Next-Generation Probiotics Targeting Clostridium difficile through Precursor-Directed Antimicrobial Biosynthesis.

Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. Clostridium difficile infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified Lactobacillus reuteri to be a promising candidate for adjunct therapy. Human-derived L. reuteri bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the pocR gene locus were potent reuterin producers, with L. reuteri 17938 inhibiting C. difficile growth at a level on par with the level of growth inhibition by vancomycin. Targeted pocR mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas treatment with either glycerol or L. reuteri alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with L. reuteri elicited changes in the composition and function of the human microbial community that preferentially targets C. difficile outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of C. difficile, and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials.

Structural and functional changes within the gut microbiota and susceptibility to Clostridium difficile infection.

Alteration of the gut microbial community structure and function through antibiotic use increases susceptibility to colonization by Clostridium difficile and other enteric pathogens. However, the mechanisms that mediate colonization resistance remain elusive. As the leading definable cause of infectious diarrhea, toxigenic C. difficile represents a burden for patients and health care systems, underscoring the need for better diagnostics and treatment strategies. Next-generation sequence data has increased our understanding of how the gut microbiota is influenced by many factors including diet, disease, aging and drugs. However, a microbial-based biomarker differentiating C. difficile infection from antibiotic-associated diarrhea has not been identified. Metabolomics profiling, which is highly responsive to changes in physiological conditions, have shown promise in differentiating subtle disease phenotypes that exhibit a nearly identical microbiome community structure, suggesting metabolite-based biomarkers may be an ideal diagnostic for identifying patients with CDI. This review focuses on the current understanding of structural and functional changes to the gut microbiota during C. difficile infection obtained from studies assessing the microbiome and metabolome of samples from patients and murine models.

Administration of probiotic kefir to mice with Clostridium difficile infection exacerbates disease.

Lifeway(®) kefir, a fermented milk product containing 12 probiotic organisms, is reported to show promise as an alternative to fecal microbiota transplantation for recurrent Clostridium difficile infection (CDI). We employed a murine CDI model to study the probiotic protective mechanisms and unexpectedly determined that kefir drastically increased disease severity. Our results emphasize the need for further independent clinical testing of kefir as alternative therapy in recurrent CDI.

Probiotics as adjunctive therapy for preventing Clostridium difficile infection - What are we waiting for?

With the end of the golden era of antibiotic discovery, the emergence of a new post-antibiotic age threatens to thrust global health and modern medicine back to the pre-antibiotic era. Antibiotic overuse has resulted in the natural evolution and selection of multi-drug resistant bacteria. One major public health threat, Clostridium difficile, is now the single leading cause of hospital-acquired bacterial infections and is by far the most deadly enteric pathogen for the U.S. POPULATION: Due to the high morbidity and mortality and increasing incidence that coincides with antibiotic use, non-traditional therapeutics are ideal alternatives to current treatment methods and also provide an avenue towards prevention. Despite the need for alternative therapies to antibiotics and the safety of most probiotics on the market, researchers are inundated with regulatory issues that hinder the translational science required to push these therapies forward. This review discusses the regulatory challenges of probiotic research, expert opinion regarding the application of probiotics to C. difficile infection and the efficacy of probiotics in preventing this disease.

Collagen-binding microbial surface components recognizing adhesive matrix molecule (MSCRAMM) of Gram-positive bacteria inhibit complement activation via the classical pathway.

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r2C1s2 was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.

Targeted protein engineering provides insights into binding mechanism and affinities of bacterial collagen adhesins.

The collagen-binding bacterial proteins, Ace and Cna, are well characterized on the biochemical and structural level. Despite overall structural similarity, recombinant forms of the Ace and Cna ligand-binding domains exhibit significantly different affinities and binding kinetics for collagen type I (CI) in vitro. In this study, we sought to understand, in submolecular detail, the bases for these differences. Using a structure-based approach, we engineered Cna and Ace variants by altering specific structural elements within the ligand-binding domains. Surface plasmon resonance-based binding analysis demonstrated that mutations that are predicted to alter the orientation of the Ace and Cna N(1) and N(2) subdomains significantly affect the interaction between the MSCRAMM (microbial surface components recognizing adhesive matrix molecule) and CI in vitro, including affinity, association/dissociation rates and binding ratio. Moreover, we utilized this information to engineer an Ace variant with an 11,000-fold higher CI affinity than the parent protein. Finally, we noted that several engineered proteins that exhibited a weak interaction with CI recognized more sites on CI, suggesting an inverse correlation between affinity and specificity.

An extracytoplasmic function sigma factor controls beta-lactamase gene expression in Bacillus anthracis and other Bacillus cereus group species.

The susceptibility of most Bacillus anthracis strains to beta-lactam antibiotics is intriguing considering that the closely related species Bacillus cereus and Bacillus thuringiensis typically produce beta-lactamases and the B. anthracis genome harbors two beta-lactamase genes, bla1 and bla2. We show that beta-lactamase activity associated with B. anthracis is affected by two genes, sigP (BA2502) and rsiP (BA2503), predicted to encode an extracytoplasmic function sigma factor and an anti-sigma factor, respectively. Deletion of the sigP-rsiP locus abolished beta-lactamase activity in a naturally occurring penicillin-resistant strain and had no effect on beta-lactamase activity in a prototypical penicillin-susceptible strain. Complementation with sigP and rsiP from the penicillin-resistant strain, but not with sigP and rsiP from the penicillin-susceptible strain, conferred constitutive beta-lactamase activity in both mutants. These results are attributed to a nucleotide deletion near the 5' end of rsiP in the penicillin-resistant strain that is predicted to result in a nonfunctional protein. B. cereus and B. thuringiensis sigP and rsiP homologues are required for inducible penicillin resistance in these species. Expression of the B. cereus or B. thuringiensis sigP and rsiP genes in a B. anthracis sigP-rsiP-null mutant confers inducible production of beta-lactamase activity, suggesting that while B. anthracis contains the genes necessary for sensing beta-lactam antibiotics, the B. anthracis sigP and rsiP gene products are not sufficient for bla induction.

plcR papR-independent expression of anthrolysin O by Bacillus anthracis.

Cholesterol-dependent cytolysins (CDCs) are secreted, pore-forming toxins that are associated with pathogenesis in a variety of gram-positive bacteria. Bacillus anthracis produces anthrolysin O (ALO), a CDC that is largely responsible for the hemolytic activity of culture supernates when the bacterium is cultured in appropriate conditions. B. cereus and B. thuringiensis, species closely related to B. anthracis, produce CDCs with significant amino acid sequence homology to ALO. Transcription of the B. cereus and B. thuringiensis CDC genes is controlled by PlcR, a transcription regulator that requires a pentapeptide derived from the papR gene product for binding to a consensus sequence (PlcR box) and transcriptional activation of downstream genes. A PlcR box precedes the B. anthracis alo gene, and the B. anthracis genome contains three plcR-like genes, one of which harbors a nonsense mutation that is predicted to result in a truncated, nonfunctional protein. We detected mRNA of alo, papR, and the three plcR-like genes in spleens of B. anthracis-infected mice, indicating gene expression in vivo. Analysis of alo transcription in batch culture revealed a potential transcription start located between the PlcR box and the translational start. Nevertheless, steady-state levels of alo transcripts and ALO protein were unaffected by deletion of papR or disruption of the PlcR box. Our data indicate that despite the presence of the transcriptionally active plcR and papR genes in B. anthracis and a PlcR box in the promoter region of the alo gene, alo expression is independent of this control system.

Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin.

We characterized the expression of a putative toxin of Bacillus anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O. We named this cytotoxin anthrolysin O (ALO). Although B. anthracis expresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely detectable amounts of hemolysin when grown in Luria-Bertani (LB) broth. Glucose supplementation of LB broth increases the amount of secreted hemolytic activity. Expression of hemolytic activity is maximal during mid- to late-log phase and decreases in the stationary phase. These observations are supported, in part, by semiquantitative reverse transcriptase PCR of alo mRNA. Hemolytic activity in growth supernatants was increased in the presence of reducing agent and almost totally inhibited in a dose-dependent manner by cholesterol; both of these activities are characteristic of a CDC toxin. A mutant of Sterne strain 7702, strain UT231, in which the alo gene was deleted and replaced by a kanamycin cassette, secreted barely detectable hemolytic activity into the growth medium. When strain UT231 was complemented in trans with native alo on a low-copy-number plasmid [strain UT231(pUTE554)], it regained the ability to secrete hemolytic activity, indicating that ALO is the major hemolysin secreted by this strain of B. anthracis in rich media in vitro. To further support the alo gene product being a hemolysin, recombinant B. anthracis ALO (rALO) purified from Escherichia coli was extremely active against washed human erythrocytes, with complete hemolysis detected at approximately 30 molecules of rALO per erythrocyte. Considering the virulence roles of CDCs for other gram-positive bacteria, we speculate that ALO may have a role in anthrax virulence.