gene expression and the intestinal weight were both increased. These data suggest that when the intestine-adipose-hypothalamic pathway is subjected to an additional energy loss (now in the adipose tissue), compensatory changes attempt to assimilate more energy. Notably, WPI and sucrose content interact to enable the component mechanisms of this pathway.

The bacteriocin bactofencin A subtly modulates gut microbial populations.

The diverse and dynamic microbiota of the gastrointestinal tract represents a vast source of bioactive substances. These include bacteriocins, which are antimicrobial peptides with the potential to modulate gut populations to impact positively on human health. Although several gut-derived bacteriocins have been isolated, there remain only a few exceptional studies in which their influence on microbial populations within the gut has been investigated. To facilitate such investigations, in vitro faecal fermentation systems can be used to simulate the anaerobic environment of the colon. In this instance, such a system was employed to explore the impact of bactofencin A, a novel broad spectrum class IId bacteriocin produced by gut isolates of Lactobacillus salivarius, on intestinal populations and overall microbial diversity. The study reveals that, although bactofencin A is a broad spectrum bacteriocin, it has a relatively subtle influence on intestinal communities, with a potentially positive impact on anaerobic populations such as Bacteroides, Clostridium and Bifidibacterium spp. The strategy taken is an important first step in investigating the merits of using bactofencin A to manipulate the gut microbiota in a beneficial way for health.

Homologues and bioengineered derivatives of LtnJ vary in ability to form D-alanine in the lantibiotic lacticin 3147.

Ltnα and Ltnß are individual components of the two-peptide lantibiotic lacticin 3147 and are unusual in that, although ribosomally synthesized, they contain d-amino acids. These result from the dehydration of l-serine to dehydroalanine by LtnM and subsequent stereospecific hydrogenation to d-alanine by LtnJ. Homologues of LtnJ are rare but have been identified in silico in Staphylococcus aureus C55 (SacJ), Pediococcus pentosaceus FBB61 (PenN), and Nostoc punctiforme PCC73102 (NpnJ, previously called NpunJ [P. D. Cotter et al., Proc. Natl. Acad. Sci. U. S. A. 102:18584-18589, 2005]). Here, the ability of these enzymes to catalyze d-alanine formation in the lacticin 3147 system was assessed through heterologous enzyme production in a ΔltnJ mutant. PenN successfully incorporated d-alanines in both peptides, and SacJ modified Ltnα only, while NpnJ was unable to modify either peptide. Site-directed mutagenesis was also employed to identify residues of key importance in LtnJ. The most surprising outcome from these investigations was the generation of peptides by specific LtnJ mutants which exhibited less bioactivity than those generated by the ΔltnJ strain. We have established that the reduced activity of these peptides is due to the inability of the associated LtnJ enzymes to generate d-alanine residues in a stereospecific manner, resulting in the presence of both d- and l-alanines at the relevant locations in the lacticin 3147 peptides.

Manipulation of charged residues within the two-peptide lantibiotic lacticin 3147.

Lantibiotics are antimicrobial peptides which contain a high percentage of post-translationally modified residues. While most attention has been paid to the role of these critical structural features, evidence continues to emerge that charged amino acids also play a key role in these peptides. Here 16 'charge' mutants of the two-peptide lantibiotic lacticin 3147 [composed of Ltnα (2+, 2-) and Ltnß (2+)] were constructed which, when supplemented with previously generated peptides, results in a total bank of 23 derivatives altered in one or more charged residues. When examined individually, in combination with a wild-type partner or, in some instances, in combination with one another, these mutants reveal the importance of charge at specific locations within Ltnα and Ltnß, confirm the critical role of the negatively charged glutamate residue in Ltnα and facilitate an investigation of the contribution of positively charged residues to the cationic Ltnß. From these investigations it is also apparent that the relative importance of the overall charge of lacticin 3147 varies depending on the target bacteria and is most evident when strains with more negatively charged cell envelopes are targeted. These studies also result in, for the first time, the creation of a derivative of a lacticin 3147 peptide (LtnßR27A) which displays enhanced specific activity.

Listeriolysin S, a novel peptide haemolysin associated with a subset of lineage I Listeria monocytogenes.

Streptolysin S (SLS) is a bacteriocin-like haemolytic and cytotoxic virulence factor that plays a key role in the virulence of Group A Streptococcus (GAS), the causative agent of pharyngitis, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. Although it has long been thought that SLS and related peptides are produced by GAS and related streptococci only, there is evidence to suggest that a number of the most notorious Gram-positive pathogenic bacteria, including Listeria monocytogenes, Clostridium botulinum and Staphylococcus aureus, produce related peptides. The distribution of the L. monocytogenes cluster is particularly noteworthy in that it is found exclusively among a subset of lineage I strains; i.e., those responsible for the majority of outbreaks of listeriosis. Expression of these genes results in the production of a haemolytic and cytotoxic factor, designated Listeriolysin S, which contributes to virulence of the pathogen as assessed by murine- and human polymorphonuclear neutrophil-based studies. Thus, in the process of establishing the existence of an extended family of SLS-like modified virulence peptides (MVPs), the genetic basis for the enhanced virulence of a proportion of lineage I L. monocytogenes may have been revealed.

Insertional mutagenesis to generate lantibiotic resistance in Lactococcus lactis.

While the potential emergence of food spoilage and pathogenic bacteria with resistance to lantibiotics is a concern, the creation of derivatives of starter cultures and adjuncts that can grow in the presence of these antimicrobials may have applications in food fermentations. Here a bank of Lactococcus lactis IL1403 mutants was created and screened, and a number of novel genetic loci involved in lantibiotic resistance were identified.

Identification of a novel two-peptide lantibiotic, haloduracin, produced by the alkaliphile Bacillus halodurans C-125.

Complete genome sequencing of the alkaliphilic bacterium Bacillus halodurans C-125 revealed the presence of several genes homologous to those involved in the production of lantibiotic peptides. Additional bioinformatic analysis identified a total of eleven genes, spanning a 15 kbp region, potentially involved in the production, modification, immunity and transport of a two-peptide lantibiotic. Having established that strain C-125 exhibited antimicrobial activity against a wide range of Gram-positive bacteria, it was demonstrated through peptide purification, MS and site-directed mutagenesis that this activity was indeed attributable to the production of a lantibiotic encoded by these genes. This antimicrobial has been designated haloduracin and represents the first occasion wherein production of two-peptide lantibiotic has been associated with a Bacillus sp. It is also the first example of a lantibiotic of any kind to be produced by an alkaliphilic species.

Two-peptide lantibiotics: a medical perspective.

Lantibiotics are ribosomally synthesised, post-translationally modified antimicrobial peptides that exhibit activity against a wide-range of Gram positive bacteria. During the last decade a number of two-peptide lantibiotics, i.e. lantibiotics that function optimally as a consequence of the synergistic activity of two peptides, have been identified, six of which (lacticin 3147, staphylococcin C55, plantaricin W, Smb, BHT-A and haloduracin) are closely related. It has been established in at least one instance, i.e. lacticin 3147, that these are extremely potent antimicrobials, which are active at nanomolar concentrations against a number of microorganisms, exhibit activity against multidrug resistant nosocomial pathogens such as MRSA and VRE and significantly, to date the development of significant levels of resistance has not been apparent. Given the similarity between lacticin 3147 and related two-peptide lantibiotics, it is likely that they too possess similarly beneficial traits and thus could potentially have medical and veterinary applications. In addition to discussing these aspects of two-peptide lantibiotic research, this review will focus on new developments in this area pertaining to studies elucidating the mechanism of action of these antimicrobials, the use of bioengineering to reveal the location of essential and variable domains therein and the potential for the use of in vivo and in vitro engineering to create derivatives with even greater activities against specific target organisms.

Complete alanine scanning of the two-component lantibiotic lacticin 3147: generating a blueprint for rational drug design.

Lantibiotics are post-translationally modified antimicrobial peptides which are active at nanomolar concentrations. Some lantibiotics have been shown to function by targeting lipid II, the essential precursor of cell wall biosynthesis. Given that lantibiotics are ribosomally synthesized and amenable to site-directed mutagenesis, they have the potential to serve as biological templates for the production of novel peptides with improved functionalities. However, if a rational approach to novel lantibiotic design is to be adopted, an appreciation of the roles of each individual amino acid (and each domain) is required. To date no lantibiotic has been subjected to such rigorous analysis. To address this issue we have carried out complete scanning mutagenesis of each of the 59 amino acids in lacticin 3147, a two-component lantibiotic which acts through the synergistic activity of the peptides LtnA1 (30 amino acids) and LtnA2 (29 amino acids). All mutations were performed in situ in the native 60 kb plasmid, pMRC01. A number of mutations resulted in the elimination of detectable bioactivity and seem to represent an invariable core within these and related peptides. Significantly however, of the 59 amino acids, at least 36 can be changed without resulting in a complete loss of activity. Many of these are clustered to form variable domains within the peptides. The information generated in this study represents a blue-print that will be critical for the rational design of lantibiotic-based antimicrobial compounds.

Overproduction of wild-type and bioengineered derivatives of the lantibiotic lacticin 3147.

Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e., ltnA1A2) is not.

Posttranslational conversion of L-serines to D-alanines is vital for optimal production and activity of the lantibiotic lacticin 3147.

As a general rule, ribosomally synthesized polypeptides contain amino acids only in the L-isoform in an order dictated by the coding DNA/RNA. Two of a total of only four examples of L to D conversions in prokaryotic systems occur in posttranslationally modified antimicrobial peptides called lantibiotics. In both examples (lactocin S and lacticin 3147), ribosomally encoded L-serines are enzymatically converted to D-alanines, giving rise to an apparent mistranslation of serine codons to alanine residues. It has been suggested that this conversion results from a two-step reaction initiated by a lantibiotic synthetase converting the gene-encoded L-serine to dehydroalanine (dha). By using lacticin 3147 as a model system, we report the identification of an enzyme, LtnJ, that is responsible for the conversion of dha to D-alanine. Deletion of this enzyme results in the residues remaining as dha intermediates, leading to a dramatic reduction in the antimicrobial activity of the producing strain. The importance of the chirality of the three D-alanines present in lacticin 3147 was confirmed when these residues were systematically substituted by L-alanines. In addition, substitution with L-threonine (ultimately modified to dehydrobutyrine), glycine, or L-valine also resulted in diminished peptide production and/or relative activity, the extent of which depended on the chirality of the newly incorporated amino acid(s).