Association Between Clostridium innocuum and Antibiotic-Associated Diarrhea in Adults and Children: A Cross-sectional Study and Comparative Genomics Analysis.

BACKGROUND: A recent study from Taiwan suggested that Clostridium innocuum may be an unrecognized cause of antibiotic-associated diarrhea (AAD) and clinically indistinguishable from Clostridioides difficile infection. Our objective was to compare C. innocuum prevalence and strain between those with AAD and asymptomatic controls. METHODS: In this cross-sectional study, we collected stool from 200 individuals with AAD and 100 asymptomatic controls. We evaluated the association between AAD and C. innocuum in stool using anaerobic culture and quantitative polymerase chain reaction (qPCR). To identify strain-specific associations with AAD, we performed whole-genome sequencing of C. innocuum isolates using Illumina MiSeq and constructed comparative genomics analyses. RESULTS: C. innocuum was isolated from stool of 126/300 (42%) subjects and more frequently from asymptomatic controls than AAD subjects (50/100 [50%] vs 76/200 [38%], respectively; P = .047). C. innocuum isolation frequency was not associated with AAD in either the adult or pediatric subgroups. C. innocuum and C. difficile were frequently co-prevalent in individuals with and without diarrhea. There were no phylogenetic differences or accessory genome associations between C. innocuum isolates from AAD subjects and asymptomatic controls. CONCLUSIONS: C. innocuum was frequently isolated and at a greater frequency in asymptomatic controls than those with AAD. We did not identify strain lineages or accessory genomic elements associated with AAD. These data highlight that differentiating C. innocuum-associated diarrhea from asymptomatic colonization, and differentiating diarrhea caused by C. difficile from C. innocuum, are clinical microbiology challenges that require additional investigation to identify host-specific factors and/or biomarkers that distinguish these conditions.

The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms.

Dispersion is an active process exhibited by Pseudomonas aeruginosa during the late stages of biofilm development or in response to various cues, including nitric oxide and glutamate. Upon cue sensing, biofilm cells employ enzymes that actively degrade the extracellular matrix, thereby allowing individual cells to become liberated. While the mechanism by which P. aeruginosa senses and relays dispersion cues has been characterized, little is known about how dispersion cue sensing mechanisms result in matrix degradation. Considering that the alginate and motility regulator AmrZ has been reported to regulate genes that play a role in dispersion, including those affecting virulence, c-di-GMP levels, Pel and Psl abundance, and motility, we asked whether AmrZ contributes to the regulation of dispersion. amrZ was found to be significantly increased in transcript abundance under dispersion-inducing conditions, with the inactivation of amrZ impairing dispersion by P. aeruginosa biofilms in response to glutamate and nitric oxide. While the overexpression of genes encoding matrix-degrading enzymes pelA, pslG, and/or endA resulted in the dispersion of wild-type biofilms, similar conditions failed to disperse biofilms formed by dtamrZ. Likewise, the inactivation of amrZ abrogated the hyperdispersive phenotype of PAO1/pJN-bdlA_G31A biofilms, with dtamrZ-impaired dispersion being independent of the expression, production, and activation of BdlA. Instead, dispersion was found to require the AmrZ-target genes napB and PA1891. Our findings indicate that AmrZ is essential for the regulation of dispersion by P. aeruginosa biofilms, functions downstream of BdlA postdispersion cue sensing, and regulates the expression of genes contributing to biofilm matrix degradation as well as napB and PA1891. IMPORTANCE In P. aeruginosa, biofilm dispersion has been well-characterized with respect to dispersion cue perception, matrix degradation, and the consequences of dispersion. While the intracellular signaling molecule c-di-GMP has been linked to many of the phenotypic changes ascribed to dispersion, including the modulation of motility and matrix production, little is known about the regulatory mechanisms leading to matrix degradation and cells actively leaving the biofilm. In this study, we report for the first time an essential role of the transcriptional regulator AmrZ and two AmrZ-dependent genes, napB, and PA1891, in the dispersion response, thereby linking dispersion cue sensing via BdlA to the regulation of matrix degradation and to the ultimate liberation of bacterial cells from the biofilm.

Clostridium innocuum: Microbiological and clinical characteristics of a potential emerging pathogen.

Clostridium innocuum is an anaerobic, gram-positive, spore-forming bacterium identified by Smith and King in 1962 after being isolated from a patient with an appendiceal abscess. Its name, C. innocuum, reflected its clinically "innocuous" nature based on observed lack of virulence in animal models of infection. Since that time, C. innocuum has been identified as both part of the normal intestinal flora and the cause of a rare, intrinsically vancomycin-resistant opportunistic infection in immunocompromised patients. More recently, reports from Taiwan suggest that C. innocuum, in addition to being a known extraintestinal pathogen, may also be a diarrheal pathogen that causes a C. difficile infection-like antibiotic-associated diarrheal illness. However, unanswered questions about the clinical relevance of C. innocuum remain. Here we review the microbiological and clinical characteristics of this emerging pathogen.

Untethering and Degradation of the Polysaccharide Matrix Are Essential Steps in the Dispersion Response of Pseudomonas aeruginosa Biofilms.

Biofilms are multicellular aggregates of bacteria that are encased in an extracellular matrix. The biofilm matrix of Pseudomonas aeruginosa PAO1 is composed of eDNA, proteins, and the polysaccharides Pel and Psl. This matrix is thought to be degraded during dispersion to liberate cells from the biofilms, with dispersion being apparent not only by single cells escaping from the biofilm but also leaving behind eroded or hollowed-out biofilm. However, little is known of the factors involved in matrix degradation. Here, we focused on the glycoside hydrolases PelA and PslG. We demonstrate that induction of pelA but not pslG expression resulted in dispersion. As Psl is tethered to the matrix adhesin CdrA, we furthermore explored the role of CdrA in dispersion. cdrA mutant biofilms were hyperdispersive, while lapG mutant biofilms were impaired in dispersion in response to glutamate and nitric oxide, indicating the presence of the surface-associated matrix protein CdrA impedes the dispersion response. In turn, insertional inactivation of cdrA enabled pslG-induced dispersion. Lowering of the intracellular c-di-GMP level via induction of PA2133 encoding a phosphodiesterase was not sufficient to induce dispersion by wild-type strains and strains overexpressing pslG, indicating that pslG-induced dispersion is independent of c-di-GMP modulation and, likely, LapG.IMPORTANCEPseudomonas aeruginosa forms multicellular aggregates or biofilms encased in a matrix. We show for the first time here that dispersion by P. aeruginosa requires the endogenous expression of pelA and pslG, leading to the degradation of both Pel and Psl polysaccharides, with PslG-induced dispersion being CdrA dependent. The findings suggested that endogenously induced Psl degradation is a sequential process, initiated by untethering of CdrA-bound Psl or CdrA-dependent cell interactions to enable Psl degradation and ultimately, dispersion. Untethering likely involves CdrA release in a manner independent of c-di-GMP modulation and thus LapG. Our findings not only provide insight into matrix degrading factors contributing to dispersion but also identify key steps in the degradation of structural components of the P. aeruginosa biofilm matrix.

Pseudomonas aeruginosa Requires the DNA-Specific Endonuclease EndA To Degrade Extracellular Genomic DNA To Disperse from the Biofilm.

The dispersion of biofilms is an active process resulting in the release of planktonic cells from the biofilm structure. While much is known about the process of dispersion cue perception and the subsequent modulation of the c-di-GMP pool, little is known about subsequent events resulting in the release of cells from the biofilm. Given that dispersion coincides with void formation and an overall erosion of the biofilm structure, we asked whether dispersion involves degradation of the biofilm matrix. Here, we focused on extracellular genomic DNA (eDNA) due to its almost universal presence in the matrix of biofilm-forming species. We identified two probable nucleases, endA and eddB, and eddA encoding a phosphatase that were significantly increased in transcript abundance in dispersed cells. However, only inactivation of endA but not eddA or eddB impaired dispersion by Pseudomonas aeruginosa biofilms in response to glutamate and nitric oxide (NO). Heterologously produced EndA was found to be secreted and active in degrading genomic DNA. While endA inactivation had little effect on biofilm formation and the presence of eDNA in biofilms, eDNA degradation upon induction of dispersion was impaired. In contrast, induction of endA expression coincided with eDNA degradation and resulted in biofilm dispersion. Thus, released cells demonstrated a hyperattaching phenotype but remained as resistant to tobramycin as biofilm cells from which they egress, indicating EndA-dispersed cells adopted some but not all of the phenotypes associated with dispersed cells. Our findings indicate for the first time a role of DNase EndA in dispersion and suggest weakening of the biofilm matrix is a requisite for biofilm dispersion.IMPORTANCE The finding that exposure to DNase I impairs biofilm formation or leads to the dispersal of early stage biofilms has led to the realization of extracellular genomic DNA (eDNA) as a structural component of the biofilm matrix. However, little is known about the contribution of intrinsic DNases to the weakening of the biofilm matrix and dispersion of established biofilms. Here, we demonstrate for the first time that nucleases are induced in dispersed Pseudomonas aeruginosa cells and are essential to the dispersion response and that degradation of matrix eDNA by endogenously produced/secreted EndA is required for P. aeruginosa biofilm dispersion. Our findings suggest that dispersing cells mediate their active release from the biofilm matrix via the induction of nucleases.

Susceptibility of Pseudomonas aeruginosa Dispersed Cells to Antimicrobial Agents Is Dependent on the Dispersion Cue and Class of the Antimicrobial Agent Used.

The biofilm life cycle is characterized by the transition of planktonic cells exhibiting high susceptibly to antimicrobial agents to a biofilm mode of growth characterized by high tolerance to antimicrobials, followed by dispersion of cells from the biofilm back into the environment. Dispersed cells, however, are not identical to planktonic cells but have been characterized as having a unique transitionary phenotype relative to biofilm and planktonic cells, with dispersed cells attaching in a manner similar to exponential-phase cells, but demonstrating gene expression patterns that are distinct from both exponential and stationary-phase planktonic cells. This raised the question whether dispersed cells are as susceptible as planktonic cells and whether the dispersion inducer or the antibiotic class affects the drug susceptibility of dispersed cells. Dispersed cells obtained in response to dispersion cues glutamate and nitric oxide (NO) were thus exposed to tobramycin and colistin. Although NO-induced dispersed cells were as susceptible to colistin and tobramycin as exponential-phase planktonic cells, glutamate-induced dispersed cells were susceptible to tobramycin but resistant to colistin. The difference in colistin susceptibility was independent of cellular c-di-GMP levels, with modulation of c-di-GMP failing to induce dispersion. Instead, drug susceptibility was inversely correlated with LPS modification system and the biofilm-specific transcriptional regulator BrlR. The susceptibility phenotype of glutamate-induced dispersed cells to colistin was found to be reversible, with dispersed cells being rendered as susceptible to colistin within 2 h postdispersion, though additional time was required for dispersed cells to display expression of genes indicative of exponential growth.

The diguanylate cyclase GcbA facilitates Pseudomonas aeruginosa biofilm dispersion by activating BdlA.

Biofilm dispersion is a highly regulated process that allows biofilm bacteria to respond to changing environmental conditions and to disseminate to new locations. The dispersion of biofilms formed by the opportunistic pathogen Pseudomonas aeruginosa is known to require a number of cyclic di-GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and the chemosensory protein BdlA, with BdlA playing a pivotal role in regulating PDE activity and enabling dispersion in response to a wide array of cues. BdlA is activated during biofilm growth via posttranslational modifications and nonprocessive cleavage in a manner that is dependent on elevated c-di-GMP levels. Here, we provide evidence that the diguanylate cyclase (DGC) GcbA contributes to the regulation of BdlA cleavage shortly after initial cellular attachment to surfaces and, thus, plays an essential role in allowing biofilm cells to disperse in response to increasing concentrations of a variety of substances, including carbohydrates, heavy metals, and nitric oxide. DGC activity of GcbA was required for its function, as a catalytically inactive variant could not rescue impaired BdlA processing or the dispersion-deficient phenotype of gcbA mutant biofilms to wild-type levels. While modulating BdlA cleavage during biofilm growth, GcbA itself was found to be subject to c-di-GMP-dependent and growth-mode-specific regulation. GcbA production was suppressed in mature wild-type biofilms and could be induced by reducing c-di-GMP levels via overexpression of genes encoding PDEs. Taken together, the present findings demonstrate that the regulatory functions of c-di-GMP-synthesizing DGCs expand beyond surface attachment and biofilm formation and illustrate a novel role for DGCs in the regulation of the reverse sessile-motile transition of dispersion.

The Pseudomonas aeruginosa diguanylate cyclase GcbA, a homolog of P. fluorescens GcbA, promotes initial attachment to surfaces, but not biofilm formation, via regulation of motility.

Cyclic di-GMP is a conserved signaling molecule regulating the transitions between motile and sessile modes of growth in a variety of bacterial species. Recent evidence suggests that Pseudomonas species harbor separate intracellular pools of c-di-GMP to control different phenotypic outputs associated with motility, attachment, and biofilm formation, with multiple diguanylate cyclases (DGCs) playing distinct roles in these processes, yet little is known about the potential conservation of functional DGCs across Pseudomonas species. In the present study, we demonstrate that the P. aeruginosa homolog of the P. fluorescens DGC GcbA involved in promoting biofilm formation via regulation of swimming motility likewise synthesizes c-di-GMP to regulate surface attachment via modulation of motility, however, without affecting subsequent biofilm formation. P. aeruginosa GcbA was found to regulate flagellum-driven motility by suppressing flagellar reversal rates in a manner independent of viscosity, surface hardness, and polysaccharide production. P. fluorescens GcbA was found to be functional in P. aeruginosa and was capable of restoring phenotypes associated with inactivation of gcbA in P. aeruginosa to wild-type levels. Motility and attachment of a gcbA mutant strain could be restored to wild-type levels via overexpression of the small regulatory RNA RsmZ. Furthermore, epistasis analysis revealed that while both contribute to the regulation of initial surface attachment and flagellum-driven motility, GcbA and the phosphodiesterase DipA act within different signaling networks to regulate these processes. Our findings expand the complexity of c-di-GMP signaling in the regulation of the motile-sessile switch by providing yet another potential link to the Gac/Rsm network and suggesting that distinct c-di-GMP-modulating signaling pathways can regulate a single phenotypic output.