Inhibition of Clinical MRSA Isolates by Coagulase Negative Staphylococci of Human Origin.

Staphylococcus aureus is frequently highlighted as a priority for novel drug research due to its pathogenicity and ability to develop antibiotic resistance. Coagulase-negative staphylococci (CoNS) are resident flora of the skin and nares. Previous studies have confirmed their ability to kill and prevent colonization by S. aureus through the production of bioactive substances. This study screened a bank of 37 CoNS for their ability to inhibit the growth of methicillin-resistant S. aureus (MRSA). Deferred antagonism assays, growth curves, and antibiofilm testing performed with the cell-free supernatant derived from overnight CoNS cultures indicated antimicrobial and antibiofilm effects against MRSA indicators. Whole genome sequencing and BAGEL4 analysis of 11 CoNS isolates shortlisted for the inhibitory effects they displayed against MRSA led to the identification of two strains possessing complete putative bacteriocin operons. The operons were predicted to encode a nukacin variant and a novel epilancin variant. From this point, strains Staphylococcus hominis C14 and Staphylococcus epidermidis C33 became the focus of the investigation. Through HPLC, a peptide identical to previously characterized nukacin KQU-131 and a novel epilancin variant were isolated from cultures of C14 and C33, respectively. Mass spectrometry confirmed the presence of each peptide in the active fractions. Spot-on-lawn assays demonstrated both bacteriocins could inhibit the growth of an MRSA indicator. The identification of natural products with clinically relevant activity is important in today's climate of escalating antimicrobial resistance and a depleting antibiotic pipeline. These findings also highlight the prospective role CoNS may play as a source of bioactive substances with activity against critical pathogens.

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.

The impact of the human microbiome in tumorigenesis, cancer progression, and biotherapeutic development.

BACKGROUND: Cancer impacts millions of lives globally each year, with approximately 10 million cancer-related deaths recorded worldwide in 2020. Mounting research has recognised the human microbiome as a key area of interest in the pathophysiology of various human diseases including cancer tumorigenesis, progression and in disease outcome. It is suggested that approximately 20% of human cancers may be linked to microbes. Certain residents of the human microbiome have been identified as potentially playing a role, including: Helicobacter pylori, Fusobacterium nucleatum, Escherichia coli, Bacteroides fragilis and Porphyromonas gingivalis. MAIN BODY: In this review, we explore the current evidence that indicate a link between the human microbiome and cancer. Microbiome compositional changes have been well documented in cancer patients. Furthermore, pathogenic microbes harbouring specific virulence factors have been implicated in driving the carcinogenic activity of various malignancies including colorectal, gastric and pancreatic cancer. The associated genetic mechanisms with possible roles in cancer will be outlined. It will be indicated which microbes have a potential direct link with cancer cell proliferation, tumorigenesis and disease progression. Recent studies have also linked certain microbial cytotoxins and probiotic strains to cancer cell death, suggesting their potential to target the tumour microenvironment given that cancer cells are integral to its composition. Studies pertaining to such cytotoxic activity have suggested the benefit of microbial therapies in oncological treatment regimes. It is also apparent that bacterial pathogenic protein products encoded for by certain loci may have potential as oncogenic therapeutic targets given their possible role in tumorigenesis. CONCLUSION: Research investigating the impact of the human microbiome in cancer has recently gathered pace. Vast amounts of evidence indicate the human microbiome as a potential player in tumorigenesis and progression. Promise in the development of cancer biomarkers and in targeted oncological therapies has also been demonstrated, although more studies are needed. Despite extensive in vitro and in vivo research, clinical studies involving large cohorts of human patients are lacking. The current literature suggests that further intensive research is necessary to validate both the role of the human microbiome in cancer, and the use of microbiome modification in cancer therapy.

Generation of Nonpolar Deletion Mutants in Listeria monocytogenes Using the "SOEing" Method.

The ability to manipulate chromosomally encoded genes is a fundamental biological tool for the analysis of gene function. Here, we provide in greater depth a protocol for the creation of nonpolar unlabelled gene deletions in Listeria monocytogenes that are facilitated by the splicing overlap extension PCR technique. For mutagenesis in L. monocytogenes, we describe the pKSV7 plasmid-based approach, which facilitates the introduction of a spliced amplicon in place of the corresponding segment of chromosomal DNA.

First evidence of production of the lantibiotic nisin P.

Nisin P is a natural nisin variant, the genetic determinants for which were previously identified in the genomes of two Streptococcus species, albeit with no confirmed evidence of production. Here we describe Streptococcus agalactiae DPC7040, a human faecal isolate, which exhibits antimicrobial activity against a panel of gut and food isolates by virtue of producing nisin P. Nisin P was purified, and its predicted structure was confirmed by nanoLC-MS/MS, with both the fully modified peptide and a variant without rings B and E being identified. Additionally, we compared its spectrum of inhibition and minimum inhibitory concentration (MIC) with that of nisin A and its antimicrobial effect in a faecal fermentation in comparison with nisin A and H. We found that its antimicrobial activity was less potent than nisin A and H, and we propose a link between this reduced activity and the peptide structure.

Potential Use of Biotherapeutic Bacteria to Target Colorectal Cancer-Associated Taxa.

The role of the gut microbiome in human health and disease is the focus of much attention. It has been widely agreed upon that our gut bacteria play a role in host immunity, nutrient absorption, digestion, metabolism, and other key drivers of health. Furthermore, certain microbial signatures and specific taxa have also been associated with the development of diseases, such as obesity; inflammatory bowel disease; and, indeed, colorectal cancer (CRC), which is the focus of this review. By extension, such taxa represent potential therapeutic targets. In particular, the emerging human pathogen Fusobacterium nucleatum represents an important agent in CRC development and its control within the gastrointestinal tract is desirable. This paper reviews the principal bacterial pathogens that have been associated with CRC to date and discusses the in vitro and human studies that have shown the potential use of biotherapeutic strains as a means of targeting CRC-associated bacteria.

Bovine Staphylococcus aureus Superantigens Stimulate the Entire T Cell Repertoire of Cattle.

Superantigens (SAgs) represent a diverse family of bacterial toxins that induce Vß-specific T cell proliferation associated with an array of important diseases in humans and animals, including mastitis of dairy cows. However, an understanding of the diversity and distribution of SAg genes among bovine Staphylococcus aureus strains and their role in the pathogenesis of mastitis is lacking. Population genomic analysis of 195 bovine S. aureus isolates representing 57 unique sequence types revealed that strains encode 2 to 13 distinct SAgs and that the majority of isolates contain 5 or more SAg genes. A genome-scale analysis of bovine reference strain RF122 revealed a complement of 11 predicted SAg genes, which were all expressed in vitro Detection of specific antibodies in convalescent cows suggests expression of 7 of 11 SAgs during natural S. aureus infection. We determined the Vß T cell activation profile for all functional SAgs encoded by RF122, revealing evidence for bovine host-specific activity among the recently identified RF122-encoded SAgs SElY and SElZ. Remarkably, we discovered that some strains have evolved the capacity to stimulate the entire T cell repertoire of cattle through an array of diverse SAgs, suggesting a key role in bovine immune evasion.

A Profile Hidden Markov Model to investigate the distribution and frequency of LanB-encoding lantibiotic modification genes in the human oral and gut microbiome.

BACKGROUND: The human microbiota plays a key role in health and disease, and bacteriocins, which are small, bacterially produced, antimicrobial peptides, are likely to have an important function in the stability and dynamics of this community. Here we examined the density and distribution of the subclass I lantibiotic modification protein, LanB, in human oral and stool microbiome datasets using a specially constructed profile Hidden Markov Model (HMM). METHODS: The model was validated by correctly identifying known lanB genes in the genomes of known bacteriocin producers more effectively than other methods, while being sensitive enough to differentiate between different subclasses of lantibiotic modification proteins. This approach was compared with two existing methods to screen both genomic and metagenomic datasets obtained from the Human Microbiome Project (HMP). RESULTS: Of the methods evaluated, the new profile HMM identified the greatest number of putative LanB proteins in the stool and oral metagenome data while BlastP identified the fewest. In addition, the model identified more LanB proteins than a pre-existing Pfam lanthionine dehydratase model. Searching the gastrointestinal tract subset of the HMP reference genome database with the new HMM identified seven putative subclass I lantibiotic producers, including two members of the Coprobacillus genus. CONCLUSIONS: These findings establish custom profile HMMs as a potentially powerful tool in the search for novel bioactive producers with the power to benefit human health, and reinforce the repertoire of apparent bacteriocin-encoding gene clusters that may have been overlooked by culture-dependent mining efforts to date.

Genome Sequence of Staphylococcus saprophyticus DPC5671, a Strain Isolated from Cheddar Cheese.

The draft genome sequence of Staphylococcus saprophyticus DPC5671, isolated from cheddar cheese, was determined. S. saprophyticus is a common Gram-positive bacterium detected on the surface of smear-ripened cheese and other fermented foods.

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.

Bacteriocin production: a relatively unharnessed probiotic trait?

Probiotics are "live microorganisms which, when consumed in adequate amounts, confer a health benefit to the host". A number of attributes are highly sought after among these microorganisms, including immunomodulation, epithelial barrier maintenance, competitive exclusion, production of short-chain fatty acids, and bile salt metabolism. Bacteriocin production is also generally regarded as a probiotic trait, but it can be argued that, in contrast to other traits, it is often considered a feature that is desirable, rather than a key probiotic trait. As such, the true potential of these antimicrobials has yet to be realised.

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.

In silico identification of bacteriocin gene clusters in the gastrointestinal tract, based on the Human Microbiome Project's reference genome database.

BACKGROUND: The human gut microbiota comprises approximately 100 trillion microbial cells which significantly impact many aspects of human physiology - including metabolism, nutrient absorption and immune function. Disturbances in this population have been implicated in many conditions and diseases, including obesity, type-2 diabetes and inflammatory bowel disease. This suggests that targeted manipulation or shaping of the gut microbiota, by bacteriocins and other antimicrobials, has potential as a therapeutic tool for the prevention or treatment of these conditions. With this in mind, several studies have used traditional culture-dependent approaches to successfully identify bacteriocin-producers from the mammalian gut. In silico-based approaches to identify novel gene clusters are now also being utilised to take advantage of the vast amount of data currently being generated by next generation sequencing technologies. In this study, we employed an in silico screening approach to mine potential bacteriocin clusters in genome-sequenced isolates from the gastrointestinal tract (GIT). More specifically, the bacteriocin genome-mining tool BAGEL3 was used to identify potential bacteriocin producers in the genomes of the GIT subset of the Human Microbiome Project's reference genome database. Each of the identified gene clusters were manually annotated and potential bacteriocin-associated genes were evaluated. RESULTS: We identified 74 clusters of note from 59 unique members of the Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria and Synergistetes. The most commonly identified class of bacteriocin was the >10 kDa class, formerly known as bacteriolysins, followed by lantibiotics and sactipeptides. CONCLUSIONS: Multiple bacteriocin gene clusters were identified in a dataset representative of the human gut microbiota. Interestingly, many of these were associated with species and genera which are not typically associated with bacteriocin production.

Impact of Environmental Factors on Bacteriocin Promoter Activity in Gut-Derived Lactobacillus salivarius.

Bacteriocin production is regarded as a desirable probiotic trait that aids in colonization and persistence in the gastrointestinal tract (GIT). Strains of Lactobacillus salivarius, a species associated with the GIT, are regarded as promising probiotic candidates and have a number of associated bacteriocins documented to date. These include multiple class IIb bacteriocins (salivaricin T, salivaricin P, and ABP-118) and the class IId bacteriocin bactofencin A, which show activity against medically important pathogens. However, the production of a bacteriocin in laboratory media does not ensure production under stressful environmental conditions, such as those encountered within the GIT. To allow this issue to be addressed, the promoter regions located upstream of the structural genes encoding the L. salivarius bacteriocins mentioned above were fused to a number of reporter proteins (green fluorescent protein [GFP], red fluorescent protein [RFP], and luciferase [Lux]). Of these, only transcriptional fusions to GFP generated signals of sufficient strength to enable the study of promoter activity in L. salivarius. While analysis of the class IIb bacteriocin promoter regions indicated relatively weak GFP expression, assessment of the promoter of the antistaphylococcal bacteriocin bactofencin A revealed a strong promoter that is most active in the absence of the antimicrobial peptide and is positively induced in the presence of mild environmental stresses, including simulated gastric fluid. Taken together, these data provide information on factors that influence bacteriocin production, which will assist in the development of strategies to optimize in vivo and in vitro production of these antimicrobials.

Nisin H Is a New Nisin Variant Produced by the Gut-Derived Strain Streptococcus hyointestinalis DPC6484.

Accumulating evidence suggests that bacteriocin production represents a probiotic trait for intestinal strains to promote dominance, fight infection, and even signal the immune system. In this respect, in a previous study, we isolated from the porcine intestine a strain of Streptococcus hyointestinalis DPC6484 that displays antimicrobial activity against a wide range of Gram-positive bacteria and produces a bacteriocin with a mass of 3,453 Da. Interestingly, the strain was also found to be immune to a nisin-producing strain. Genome sequencing revealed the genetic determinants responsible for a novel version of nisin, designated nisin H, consisting of the nshABTCPRKGEF genes, with transposases encoded between nshP and nshR and between nshK and nshG. A similar gene cluster is also found in S. hyointestinalis LMG14581. Notably, the cluster lacks an equivalent of the nisin immunity gene, nisI. Nisin H is proposed to have the same structure as the prototypical nisin A but differs at 5 amino acid positions-Ile1Phe (i.e., at position 1, nisin A has Ile while nisin H has Phe), Leu6Met, Gly18Dhb (threonine dehydrated to dehydrobutyrine), Met21Tyr, and His31Lys--and appears to represent an intermediate between the lactococcal nisin A and the streptococcal nisin U variant of nisin. Purified nisin H inhibits a wide range of Gram-positive bacteria, including staphylococci, streptococci, Listeria spp., bacilli, and enterococci. It represents the first example of a natural nisin variant produced by an intestinal isolate of streptococcal origin.

A single natural nucleotide mutation alters bacterial pathogen host tropism.

The capacity of microbial pathogens to alter their host tropism leading to epidemics in distinct host species populations is a global public and veterinary health concern. To investigate the molecular basis of a bacterial host-switching event in a tractable host species, we traced the evolutionary trajectory of the common rabbit clone of Staphylococcus aureus. We report that it evolved through a likely human-to-rabbit host jump over 40 years ago and that only a single naturally occurring nucleotide mutation was required and sufficient to convert a human-specific S. aureus strain into one that could infect rabbits. Related mutations were identified at the same locus in other rabbit strains of distinct clonal origin, consistent with convergent evolution. This first report of a single mutation that was sufficient to alter the host tropism of a microorganism during its evolution highlights the capacity of some pathogens to readily expand into new host species populations.

Genome Sequence of the Heteropolysaccharide-Producing Strain Lactobacillus mucosae DPC 6426.

Exopolysaccharide-synthesizing Lactobacillus mucosae DPC 6426 is a heterofermentative strain, which has demonstrated cholesterol-lowering properties in an animal model of lipid-driven atherosclerosis. The genome revealed a plethora of homologues linked to carbohydrate metabolism and mucin binding.

Genome analysis of Cronobacter phage vB_CsaP_Ss1 reveals an endolysin with potential for biocontrol of Gram-negative bacterial pathogens.

Bacteriophages and their derivatives are continuously gaining impetus as viable alternative therapeutic agents to control harmful multidrug-resistant bacterial pathogens, particularly in the food industry. The reduced efficacy of conventional antibiotics has resulted in a quest to find novel alternatives in the war against infectious disease. This study describes the full-genome sequence of Cronobacter phage vB_CsaP_Ss1, with subsequent cloning and expression of its endolysin, capable of hydrolysing Gram-negative peptidoglycan. Cronobacter phage vB_CsaP_Ss1 is composed of 42 205 bp of dsDNA with a G+C content of 46.1 mol%. A total of 57 ORFs were identified of which 18 could be assigned a putative function based on similarity to characterized proteins. The genome of Cronobacter phage vB_CsaP_Ss1 showed little similarity to any other bacteriophage genomes available in the database and thus was considered unique. In addition, functional analysis of the predicted endolysin (LysSs1) was also investigated. Zymographic experiments demonstrated the hydrolytic activity of LysSs1 against Gram-negative peptidoglycan, and this endolysin thus represents a novel candidate with potential for use against Gram-negative pathogens.

Generation of nonpolar deletion mutants in Listeria monocytogenes using the "SOEing" method.

The ability to manipulate chromosomally encoded genes is a fundamental biological tool for the analysis of gene function. Here, we provide in greater depth protocols for the creation of nonpolar unlabeled gene deletions in Listeria (L.) monocytogenes that are facilitated by the splicing overlap extension PCR technique. For mutagenesis in L. monocytogenes, we describe two different plasmid-based approaches, which facilitate the introduction of this spliced amplicon in place of the corresponding segment of chromosomal DNA: the pKSV7 system and the pORI280/pVE6007 system.