Search

Human microbiomes are essential to health throughout the lifespan and are increasingly recognized and studied for their roles in metabolic, immunological and neurological processes. Although the full complexity of these microbial communities is not fully understood, their clinical and industrial exploitation is well advanced and expanding, needing greater oversight guided by a consensus from the research community. One of the most controversial issues in microbiome research is the definition of a ‘healthy’ human microbiome. This concept is complicated by the microbial variability over different spatial and temporal scales along with the challenge of applying a unified definition to the spectrum of healthy microbiome configurations. In this Perspective, we examine the progress made and the key gaps that remain to be addressed to fully harness the benefits of the human microbiome. We propose a road map to expand our knowledge of the microbiome–health relationship, incorporating epidemiological approaches informed by the unique ecological characteristics of these communities.

Bacteriocins are potent antimicrobial peptides that are produced by bacteria. Since their discovery almost a century ago, diverse peptides have been discovered and described, and some are currently used as commercial food preservatives. Many bacteriocins exhibit extensively post-translationally modified structures encoded on complex gene clusters, whereas others have simple linear structures. The molecular structures, mechanisms of action and resistance have been determined for a number of bacteriocins, but most remain incompletely characterized. These gene-encoded peptides are amenable to bioengineering strategies and heterologous expression, enabling metagenomic mining and modification of novel antimicrobials. The ongoing global antimicrobial resistance crisis demands that novel therapeutics be developed to combat infectious pathogens. New compounds that are target-specific and compatible with the resident microbiota would be valuable alternatives to current antimicrobials. As bacteriocins can be broad or narrow spectrum in nature, they are promising tools for this purpose. However, few bacteriocins have gone beyond preclinical trials and none is currently used therapeutically in humans. In this Review, we explore the broad diversity in bacteriocin structure and function, describe identification and optimization methods and discuss the reasons behind the lack of translation beyond the laboratory of these potentially valuable antimicrobials.

CrAssphage and related viruses of the order Crassvirales(hereafter referred to as crassviruses) were originally discovered by cross-assembly of metagenomic sequences. They are the most abundant viruses in the human gut, are found in the majority of individual gut viromes, and account for up to 95% of the viral sequences in some individuals1–4. Crassviruses are likely to have major roles in shaping the composition and functionality of the human microbiome, but the structures and roles of most of the virally encoded proteins are unknown, with only generic predictions resulting from bioinformatic analyses4,5. Here we present a cryo-electron microscopy reconstruction of Bacteroides intestinalisvirus ΦcrAss0016, providing the structural basis for the functional assignment of most of its virion proteins. The muzzle protein forms an assembly about 1 MDa in size at the end of the tail and exhibits a previously unknown fold that we designate the ‘crass fold’, that is likely to serve as a gatekeeper that controls the ejection of cargos. In addition to packing the approximately 103 kb of virus DNA, the ΦcrAss001 virion has extensive storage space for virally encoded cargo proteins in the capsid and, unusually, within the tail. One of the cargo proteins is present in both the capsid and the tail, suggesting a general mechanism for protein ejection, which involves partial unfolding of proteins during their extrusion through the tail. These findings provide a structural basis for understanding the mechanisms of assembly and infection of these highly abundant crassviruses.

Live dietary microbes have been hypothesized to contribute to human health but direct evidence is lacking.

Graphical abstract:

Streptococcus pneumoniae(pneumococcus) is a multidrug-resistant pathogen associated with pneumonia, otitis media, meningitis and other severe complications that are currently a global threat to human health. The World Health Organization listed Pneumococcusas the fourth of twelve globally prioritized pathogens. Identifying alternatives to antibiotic therapies is urgently needed to combat Pneumococcus. Bacteriophage-derived endolysins can be used as alternative therapeutics due to their bacterial cell wall hydrolyzing capability. In this study, S. pneumoniaephage genomes were screened to create a database of endolysins for molecular modelling and diversity analysis of these lytic proteins. A total of 89 lytic proteins were curated from 81 phage genomes and categorized into eight groups corresponding to their different enzymatically active (EAD) domains and cell wall binding (CBDs) domains. We then constructed three-dimensional structures that provided insights into these endolysins. Group I, II, III, V, and VI endolysins showed conserved catalytic and ion-binding residues similar to existing endolysins available in the Protein Data Bank. While performing structural and sequence analysis with template lysin, an additional cell wall binding repeat was observed in Group II lysin, which was not previously known. Molecular docking performed with choline confirmed the existence of this additional repeat. Group III endolysins showed 99.16 % similarity to LysME-EF1, a lysin derived from Enterococcus faecalis. Furthermore, the comparative computational analysis revealed the existence of CBDs in Group III lysin. This study provides the first insight into the molecular and diversity analysis of S. pneumoniaephage endolysins that could be valuable for developing novel lysin-based therapeutics.

Supplemental Digital Content is available in the text.Improving diversity in residency programs has been increasingly emphasized as a means to address gender, racial, and ethnic disparities in medicine. However, limited attention has been given to the potential benefits of training physicians with differences other than gender or race and ethnicity. Americans with a disability represent about 27% of the U.S. population, whereas 1%–3% of physician trainees report having a disability. In 2013, a national survey identified only 86 physicians or trainees reporting deafness or hearing loss as a disability. To date, there are no published strategies on how to create an inclusive program for Deaf trainees. Herein, the authors report on the development of a Deaf and American Sign Language (ASL) inclusive residency program that can serve as an academic model for other programs, in any medical specialty, seeking to create an accessible training program for Deaf physicians and that can be adapted for trainees with other disabilities. In March 2017, the radiation oncology residency program at Johns Hopkins University matched an ASL-signing Deaf resident who would begin the program in July 2018. In preparation, department leadership engaged key stakeholders and leaders within the university’s health system and among the department faculty, residents, and staff as well as the incoming resident to create an ASL inclusive program. A 5-step transition process for the training program was ultimately developed and implemented. The authors focused on engaging the Deaf trainee and interpreters, engaging health system and departmental leadership, contracting a training consultant and developing oral and written training materials for faculty and staff, and optimizing the workspace via accommodations. Through collaborative preparation, a Deaf and ASL-signing resident was successfully integrated into the residency program. The proposed 5-step transition process provides an effective, engaging model to encourage other institutions that are seeking to employ similar inclusivity initiatives.

Bacteriophages (phages) are often described as obligate predators of their bacterial hosts, and phage predation is one of the leading forces controlling the density and distribution of bacterial populations. Every 48 h half of all bacteria on Earth are killed by phages. Efficient killing also forms the basis of phage therapy in humans and animals and the use of phages as food preservatives. In turn, bacteria have a plethora of resistance systems against phage attack, but very few bacterial species, if any, have entirely escaped phage predation. However, in complex communities and environments such as the human gut, this antagonistic model of attack and counter-defence does not fully describe the scope of phage–bacterium interactions. In this Review, we explore some of the more mutualistic aspects of phage–bacterium interactions in the human gut, and we suggest that the relationship between phages and their bacterial hosts in the gut is best characterized not as a fight to the death between enemies but rather as a mutualistic relationship between partners.

Abstract: Lacticin 3147 is a dual-acting two-peptide bacteriocin which is generally active against Gram-positive bacteria, including Listeria monocytogenesand antimicrobial-resistant bacteria such as Closteroides difficilein the colon. L. monocytogenesinfections can cause life-long effects in the elderly and vulnerable and can cause severe complications in pregnant women. C. difficilecauses one of the most common healthcare-associated infections and can be fatal in vulnerable groups such as the elderly. Although lacticin 3147 is degraded by intestinal proteases and has poor aqueous solubility, encapsulation of the bacteriocin could enable its use as an antimicrobial for treating these bacterial infections locally in the gastrointestinal tract. Lacticin 3147 displayed activity in aqueous solutions at a range of pH values and in gastric and intestinal fluids. Exposure to trypsin and α-chymotrypsin resulted in complete inactivation, implying that lacticin 3147 should be protected from these enzymes to achieve successful local delivery to the gastrointestinal tract. The amount of lacticin 3147 dissolved, i.e. its solution concentration, in water or buffered solutions at pH 1.6 and 7.4 was low and varied with time but increased and was stabilized in gastrointestinal fluids by the phospholipid and bile salt components present. Thus, the feasibility of a solid lipid nanoparticle (SLN) delivery system for local administration of lacticin 3147 was investigated. Bacteriocin activity was observed after encapsulation and release from a lipid matrix. Moreover, activity was seen after exposure to degrading enzymes. Further optimization of SLN delivery systems could enable the successful pharmaceutical development of active lacticin 3147 as an alternative to traditional antibiotics. Graphical abstract:

An expert panel was convened in September 2019 by The International Scientific Association for Probiotics and Prebiotics (ISAPP) to develop a definition for fermented foods and to describe their role in the human diet. Although these foods have been consumed for thousands of years, they are receiving increased attention among biologists, nutritionists, technologists, clinicians and consumers. Despite this interest, inconsistencies related to the use of the term ‘fermented’ led the panel to define fermented foods and beverages as “foods made through desired microbial growth and enzymatic conversions of food components”. This definition, encompassing the many varieties of fermented foods, is intended to clarify what is (and is not) a fermented food. The distinction between fermented foods and probiotics is further clarified. The panel also addressed the current state of knowledge on the safety, risks and health benefits, including an assessment of the nutritional attributes and a mechanistic rationale for how fermented foods could improve gastrointestinal and general health. The latest advancements in our understanding of the microbial ecology and systems biology of these foods were discussed. Finally, the panel reviewed how fermented foods are regulated and discussed efforts to include them as a separate category in national dietary guidelines.

In 2019, the International Scientific Association for Probiotics and Prebiotics (ISAPP) convened a panel of experts specializing in nutrition, microbial physiology, gastroenterology, paediatrics, food science and microbiology to review the definition and scope of postbiotics. The term ‘postbiotics’ is increasingly found in the scientific literature and on commercial products, yet is inconsistently used and lacks a clear definition. The purpose of this panel was to consider the scientific, commercial and regulatory parameters encompassing this emerging term, propose a useful definition and thereby establish a foundation for future developments. The panel defined a postbiotic as a “preparation of inanimate microorganisms and/or their components that confers a health benefit on the host”. Effective postbiotics must contain inactivated microbial cells or cell components, with or without metabolites, that contribute to observed health benefits. The panel also discussed existing evidence of health-promoting effects of postbiotics, potential mechanisms of action, levels of evidence required to meet the stated definition, safety and implications for stakeholders. The panel determined that a definition of postbiotics is useful so that scientists, clinical triallists, industry, regulators and consumers have common ground for future activity in this area. A generally accepted definition will hopefully lead to regulatory clarity and promote innovation and the development of new postbiotic products.

Aim:To determine if bacteriocins improve antibiotic efficacy. Materials & methods:Deferred antagonism assays identified bacteriocins with activity. Growth curves and time kill assays demonstrated bactericidal activity of antimicrobial combinations, and checkerboard assays confirmed synergy. Methicillin-resistant Staphylococcus aureus-infected porcine skin model determined ex vivoefficacy. Results:Subinhibitory concentrations of lacticin with penicillin or vancomycin resulted in complete growth inhibition of strains and the improved inhibitory effect was apparent after 1 h. Nisin with methicillin proved more effective against methicillin-resistant Staphylococcus aureusthan either antimicrobial alone, revealing partial synergy and significantly reduced pathogen numbers on porcine skin after 3 h compared with minimal inhibition for either antimicrobial alone. Conclusion:Nisin Z and lacticin 3147 may support the use of certain antibiotics and revive ineffective antibiotics.

ABSTRACTAlthough many recent studies have examined associations between the gut microbiome and COVID-19 disease severity in individual patient cohorts, questions remain on the robustness across international cohorts of the biomarkers they reported. Here, we performed a meta-analysis of eight shotgun metagenomic studies of COVID-19 patients (comprising 1,023 stool samples) and 23 > 16S rRNA gene amplicon sequencing (16S) cohorts (2,415 total stool samples). We found that disease severity (as defined by the WHO clinical progression scale) was associated with taxonomic and functional microbiome differences. This alteration in gut microbiome configuration peaks at days 7–30 post diagnosis, after which the gut microbiome returns to a configuration that becomes more similar to that of healthy controls over time. Furthermore, we identified a core set of species that were consistently associated with disease severity across shotgun metagenomic and 16S cohorts, and whose abundance can accurately predict disease severity category of SARS-CoV-2 infected subjects, with Actinomyces orisabundance predicting population-level mortality rate of COVID-19. Additionally, we used relational diet-microbiome databases constructed from cohort studies to predict microbiota-targeted diet patterns that would modulate gut microbiota composition toward that of healthy controls. Finally, we demonstrated the association of disease severity with the composition of intestinal archaeal, fungal, viral, and parasitic communities. Collectively, this study has identified robust COVID-19 microbiome biomarkers, established accurate predictive models as a basis for clinical prognostic tests for disease severity, and proposed biomarker-targeted diets for managing COVID-19 infection.

ABSTRACTA high-fat (HF) diet reduces resistance to the foodborne pathogen Listeria monocytogenes. We demonstrate that short-term gavage with A. muciniphilaincreases resistance to oral and systemic L. monocytogenesinfection in mice fed a HF diet. A. muciniphilareduced inflammation in the gut and liver of mice fed a high-fat diet prior to infection and reduced inflammatory cell infiltration in the ileum to levels similar to mice fed a low-fat (LF) diet. Akkermansiaadministration had minimal impacts upon the microbiota and microbial metabolites and did not affect individual taxa or impact the Bacteroidetes to Firmicutes ratio. In summary, A. muciniphilaincreased resistance to L. monocytogenesinfection in mice fed a HF diet by moderating immune/physiological effects through specific interaction between A. muciniphilaand the host gut.

ABSTRACTRuminococcus gnavusis a prevalent gut microbe reported to occur in higher abundance among individuals with inflammatory bowel disease (IBD). This study reports the isolation and characterization of six bacteriophages (phages) isolated from human fecal material and environmental samples that infect this species. Isolated phages have a siphovirus morphology, with genomes ranging between 36.5 and 37.8 kbp. Genome analysis indicates that the phages have a temperate lifestyle, which was confirmed by their ability to form lysogens on their host bacterial species. In contrast to the finding that phages lyse their host in liquid medium, results from a mouse trial indicate these phages can co-exist with the host bacterium in the gut without causing a significant reduction of R. gnavus. The bacterial counts in the feces of phage-treated mice did not significantly differ in the presence of phage. Furthermore, analysis of publicly available gut virome sequence data indicates a high abundance of these phages among individuals suffering from IBD. This work provides the first insight into how phages interact with R. gnavusin the human gut microbiome.

ABSTRACTProbiotics are used for both generally healthy consumers and in clinical settings. However, theoretical and proven adverse events from probiotic consumption exist. New probiotic strains and products, as well as expanding use of probiotics into vulnerable populations, warrants concise, and actionable recommendations on how to work toward their safe and effective use. The International Scientific Association for Probiotics and Prebiotics convened a meeting to discuss and produce evidence-based recommendations on potential acute and long-term risks, risks to vulnerable populations, the importance for probiotic product quality to match the needs of vulnerable populations, and the need for adverse event reporting related to probiotic use. The importance of whole genome sequencing, which enables determination of virulence, toxin, and antibiotic resistance genes, as well as clear assignment of species and strain identity, is emphasized. We present recommendations to guide the scientific and medical community on judging probiotic safety.

ABSTRACTConflicting evidence exists on the association between consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and symptomatic worsening of inflammatory bowel disease (IBD). We hypothesized that the heterogeneous prevalence of pathobionts [e.g., adherent-invasive Escherichia coli(AIEC)], might explain this inconsistent NSAIDs/IBD correlation. Using IL10−/−mice, we found that NSAID aggravated colitis in AIEC-colonized animals. This was accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis, and pyroptosis, features not seen in mice exposed to AIEC or NSAID alone, revealing an AIEC/NSAID synergistic effect. Inhibition of NLRP3 or Caspase-8 activity ameliorated colitis, with reduction in NLRP3 inflammasome activation, cell death markers, activated T-cells and macrophages, improved histology, and increased abundance of Clostridiumcluster XIVa species. Our findings provide new insights into how NSAIDs and an opportunistic gut-pathobiont can synergize to worsen IBD symptoms. Targeting the NLRP3 inflammasome or Caspase-8 could be a potential therapeutic strategy in IBD patients with gut inflammation, which is worsened by NSAIDs.

ABSTRACTGlyphosate, the active ingredient in the broad-spectrum herbicide RoundupTM, has been a topic of discussion for decades due to contradictory reports of the effect of glyphosate on human health. Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of the shikimic pathway producing aromatic amino acids in plants, a mechanism that suggests that the herbicide would not affect humans as this pathway is not found in mammals. However, numerous studies have implicated glyphosate exposure in the manifestation of a variety of disorders in the human body. This review specifically outlines the potential effect of glyphosate exposure on the composition and functionality of the gut microbiome. Evidence has been building behind the hypothesis that the composition of each individual gut microbiota significantly impacts health. For this reason, the potential of glyphosate to inhibit the growth of beneficial microbes in the gut or alter their functionality is an important topic that warrants further consideration.

The gut microbiome is fundamental to human health and development. Altered microbiomes have been associated with many diseases. However, variation between individuals, environmental effects, and a lack of standardization across studies makes differentiation between health and disease challenging. Large-scale population cohorts in different countries will be required to match disease subjects with healthy controls, whereas standardized, reproducible pipelines for analysis are required to compare findings between studies. Despite this, several conditions have already demonstrated great promise for developing microbiome-based biomarkers as well as providing a gateway into integrated personalized medicine.

For almost all of human evolution our food and water has contained large numbers of microbes. Our immune systems evolved to cope with this daily intake, and our microbiomes (the collection of microbes on and in the human body) are increasingly recognized as playing an important role in human health. However, in recent times we have gone to great lengths to eliminate microbes from our diets, using food processing, water purification and hygiene to reduce our exposure. But has this come at a cost? Could our immune systems, primed to deal with trillions of microbes with every meal, be struggling to cope with their absence? Could this be a factor in the rise of modern inflammatory diseases in which the immune system misbehaves in response to dietary antigens or to our own epithelial cells? Perhaps we need to go back to consuming large numbers of (safe) microbes every day — a microbial RDA?

Bacteriophages, which lost out to antibiotic therapy in the past, may be poised to make a comeback. Once discarded because of their narrow activity spectrum, it can now be viewed as a major advantage that these intracellular, self-replicating entities can exert their killing effect with minimal damage to the commensal microbiome. In eastern Europe, phages continue to be used both prophylactically and therapeutically to treat infections. More recently, much needed regulated clinical trials are underway with a view to restoring phage therapy as a tool for mainstream medicine, although current regulations may impede their full potential. One hundred years after their discovery, and amid an antibiotic resistance crisis, we must ask, what can be done to harness their full antibacterial potential?

•Bioengineering can generate novel bacteriocin variants for specific purposes.•Genome mining has identified new bacteriocin biosynthetic gene clusters.•Bioengineered bateriocins show great promise as synergists in hurdle technology.•Bacteriocin producing strains which have been tailored through food-grade methods can be directly added to food.

The purpose of this study was to analyze the patterns of care of local therapies and their impact on overall survival (OS) among elderly patients with early-stage non-small cell lung cancer (NSCLC) in the USA. The National Cancer Database was queried for patients at least age 80 years with NSCLC diagnosed in 2004–2013 with clinical stage T1–3N0M0. Local therapy was analyzed over time and by age. Multivariable (MVA) models were performed to investigate the impact of prognostic factors on OS. Among 40,561 patients meeting inclusion criteria, 17,418 (43%), 13,008 (32%), and 10,135 (25%) of patients underwent surgical resection, radiotherapy, and observation, respectively, as their initial mode of local therapy. Overtime, while the utilization of surgical managements generally remained stable, the utilization of conventionally fractionated radiotherapy and observation decreased in favor of stereotactic body radiotherapy (SBRT, p< 0.001). Among operable patients (n= 16,377), after MVA several factors were associated with OS including the choice of local therapy favoring resection over conventionally fractionated radiotherapy and observation (HR compared to lobectomy 1.362, and 2.656, respectively, each p< 0.001). In contrast, there was no statistical difference in OS between resection and SBRT among operable patients (HR for SBRT 1.128, p= 0.156). The utilization of SBRT as the definitive local therapy in elderly patients with early-stage NSCLC is increasing in the USA. Given its generally favorable toxicity profile, SBRT should be considered in the substantial proportion of elderly patients still not receiving any definitive local therapy. Among medically operable elderly patients, OS was similar between resection and SBRT.

Antibiotics have revolutionised the treatment of infectious disease and improved the lives of billions of people worldwide over many decades. With the rise in antimicrobial resistance (AMR) and corresponding lack of antibiotic development, we find ourselves in dire need of alternative treatments. Bacteriocins are a class of bacterially produced, ribosomally synthesised, antimicrobial peptides that may be narrow or broad in their spectra of activity. Animal models have demonstrated the safety and efficacy of bacteriocins in treating a broad range of infections; however, one of the principal drawbacks has been their relatively narrow spectra when compared with small-molecule antibiotics. In an era where we are beginning to appreciate the role of the microbiota in human and animal health, the fact that bacteriocins cause much less collateral damage to the host microbiome makes them a highly desirable therapeutic. This review makes a case for the implementation of bacteriocins as therapeutic antimicrobials, either alone or in combination with existing antibiotics to alleviate the AMR crisis and to lessen the impact of antibiotics on the host microbiome.

We sought to evaluate the pattern of disease progression and survival of patients with multilesion glioblastoma (GBM) in a modern patient cohort. We retrospectively collected the clinical data of patients over 2004–2014 with newly diagnosed GBM. Following resection (or biopsy), all patients received radiotherapy to 60 Gy in 30 fractions with concurrent and adjuvant temozolomide. Preoperative MRIs were reviewed by a single diagnostic radiologist blinded to treatment outcomes and categorized as single focus or multilesion (multifocal/multicentric). Multifocal tumors were defined as noncontiguous enhancement along known neural pathways and multicentric tumors were defined as noncontiguous enhancement along disjointed pathways (i.e., different lobes). Univariate and multivariate (MVA) analyses were performed to investigate factors prognostic of overall survival. Of 155 eligible patients, 30 presented with a multilesion tumor focus (19.4%). Of the patients that developed recurrent disease before death (n= 123), the predominant pattern of failure was local (80%) and did not differ by focality (p= 0.18). Unifocal, multifocal, and multicentric tumors demonstrated similar actuarial disease progression (p= 0.12). Median survival was inferior in patients with multifocal and multicentric disease compared to unifocal disease (11.5, 7.1, and 18.0 months, respectively, p= 0.009). On MVA, factors prognostic of overall survival included age ≥70 (HR 3.89, p< 0.001), MGMT promoter hypermethylation (HR 0.21, p= 0.004), IDH1/2 mutation status (p= 0.044), and multicentric tumors (HR 7.18, p= 0.002). Extent of tumor resection did not impact OS (p= 0.308). We identified unifocal, multifocal, and multicentric GBM entities with differing overall survival following modern chemo-radiotherapy techniques despite similar pattern of disease progression.

ABSTRACTAn optimally operating microbiome supports protective, metabolic, and immune functions, but disruptions produce metabolites and toxins which can be involved in many conditions. Probiotics have the potential to manage these. However, their use in vulnerable people is linked to possible safety concerns and maintaining their viability is difficult. Interest in postbiotics is therefore increasing. Postbiotics contain inactivated microbial cells or cell components, thus are more stable and exert similar health benefits to probiotics. To review the evidence for the clinical benefits of postbiotics in highly prevalent conditions and consider future potential areas of benefit. There is growing evidence revealing the diverse clinical benefits of postbiotics in many prevalent conditions. Postbiotics could offer a novel therapeutic approach and may be a safer alternative to probiotics. Establishing interaction mechanisms between postbiotics and commensal microorganisms will improve the understanding of potential clinical benefits and may lead to targeted postbiotic therapy.

ABSTRACTPediocin PA-1 is a class IIa bacteriocin that is particularly effective against the foodborne pathogen Listeria monocytogenes. The loss of activity of PA-1 pediocin due to methionine oxidation is one of the challenges that limit the wider application of the bacteriocin. In this study, we heterologously expressed an oxidation resistant form of pediocin PA-1, i.e., pediocin M31L, and compared its activity to that of native pediocin PA-1 and to penocin A, a pediocin-like bacteriocin that displays a narrower antimicrobial spectrum. Minimal inhibitory concentration assays revealed that pediocin M31L was as effective as PA-1 and more effective than synthetic penocin A against Listeriawith negligible activity against a range of obligate anaerobic commensal gut bacterial species. The anti-Listeriaactivity of these pediocins was also assessed in a simulated human distal colon model assay using the L. monocytogenes, spiked at 6.5 0.13 Log CFU/mL, as a bioindicator. At 24 h, pediocin M31L and penocin A (2.6 μM) reduced Listeriacounts to 3.5 0.4 and 3.64 0.62 Log CFU/mL, respectively, whereas Listeriacounts were considerably higher, i.e. 7.75 0.43 Log CFU/mL, in the non-bacteriocin-containing control. Ultimately, it was established that synthetic penocin A and the stable pediocin M31L derivative, heterologously produced, display effective anti-Listeriaactivity in a human gut environment.

ABSTRACTThe Gram positive intracellular pathogen Listeria monocytogenesrepresents a promising vaccine or therapeutic DNA delivery vector that has been successfully administered to humans in clinical trials. However in generating Listeriamutants with therapeutic potential it is important to balance safety attenuation with efficacy. Here we show that L. monocytogenesmutants with a reduced capacity for murine gallbladder replication are capable of stimulating T cell responses in mice and protecting vaccinated animals from secondary challenge. Mutation of L. monocytogenesgenes lmo2566or lmo0598resulted in significant attenuation in the murine model yet mutants retained a capacity for intracellular growth and stimulation of T cell responses against key Listeriaepitopes (LLO91-99and P60217-225). Importantly the mutants showed a reduced capacity for growth in the gallbladders of vaccinated mice as well as significantly reduced faecal shedding indicating that this approach generates live Listeria-based vector delivery systems with a reduced capacity for the spread of live genetically modified microorganisms into the natural environment.

Endolysins (lysins) are bacteriophage-encoded enzymes that have evolved to degrade specific bonds within the bacterial cell wall. These enzymes represent a novel class of antibacterial agents against infectious pathogens, especially in light of multidrug-resistant bacteria, which have made antibiotic therapy increasingly redundant. Lysins have been used successfully to eliminate/control bacterial pathogens in various anatomical locations in mouse and other animal models. Engineering tactics have also been successfully applied to improve lysin function. This review discusses the structure and function of lysins. It highlights protein-engineering tactics utilised to improve lysin activity. It also reviews the applications of lysins towards food biopreservation, therapeutics, bioifilm elimination and diagnostics.

ABSTRACTWe propose a mechanism of action for the betL* mutation which is based on DNA topology. Removing a single thymine residue from the betLσApromoter's −10 and −35 spacer results in a ‘twist’-mediated activation of transcription which accounts for the osmotolerance phenotype observed for strains expressing betL*.

Staphylococcus aureus is a major cause of infection in humans and animals, causing a wide variety of diseases, from local inflammations to fatal sepsis. The bacterium is commonly multi-drug resistant and thus many front-line antibiotics have been rendered ineffective for treating such infections. Research on murein/peptidoglycan hydrolases, derived from bacterial viruses (bacteriophages), has demonstrated that such proteins are attractive candidates for development as novel antibacterial agents for combatting Gram-positive pathogens. Here we review the research produced to-date on the bacteriophage-derived CHAPKmurein peptidase. Initially, we sequenced and annotated the genome of anti-staphylococcal bacteriophage K and cloned the gene for the bacteriophage endolysin, a murein hydrolase which plays a role in cell killing during the bacteriophage life cycle. An highly active domain of the enzyme, a cysteine, histidine-dependent amido hydrolase/peptidase (CHAPK), was cloned, overexpressed in E. coli and purified. This CHAPKenzyme was demonstrated to rapidly lyse several strains of methicillin resistant S. aureus and both disrupted and prevented the formation of a staphylococcal biofilm. The staphylolytic activity of the peptidase was demonstrated in vivo using a mouse model, without adverse effects on the animals. The crystal structure of the enzyme was elucidated, revealing a calcium ion close to the active site. Site-directed mutagenesis indicated that this calcium ion is involved in the catalytic mechanism of the enzyme. The crystal structure of this enzyme is a valuable source of information for efficient engineering of this and similar CHAP-domain-containing proteins. Overall, the data collected to date on CHAPKhas demonstrated its strong potential as a novel therapeutic candidate for treatment of staphylococcal infections and has provided us with insight into the fundamental enzymatic mechanisms of CHAP domaincontaining peptidoglycan hydrolases.

The sactibiotics are a recently designated subclass of bacteriocins that contain characteristic cysteine sulphur to α -carbon linkages mediated through post-translational modifications. They are a relatively small subclass of bacteriocins compared to the most thoroughly studied lantibiotics. The sactibiotics that have been extensively studied thus far are thuricin CD, subtilosin A, thurincin H, and propionicin F. Despite their recent discovery, there have already been significant advances made in the study of sactibiotics, most notably the discovery of the narrow spectrum anti-Clostridium difficile sactibiotic, thuricin CD. In addition, scientists have gained insights into the mechanisms of action of the sactibiotic subtilosin A, which targets Listeria monocytogenes, Gardnerella vaginalis, and other pathogens. Also, the development of heterologous host systems and homologous expression and site-directed mutagenesis systems for the sactibiotic thurincin H have opened up many opportunities for further studies on this sactibiotic. These and other recent studies concerning the molecular biology, 3D structural elucidation, mode of action, self-protection mechanisms, and antimicrobial spectrum of the sactibiotic subgroup of bacteriocins are discussed in this review.

The lantibiotics are a class of bacterially produced antimicrobial peptides (bacteriocins) that contain several unusual amino acids resulting from enzyme-mediated post-translational modifications. They exhibit high specific activity against Gram-positive targets, including many antibiotic-resistant pathogens, and consequently have been investigated with a view to their application as antimicrobials in both the food and medical arenas. Importantly, the gene-encoded nature of lantibiotics makes them more amenable to bioengineering strategies to further enhance their antimicrobial and physicochemical properties. However, although the bioengineering of lantibiotics has been underway for over 2 decades, significant progress has only been reported in recent years. This review charts recent developments with regard to the implementation of bioengineering strategies to enhance the functional characteristics of the prototypical and most studied lantibiotic nisin.