Bacterial lipopolysaccharide modulates immune response in the colorectal tumor microenvironment.

Immune responses can have opposing effects in colorectal cancer (CRC), the balance of which may determine whether a cancer regresses, progresses, or potentially metastasizes. These effects are evident in CRC consensus molecular subtypes (CMS) where both CMS1 and CMS4 contain immune infiltrates yet have opposing prognoses. The microbiome has previously been associated with CRC and immune response in CRC but has largely been ignored in the CRC subtype discussion. We used CMS subtyping on surgical resections from patients and aimed to determine the contributions of the microbiome to the pleiotropic effects evident in immune-infiltrated subtypes. We integrated host gene-expression and meta-transcriptomic data to determine the link between immune characteristics and microbiome contributions in these subtypes and identified lipopolysaccharide (LPS) binding as a potential functional mechanism. We identified candidate bacteria with LPS properties that could affect immune response, and tested the effects of their LPS on cytokine production of peripheral blood mononuclear cells (PBMCs). We focused on Fusobacterium periodonticum and Bacteroides fragilis in CMS1, and Porphyromonas asaccharolytica in CMS4. Treatment of PBMCs with LPS isolated from these bacteria showed that F. periodonticum stimulates cytokine production in PBMCs while both B. fragilis and P. asaccharolytica had an inhibitory effect. Furthermore, LPS from the latter two species can inhibit the immunogenic properties of F. periodonticum LPS when co-incubated with PBMCs. We propose that different microbes in the CRC tumor microenvironment can alter the local immune activity, with important implications for prognosis and treatment response.

First-in-class Microbial Ecosystem Therapeutic 4 (MET4) in combination with immune checkpoint inhibitors in patients with advanced solid tumors (MET4-IO trial).

BACKGROUND: The intestinal microbiome has been associated with response to immune checkpoint inhibitors (ICIs) in humans and causally implicated in ICI responsiveness in animal models. Two recent human trials demonstrated that fecal microbiota transplant (FMT) from ICI responders can rescue ICI responses in refractory melanoma, but FMT has specific limitations to scaled use. PATIENTS AND METHODS: We conducted an early-phase clinical trial of a cultivated, orally delivered 30-species microbial consortium (Microbial Ecosystem Therapeutic 4, MET4) designed for co-administration with ICIs as an alternative to FMT and assessed safety, tolerability and ecological responses in patients with advanced solid tumors. RESULTS: The trial achieved its primary safety and tolerability outcomes. There were no statistically significant differences in the primary ecological outcomes; however, differences in MET4 species relative abundance were evident after randomization that varied by patient and species. Increases in the relative abundance of several MET4 taxa, including Enterococcus and Bifidobacterium, taxa previously associated with ICI responsiveness, were observed and MET4 engraftment was associated with decreases in plasma and stool primary bile acids. CONCLUSIONS: This trial is the first report of the use of a microbial consortium as an alternative to FMT in advanced cancer patients receiving ICI and the results justify the further development of microbial consortia as a therapeutic co-intervention for ICI treatment in cancer.

Quantitative proteomic profiling of shake flask versus bioreactor growth reveals distinct responses of Agrobacterium tumefaciens for preparation in molecular pharming.

The preparation of Agrobacterium tumefaciens cultures with strains encoding proteins intended for therapeutic or industrial purposes is an important activity prior to treatment of plants for transient expression of valuable protein products. The rising demand for biologic products such as these underscores the expansion of molecular pharming and warrants the need to produce transformed plants at an industrial scale. This requires large quantities of A. tumefaciens culture, which is challenging using traditional growth methods (e.g., shake flask). To overcome this limitation, we investigate the use of bioreactors as an alternative to shake flasks to meet production demands. Here, we observe differences in bacterial growth among the tested parameters and define conditions for consistent bacterial culturing between shake flask and bioreactor. Quantitative proteomic profiling of cultures from each growth condition defines unique growth-specific responses in bacterial protein abundance and highlights the functional roles of these proteins, which may influence bacterial processes important for effective agroinfiltration and transformation. Overall, our study establishes and optimizes comparable growth conditions for shake flask versus bioreactors and provides novel insights into fundamental biological processes of A. tumefaciens influenced by such growth conditions.

Development of the faecal microbiota in foals.

REASONS FOR PERFORMING STUDY: The intestinal microbiota is a complex polymicrobial ecosystem that exerts extremely important roles in the development and maintenance of health. Recently, as new sequencing technologies have become more available, there has been a revolution in the understanding of the equine intestinal microbiota. However, studies characterising the pioneer intestinal bacteria colonising foals and its development over time are still limited. OBJECTIVES: The objectives of this study were to characterise the intestinal bacterial colonisation of newborn foals and to follow individual animals over time until age 9 months. STUDY DESIGN: Longitudinal study. METHODS: Eleven pregnant mares from one farm were enrolled and faecal samples were collected longitudinally from mares and foals during their first day post partum and again periodically until foals were age 9 months. The V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. RESULTS: Newborn foals had a rich and diverse bacterial community, which was mainly comprised of the Firmicutes phylum with several low abundant genera being unique at this age. Foals aged 2-30 days had significantly decreased diversity compared to older animals, with the majority of organisms classified as Akkermansia spp. After 60 days of life, the intestinal microbiota structure tended to remain stable, but differences in community membership were still present between 9-month-old animals and mature mares. Several differences at the phylum level were observed between different ages, including a higher abundance of Fibrobacteres after weaning. CONCLUSIONS: The intestinal microbiota of the equine newborn is already complex by the first day of life. Microbiota adaptation occurs during the first month and the microbiota of foals older than 60 days resemble the mother's microbiota, although differences in community membership are still present.

Using bugs as drugs: Microbial ecosystem therapeutics.

The human gut harbours a dense and highly diverse microbial ecosystem-the microbiota-that plays an important role in the maintenance of health. Modern lifestyle practices, including widespread antibiotic use, have degraded microbiota diversity, compromising the integrity of this vital ecosystem and creating susceptibility to diseases such as Clostridium difficile infection. Treatment of patients to restore the diversity of the gut microbiota offers a logical solution to disease. Although fecal microbial therapy (FMT) has started to gain traction as an effective method to effect this restoration, it is not without risks and there are significant barriers to its implementation in the clinic. Some of the risks and challenges with FMT are addressed by microbial ecosystem therapeutics (MET), an alternative approach to FMT that uses selected, defined microbial ecosystems to redress microbiota balance and functionality. The time has come for the use of bugs as drugs.

Stability and biological activity of wild blueberry (Vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion.

Wild blueberries are rich in polyphenols and have several potential health benefits. Understanding the factors that affect the bioaccessibility and bioavailability of polyphenols is important for evaluating their biological significance and efficacy as functional food ingredients. Since the bioavailability of polyphenols such as anthocyanins is generally low, it has been proposed that metabolites resulting during colonic fermentation may be the components that exert health benefits. In this study, an in vitro gastrointestinal model comprising sequential chemostat fermentation steps that simulate digestive conditions in the stomach, small intestine and colon was used to investigate the breakdown of blueberry polyphenols. The catabolic products were isolated and biological effects tested using a normal human colonic epithelial cell line (CRL 1790) and a human colorectal cancer cell line (HT 29). The results showed a high stability of total polyphenols and anthocyanins during simulated gastric digestion step with approximately 93% and 99% of recovery, respectively. Intestinal digestion decreased polyphenol- and anthocyanin- contents by 49% and 15%, respectively, by comparison to the non-digested samples. During chemostat fermentation that simulates colonic digestion, the complex polyphenol mixture was degraded to a limited number of phenolic compounds such as syringic, cinnamic, caffeic, and protocatechuic acids. Only acetylated anthocyanins were detected in low amounts after chemostat fermentation. The catabolites showed lowered antioxidant activity and cell growth inhibition potential. Results suggest that colonic fermentation may alter the biological activity of blueberry polyphenols.

Microbial ecosystems therapeutics: a new paradigm in medicine?

Increasing evidence indicates that the complex microbial ecosystem of the human intestine plays a critical role in protecting the host against disease. This review discusses gut dysbiosis (here defined as a state of imbalance in the gut microbial ecosystem, including overgrowth of some organisms and loss of others) as the foundation for several diseases, and the applicability of refined microbial ecosystem replacement therapies as a future treatment modality. Consistent with the concept of a 'core' microbiome encompassing key functions required for normal intestinal homeostasis, 'Microbial Ecosystem Therapeutics' (MET) would entail replacing a dysfunctional, damaged ecosystem with a fully developed and healthy ecosystem of 'native' intestinal bacteria. Its application in treating Clostridium difficile infection is discussed and possible applications to other diseases such as ulcerative colitis, obesity, necrotising enterocolitis, and regressive-type autism are reviewed. Unlike conventional probiotic therapies that are generally limited to a single strain or at most a few strains of bacteria 'Microbial Ecosystem Therapeutics' would utilise whole bacterial communities derived directly from the human gastrointestinal tract. By taking into account the intrinsic needs of the entire microbial ecosystem, MET would emphasise the rational design of healthy, resilient and robust microbial communities that could be used to maintain or restore human health. More than simply a new probiotic treatment, this emerging paradigm in medicine may lead to novel strategies in treating and managing a wide variety of human diseases.

Avian- and mammalian-derived antibodies against adherence-associated proteins inhibit host cell colonization by Escherichia coli O157:H7.

AIM: To evaluate the potential for polyclonal antibodies targeting enterohaemorrhagic Escherichia coli (EHEC) virulence determinants to prevent colonization of host cells by E. coli O157:H7. METHODS AND RESULTS: Rats and laying hens were immunized with recombinant proteins from E. coli O157:H7, EspA, C-terminal intimin or EscF. Rat antisera (IgG) or chicken egg powders (IgY) were assessed for their ability to inhibit growth and colonization-associated processes of E. coli O157:H7. Mammalian antisera with antibodies to intimin, EspA or EscF effectively reduced adherence of the pathogen to HeLa cells (P<0.05) and prevented type III secretion of Tir. Similarly, HeLa cells treated with chicken egg powder containing antibodies against intimin or EspA were protected from EHEC adherence (P<0.05). Neither egg nor rat antibody preparations had any antibacterial effect on the growth of EHEC (P>0.05). CONCLUSIONS: Antibody preparations targeting EHEC adherence-associated factors were effective at preventing adhesion and intimate colonization-associated events. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates that immunotherapy with anti-adherence antibodies can reduce E. coli O157:H7 colonization of host cells. Passive immunization with specific antibodies may have the potential to reduce E. coli O157:H7 colonization in hosts such as cattle or humans.

An efficient system for markerless gene replacement applicable in a wide variety of enterobacterial species.

We describe an improved allelic-exchange method for generating unmarked mutations and chromosomal DNA alterations in enterobacterial species. Initially developed for use in Salmonella enterica, we have refined the method in terms of time, simplicity, and efficiency. We have extended its use into related bacterial species that are more recalcitrant to genetic manipulations, including enterohemorrhagic and enteropathogenic Escherichia coli and Vibrio parahaemolyticus. Data from over 50 experiments are presented including gene inactivations, site-directed mutagenesis, and promoter exchanges. In each case, desired mutations were identified by polymerase chain reaction screening typically from as few as 10-20 colonies up to a maximum of 300 colonies. The method does not require antibiotic nor nutritional markers in target genes and works efficiently in wild-type strains, obviating the need for specialized hosts or genetic systems. The use is simple, requiring basic laboratory materials, and represents an alternative to existing methods for gene manipulation in the Enterobacteriaceae.

Adhesion of Salmonella enterica var Enteritidis strains lacking fimbriae and flagella to rat ileal explants cultured at the air interface or submerged in tissue culture medium.

Rat ileal air interface and submerged explant models were developed and used to compare the adhesion of Salmonella enterica var Enteritidis wild-type strains with that of their isogenic single and multiple deletion mutants. The modified strains studied were defective for fimbriae, flagella, motility or chemotaxis and binding was assessed on tissues with and without an intact mucus layer. A multiple afimbriate/aflagellate (fim-/fla-) strain, a fimbriate but aflagellate (fla-) strain and a fimbriate/flagellate but non-motile (mot-) strain bound significantly less extensively to the explants than the corresponding wild-type strains. With the submerged explant model this difference was evident in tissues with or without a mucus layer, whereas in the air interface model it was observed only in tissues with an intact mucus layer. A smooth swimming chemotaxis-defective (che-) strain and single or multiple afimbriate strains bound to explants as well as their corresponding wild-type strain. This suggests that under the present experimental conditions fimbriae were not essential for attachment of S. enterica var Enteritidis to rat ileal explants. However, the possession of active flagella did appear to be an important factor in enabling salmonellae to penetrate the gastrointestinal mucus layer and attach specifically to epithelial cells.

Colonisation of the chicken caecum by afimbriate and aflagellate derivatives of Salmonella enterica serotype Enteritidis.

A semi-quantitative cloacal-swab method was used as an indirect measure of caecal colonisation of one-day old and five-day old chicks after oral dosing with wild-type Salmonella enterica serovar Enteritidis PT4 and genetically defined isogenic derivatives lacking the ability to elaborate flagella or fimbriae. Birds of both ages were readily and persistently colonised by all strains although there was a decline in shedding by the older birds after about 21 days. There were no significant differences in shedding of wild-type or mutants in single-dose experiments. In competition experiments, in which five-day old birds were dosed orally with wild-type and mutants together, shedding of non-motile derivatives was significantly lower than wild-type. At 35 days post infection, birds were sacrificed and direct counts of mutants and wild-type from each caecum were determined. Whilst there appeared to be poor correlation between direct counts and the indirect swab method, the overall trends shown by these methods of assessment indicated that flagella and not fimbriae were important in caecal colonisation in these models.

Virulence of Salmonella enterica serotype Enteritidis aflagellate and afimbriate mutants in a day-old chick model.

Certain fimbriae and the flagellae of Salmonella enterica serovar Typhimurium have been shown to contribute to attachment and invasion of gut epithelium in the murine typhoid infection model and to contribute to pathogenesis in the chick. However, little is known of the role these organelles play in Enteritidis poultry infections and, to study this, day-old chicks were dosed orally in separate experiments with defined multiply afimbriate and/or aflagellate mutant strains of Enteritidis. The colonization and invasion characteristics of each mutant were compared with those of the isogenic wild type strain by the determination of the number of bacteria recovered from livers and spleens at known time points post infection. Compared with wild type Enteritidis, a mutant unable to express flagella but retaining the genetic potential to express fimbriae was recovered post mortem from livers and spleens in significantly reduced numbers compared to the isogenic wild-type at all time points post infection (P < 0.001). Conversely, a flagellate but multiply afimbriate mutant (defective for the elaboration of five different fimbrial types) and a flagellate but non-motile 'paralysed' mutant were recovered from livers and spleens in similar numbers to the wild-type. The data suggested that Enteritidis flagella, but not fimbriae, played an important role in pathogenesis in the chick model and that the flagellar apparatus itself and not motility per se contributed significantly to this role.

Fimbriae- and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella enteritidis.

Salmonella enteritidis expresses flagella and several finely regulated fimbriae, including SEF14, SEF17 and SEF21 (type 1). A panel of mutants was prepared in three strains of S. enteritidis to elucidate the role of these surface appendages in the association with and invasion of cultured epithelial cells. In all assays, the naturally occurring regulatory-defective strain 27655R associated with tissue culture cells significantly more than wild-type progenitor strains LA5 and S1400/94. Compared with wild-type strains, SEF14 mutants had no effect on association and invasion, whereas SEF17, SEF21 and aflagellate mutants showed significant reductions in both processes. Histological examination suggested a role for SEF17 in localized, aggregative adherence, which could be specifically blocked by anti-SEF17 sera and purified SEF17 fimbriae. SEF21-mediated association was neutralized by mannose and a specific monoclonal antibody, although to observe enhanced association it was necessary for the bacteria to be in fimbriate phase prior to infection. Additionally, aflagellate mutants associated and invaded less than motile bacteria. This study demonstrated the potential for multifactorial association and invasion of epithelial cells which involved SEF17 and SEF21 fimbriae, and flagella-mediated motility.

The variant rpoS allele of S. enteritidis strain 27655R does not affect virulence in a chick model nor constitutive curliation but does generate a cold-sensitive phenotype.

Adherence of pathogenic Escherichia coli and Salmonella spp. to host cells is in part mediated by curli fimbriae which, along with other virulence determinants, are positively regulated by RpoS. Interested in the role and regulation of curli (SEF17) fimbriae of Salmonella enteritidis in poultry infection, we tested the virulence of naturally occurring S. enteritidis PT4 strains 27655R and 27655S which displayed constitutive and null expression of curli (SEF17) fimbriae, respectively, in a chick invasion assay and analysed their rpoS alleles. Both strains were shown to be equally invasive and as invasive as a wild-type phage type 4 strain and an isogenic derivative defective for the elaboration of curli. We showed that the rpoS allele of 27655S was intact even though this strain was non-curliated and we confirmed that a S. enteritidis rpoS::strr null mutant was unable to express curli, as anticipated. Strain 27655R, constitutively curliated, possessed a frameshift mutation at position 697 of the rpoS coding sequence which resulted in a truncated product and remained curliated even when transduced to rpoS::strr. Additionally, rpoS mutants are known to be cold-sensitive, a phenotype confirmed for strain 27655R. Collectively, these data indicated that curliation was not a significant factor for pathogenesis of S. enteritidis in this model and that curliation of strains 27655R and 27655S was independent of RpoS. Significantly, strain 27655R possessed a defective rpoS allele and remained virulent. Here was evidence that supported the concept that different naturally occurring rpoS alleles may generate varying virulence phenotypic traits.

SEF17 fimbriae are essential for the convoluted colonial morphology of Salmonella enteritidis.

Salmonella enteritidis isolated from poultry infections generated a convoluted colonial morphology after 48 h growth on colonisation factor antigen (CFA) agar at 25 degrees C. A mutant S. enteritidis defective for the elaboration of the SEF17 fimbrial antigen, in which the agf gene cluster was inactivated by insertion of an ampicillin resistance gene cassette, and other wild-type S. enteritidis transduced to this genotype failed to produce convoluted colonies. However, growth of SEF17- mutants at 25 degrees C on CFA agar supplemented with 0.001% Congo red resulted in partial recovery of the phenotype. Immunoelectron microscopy demonstrated that copious amounts of the SEF17 fimbrial antigen were present in the extracellular matrix of convoluted colonies of wild-type virulent S. enteritidis isolates. Bacteria were often hyperflagellated also. Immunoelectron microscopy of SEF17- mutants grown on CFA agar+0.001% Congo red demonstrated the elaboration of an as yet undefined fimbrial structure. Isolates of S. enteritidis which were described previously as avirulent and sensitive to environmental stress failed to express SEF17 or produce convoluted colonies. These data indicate an essential role for SEF17, and possibly for another fimbria and flagella, in the generation of the convoluted colonial phenotype. The relationship between virulence and colonial phenotype is discussed.

Salmonella enterica var Typhimurium and Salmonella enterica var Enteritidis express type 1 fimbriae in the rat in vivo.

In a series of experiments rats were dosed with purified type 1 fimbriae from Salmonella enterica var Enteritidis or with fimbriated cultures of either S. enterica var Typhimurium or S. enterica var Enteritidis. Paraffin-wax embedded histological sections of jejunal and ileal tissue were taken and stained by the streptavidin biotin complex (sABC) staining technique for the detection of salmonella and type 1 fimbriae. On oral infection with Enteritidis and Typhimurium both bacteria were shown to be closely associated with the rat ileal epithelium and expressed type 1 fimbriae, thus clearly demonstrating that type 1 fimbriae are expressed by salmonellae in vivo. Moreover, association with the ileum was also shown to occur when purified type 1 fimbriae were orally administered to rats. Our results suggest that type 1 fimbriae alone or in combination with other fimbriae may play an important role in the early stages of infection with these pathogenic bacteria.

Expression of SEF17 fimbriae by Salmonella enteritidis.

Specific immunological reagents were used to investigate the expression of SEF17 fimbriae by cultured strains of Salmonella enteritidis. Most strains of Salm. enteritidis tested expressed SEF17 when cultured at temperatures of 18-30 degrees C. However, two wild-type strains produced SEF17 when also grown at 37 degrees C and 42 degrees C. Colonization factor antigen agar was the optimum medium for SEF17 expression, whereas Drigalski and Sensitest agars poorly supported SEF17 production. Very fine fimbriae produced by a strain of Salm. typhimurium were specifically and strongly labelled by SEF17 monoclonal and polyclonal antibodies, indicating considerable antigenic conservation between the two. Curli fimbriae from Escherichia coli were similarly labelled. The production of these fimbriae correlated with the binding of fibronectin by the organism. Congo red binding by cultured bacteria was not a reliable criterion for the expression of SEF17 fimbriae.

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis.

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.