RNA-based regulation in bacteria-phage interactions.

Interactions of bacteria with their viruses named bacteriophages or phages shape the bacterial genome evolution and contribute to the diversity of phages. RNAs have emerged as key components of several anti-phage defense systems in bacteria including CRISPR-Cas, toxin-antitoxin and abortive infection. Frequent association with mobile genetic elements and interplay between different anti-phage defense systems are largely discussed. Newly discovered defense systems such as retrons and CBASS include RNA components. RNAs also perform their well-recognized regulatory roles in crossroad of phage-bacteria regulatory networks. Both regulatory and defensive function can be sometimes attributed to the same RNA molecules including CRISPR RNAs. This review presents the recent advances on the role of RNAs in the bacteria-phage interactions with a particular focus on clostridial species including an important human pathogen, Clostridioides difficile.

Chronic Diarrhea Caused by a Klebsiella oxytoca Toxin Producer Strain Following Antibiotic-Associated Hemorrhagic Colitis: Successful Treatment by Fecal Microbiota Transplant.

Klebsiella oxytoca is a gram-negative bacterium found in fecal microbiota and known to cause several infections in humans, including antibiotic-associated hemorrhagic colitis. We present here a case of colitis caused by K. oxytoca toxin-producing strains that evolved in chronic diarrhea successfully treated by fecal microbiota transplant.

Inactivation of the riboswitch-controlled GMP synthase GuaA in Clostridioides difficile is associated with severe growth defects and poor infectivity in a mouse model of infection.

Clostridioides difficile is the main cause of nosocomial antibiotic-associated diarrhoea. There is a need for new antimicrobials to tackle this pathogen. Guanine riboswitches have been proposed as promising new antimicrobial targets, but experimental evidence of their importance in C. difficile is missing. The genome of C. difficile encodes four distinct guanine riboswitches, each controlling a single gene involved in purine metabolism and transport. One of them controls the expression of guaA, encoding a guanosine monophosphate (GMP) synthase. Here, using in-line probing and GusA reporter assays, we show that these riboswitches are functional in C. difficile and cause premature transcription termination upon binding of guanine. All riboswitches exhibit a high affinity for guanine characterized by Kd values in the low nanomolar range. Xanthine and guanosine also bind the guanine riboswitches, although with less affinity. Inactivating the GMP synthase (guaA) in C. difficile strain 630 led to cell death in minimal growth conditions, but not in rich medium. Importantly, the capacity of a guaA mutant to colonize the mouse gut was significantly reduced. Together, these results demonstrate the importance of de novo GMP biosynthesis in C. difficile during infection, suggesting that targeting guanine riboswitches with analogues could be a viable therapeutic strategy.

Pathophysiology of Vaginal Erosions in Women Using Pessary: A Pilot Study Examining Vaginal Microbiota.

OBJECTIVE: Many women with pelvic organ prolapse opt for a pessary, and some of these women develop erosions of the vaginal mucosa. Ongoing erosions might lead to the discontinuation of this otherwise effective, non-invasive, and inexpensive treatment. The objectives of this study were to investigate the differences in vaginal pH and variations of the vaginal microbiota among pessary and non-pessary users. METHODS: For this descriptive observational study, 30 women, followed in our urogynecology clinic, were recruited to form 3 equal groups: 2 groups of women using a pessary (with and without erosions) and 1 control group of women not using a pessary. Vaginal pH was measured distally and next to the erosion. Vaginal swabs were used to investigate the vaginal microbiota by sequencing the V4 region of the 16S ribosomal RNA gene and analyzing the data with Qiime2. Descriptive statistics were reported using the median values. Vaginal pH comparisons between groups were made using a Kruskal-Wallis test with Dunn's correction for multiple comparisons. RESULTS: The pH of the vagina was more alkaline in women with erosions compared with women in the other 2 groups (P < 0.01). Also, the pH of the distal vagina was not different from the pH next to the erosion (P = 0.25). Patients with erosions displayed significant differences in their vaginal microbiota, which contained a much greater bacterial diversity with an increase in gram-negative bacteria (e.g., Bacteroidetes, Actinobacteria) and a decrease in lactobacilli. CONCLUSION: In our study, women with vaginal erosions had significantly higher vaginal pH and more complex vaginal microbiota than women in the control groups. Treatments focusing on lowering the vaginal pH and/or re-establishing the vaginal microbiota should be considered.

Multilocus Variable-Number Tandem-Repeat Analysis of Clostridioides difficile Clusters in Ribotype 027 Isolates and Lack of Association with Clinical Outcomes.

The epidemiology of Clostridioides difficile infection (CDI) has drastically changed since the emergence of the epidemic strain BI/NAP1/027, also known as ribotype 027 (R027). However, the relationship between the infecting C. difficile strain and clinical outcomes is still debated. We hypothesized that certain subpopulations of R027 isolates could be associated with unfavorable outcomes. We applied high-resolution multilocus variable-number tandem-repeat analysis (MLVA) to characterize C. difficile R027 isolates collected from confirmed CDI patients recruited across 10 Canadian hospitals from 2005 to 2008. PCR ribotyping was performed first to select R027 isolates that were then analyzed by MLVA (n = 450). Complicated CDI (cCDI) was defined by the occurrence of any of admission to an intensive care unit, colonic perforation, toxic megacolon, colectomy, and if CDI was the cause or contributed to death within 30 days after enrollment. Three major MLVA clusters were identified, MC-1, MC-3, and MC-10. MC-1 and MC-3 were exclusive to Quebec centers, while MC-10 was found only in Ontario. Fewer cases infected with MC-1 developed cCDI (4%) than those infected with MC-3 and MC-10 (15% and 16%, respectively), but a statistically significant difference was not reached. Our data did not identify a clear association between subpopulations of R027 and different clinical outcomes; however, the data confirmed the utility of MLVA's higher discrimination potential to better characterize CDI populations in an epidemiological analysis. For a patient with CDI, the progression toward an unfavorable outcome is a complex process that probably includes several interrelated strain and host characteristics.

High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages in Clostridioides difficile.

Clostridioides difficile (formerly Clostridium difficile) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in C. difficile so far, with a genome of 131 kbp. It shares morphological and genomic similarity with other large siphophages, like phage 949, infecting Lactococcus lactis, and phage c-st, infecting Clostridium botulinum A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and a cas3 gene. We screened 2,584 C. difficile genomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected in C. difficile strains corresponding to 21 different multilocus sequence type groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements.IMPORTANCEClostridioides difficile is a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could explain the apparent differences observed in the virulence of different C. difficile strains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infect C. difficile so far. Screening 2,584 C. difficile genomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple-genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could influence bacterial biology and fitness. Our study further supports the relevance of studying phages in C. difficile to better understand the epidemiology of this clinically important human pathogen.

The Contribution of Bacteriophages to the Biology and Virulence of Pathogenic Clostridia.

Bacteriophages are key players in the evolution of most bacteria. Temperate phages have been associated with virulence of some of the deadliest pathogenic bacteria. Among the most notorious cases, the genes encoding the botulinum neurotoxin produced by Clostridium botulinum types C and D and the α-toxin (TcnA) produced by Clostridium novyi are both encoded within prophage genomes. Clostridium difficile is another important human pathogen and the recent identification of a complete binary toxin locus (CdtLoc) carried on a C. difficile prophage raises the potential for horizontal transfer of toxin genes by mobile genetic elements. Although the TcdA and TcdB toxins produced by C. difficile have never been found outside the pathogenicity locus (PaLoc), some prophages can still influence their production. Prophages can alter the expression of several metabolic and regulatory genes in C. difficile, as well as cell surface proteins such as CwpV, which confers phage resistance. Homologs of an Agr-like quorum sensing system have been identified in a C. difficile prophage, suggesting that it could possibly participate in cell-cell communication. Yet, other C. difficile prophages contain riboswitches predicted to recognize the secondary messenger molecule c-di-GMP involved in bacterial multicellular behaviors. Altogether, recent findings on clostridial phages underline the diversity of mechanisms and intricate relationship linking phages with their host. Here, milestone discoveries linking phages and virulence of some of the most pathogenic clostridial species will be retraced, with a focus on C. botulinum, C. novyi, C. difficile, and Clostridium perfringens phages, for which evidences are mostly available.

Ileal antimicrobial peptide expression is dysregulated in old age.

In an effort to understand the mechanisms underlying the high prevalence of gastrointestinal tract disorders in old age, we investigated the expression of intestinal antimicrobial peptides in the terminal small intestine of aged mice. Our results show that old mice have reduced transcript levels of ileal α-defensins and lysozyme, two important types of intestinal antimicrobial peptides produced by Paneth cells. In contrast, expression of the C-type lectins Reg3b and Reg3g, as well as ß-defensin 1, angiogenin 4 and Relmb, which are made by several epithelial cell types, was significantly upregulated in aged animals suggesting an ongoing response to epithelial distress. Those changes in antimicrobial peptide gene expression associated with histological damage of the ileal epithelium and subtle modifications in the composition of the commensal microbiota. Our findings suggest that dysregulation of antimicrobial peptides expression is a feature of homeostasis disruption in the aged intestine and may contribute to geriatric gastrointestinal dysfunction.

The Clostridium difficile cell wall protein CwpV confers phase-variable phage resistance.

Bacteriophages are present in virtually all ecosystems, and bacteria have developed multiple antiphage strategies to counter their attacks. Clostridium difficile is an important pathogen causing severe intestinal infections in humans and animals. Here we show that the conserved cell-surface protein CwpV provides antiphage protection in C. difficile. This protein, for which the expression is phase-variable, is classified into five types, each differing in their repeat-containing C-terminal domain. When expressed constitutively from a plasmid or the chromosome of locked 'ON' cells of C. difficile R20291, CwpV conferred antiphage protection. Differences in the level of phage protection were observed depending on the phage morphological group, siphophages being the most sensitive with efficiency of plaquing (EOP) values of < 5 × 10(-7) for phages ϕCD38-2, ϕCD111 and ϕCD146. Protection against the myophages ϕMMP01 and ϕCD52 was weaker, with EOP values between 9.0 × 10(-3) and 1.1 × 10(-1). The C-terminal domain of CwpV carries the antiphage activity and its deletion, or part of it, significantly reduced the antiphage protection. CwpV does not affect phage adsorption, but phage DNA replication is prevented, suggesting a mechanism reminiscent of superinfection exclusion systems normally encoded on prophages. CwpV thus represents a novel ubiquitous host-encoded and phase-variable antiphage system in C. difficile.

Cyclic di-GMP riboswitch-regulated type IV pili contribute to aggregation of Clostridium difficile.

Clostridium difficile is an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration in C. difficile was recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes, pilA1 (CD3513) and pilB1 (CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions. pilA1 is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-round in vitro transcription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation in C. difficile.

Factors Associated With Complications of Clostridium difficile Infection in a Multicenter Prospective Cohort.

BACKGROUND: Clostridium difficile infection (CDI) is the most common cause of nosocomial infectious diarrhea and may result in severe complications including death. We conducted a prospective study to identify risk factors for complications of CDI (cCDI). METHODS: Adult inpatients with confirmed CDI in 10 Canadian hospitals were enrolled and followed for 90 days. Potential risk factors were measured within 24 hours of diagnosis. Isolates were typed by polymerase chain reaction ribotyping. cCDI was defined as 1 or more of the following: colonic perforation, toxic megacolon, colectomy, admission to an intensive care unit for cCDI, or if CDI contributed to death within 30 days of enrollment. Risk factors for cCDI were investigated by logistic regression. RESULTS: A total of 1380 patients were enrolled. cCDI was observed in 8% of patients. The ribotype was identified in 922 patients, of whom 52% were infected with R027. Age ≥ 80 years, heart rate >90/minute, respiratory rate >20/minute, white cell count <4 × 10(9)/L or ≥ 20 × 10(9)/L, albumin <25 g/L, blood urea nitrogen >7 mmol/L, and C-reactive protein ≥ 150 mg/L were independently associated with cCDI. A higher frequency of cCDI was observed among R027-infected patients (10.9% vs 7.2%), but the association was not significant in adjusted analysis. CONCLUSIONS: CDI complications were associated with older age, abnormal blood tests, and abnormal vital signs. These factors, which are readily available to clinicians at the time of diagnosis, could be used for outcome prediction and risk stratification to select patients who may need closer monitoring or more aggressive therapy.

Global transcriptional response of Clostridium difficile carrying the CD38 prophage.

Clostridium difficile is one of the most dangerous pathogens in hospital settings. Most strains of C. difficile carry one or more prophages, and some of them, like CD38-2 and CD119, can influence the expression of toxin genes. However, little is known about the global host response in the presence of a given prophage. In order to fill this knowledge gap, we used high-throughput RNA sequencing (RNA-seq) to conduct a genome-wide transcriptomic analysis of the epidemic C. difficile strain R20291 carrying the CD38-2 prophage. A total of 39 bacterial genes were differentially expressed in the R20291 lysogen, 26 of them being downregulated. Several of the regulated genes encode transcriptional regulators and phosphotransferase system (PTS) subunits involved in glucose, fructose, and glucitol/sorbitol uptake and metabolism. CD38-2 also upregulated the expression of a group of regulatory genes located in phi-027, a resident prophage common to most ribotype 027 isolates. The most differentially expressed gene was that encoding the conserved phase-variable cell wall protein CwpV, which was upregulated 20-fold in the lysogen. Quantitative PCR and immunofluorescence showed that the increased cwpV expression results from a greater proportion of cells actively transcribing the gene. Indeed, 95% of f lysogenic cells express cwpV, as opposed to only 5% of wild-type cells. Furthermore, the higher proportion of cells expressing cwpV results from a higher frequency of recombination of the genetic switch controlling phase variation, which we confirmed to be dependent on the host-encoded recombinase RecV. In summary, CD38-2 interferes with phase variation of the surface protein CwpV and the expression of metabolic genes.

Characterization of temperate phages infecting Clostridium difficile isolates of human and animal origins.

Clostridium difficile is a Gram-positive pathogen infecting humans and animals. Recent studies suggest that animals could represent potential reservoirs of C. difficile that could then transfer to humans. Temperate phages contribute to the evolution of most bacteria, for example, by promoting the transduction of virulence, fitness, and antibiotic resistance genes. In C. difficile, little is known about their role, mainly because suitable propagating hosts and conditions are lacking. Here we report the isolation, propagation, and preliminary characterization of nine temperate phages from animal and human C. difficile isolates. Prophages were induced by UV light from 58 C. difficile isolates of animal and human origins. Using soft agar overlays with 27 different C. difficile test strains, we isolated and further propagated nine temperate phages: two from horse isolates (ΦCD481-1 and ΦCD481-2), three from dog isolates (ΦCD505, ΦCD506, and ΦCD508), and four from human isolates (ΦCD24-2, ΦCD111, ΦCD146, and ΦCD526). Two phages are members of the Siphoviridae family (ΦCD111 and ΦCD146), while the others are Myoviridae phages. Pulsed-field gel electrophoresis and restriction enzyme analyses showed that all of the phages had unique double-stranded DNA genomes of 30 to 60 kb. Phages induced from human C. difficile isolates, especially the members of the Siphoviridae family, had a broader host range than phages from animal C. difficile isolates. Nevertheless, most of the phages could infect both human and animal strains. Phage transduction of antibiotic resistance was recently reported in C. difficile. Our findings therefore call for further investigation of the potential risk of transduction between animal and human C. difficile isolates.

Prevention of Clostridium difficile spore formation by sub-inhibitory concentrations of tigecycline and piperacillin/tazobactam.

BACKGROUND: Sporulation of Clostridium difficile during infection and persistence of spores within the gut could partly explain treatment failures and recurrence. However, the influence of antibiotics on sporulation is unclear. The objective of our study was to evaluate the impact of ciprofloxacin, metronidazole, piperacillin/tazobactam, tigecycline, and vancomycin on C. difficile sporulation in vitro. METHODS: The reference strains ATCC 9689, 630, VPI 10463, and seven other clinical isolates of C. difficile were used, including three epidemic NAP1/027 isolates. Minimum inhibitory concentrations (MIC) were determined and sporulation was assessed after growth in the absence or presence of ≤0.5x MIC concentrations of each antibiotic. RESULTS: All strains were sensitive to the antibiotics tested, except ribotype 027 isolates that were resistant to ciprofloxacin (MIC = 128 mg/L). Metronidazole and vancomycin generally did not significantly affect spore production in C. difficile, although vancomycin slightly affected sporulation of a few isolates. Ciprofloxacin inhibited sporulation of ribotype 027 isolates mainly. Interestingly, sub-MIC concentrations of piperacillin/tazobactam reduced spore formation in several isolates. However, the most striking observation was made with tigecycline, with an important reduction of spore formation in most isolates. CONCLUSIONS: The capacity of C. difficile to sporulate can be significantly affected by certain antibiotics. The reduced sporulation observed with tigecycline and piperacillin/tazobactam might explain why these antibiotics are generally associated with lower risk of C. difficile infections. In addition, the inhibition of sporulation might partly explain the apparent efficacy of tigecycline for treatment of patients with recurrent infection.

c-di-GMP turn-over in Clostridium difficile is controlled by a plethora of diguanylate cyclases and phosphodiesterases.

Clostridium difficile infections have become a major healthcare concern in the last decade during which the emergence of new strains has underscored this bacterium's capacity to cause persistent epidemics. c-di-GMP is a bacterial second messenger regulating diverse bacterial phenotypes, notably motility and biofilm formation, in proteobacteria such as Vibrio cholerae, Pseudomonas aeruginosa, and Salmonella. c-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a conserved GGDEF domain. It is degraded by phosphodiesterases (PDEs) that contain either an EAL or an HD-GYP conserved domain. Very little is known about the role of c-di-GMP in the regulation of phenotypes of Gram-positive or fastidious bacteria. Herein, we exposed the main components of c-di-GMP signalling in 20 genomes of C. difficile, revealed their prevalence, and predicted their enzymatic activity. Ectopic expression of 31 of these conserved genes was carried out in V. cholerae to evaluate their effect on motility and biofilm formation, two well-characterized phenotype alterations associated with intracellular c-di-GMP variation in this bacterium. Most of the predicted DGCs and PDEs were found to be active in the V. cholerae model. Expression of truncated versions of CD0522, a protein with two GGDEF domains and one EAL domain, suggests that it can act alternatively as a DGC or a PDE. The activity of one purified DGC (CD1420) and one purified PDE (CD0757) was confirmed by in vitro enzymatic assays. GTP was shown to be important for the PDE activity of CD0757. Our results indicate that, in contrast to most Gram-positive bacteria including its closest relatives, C. difficile encodes a large assortment of functional DGCs and PDEs, revealing that c-di-GMP signalling is an important and well-conserved signal transduction system in this human pathogen.

Protection against Clostridioides difficile disease by a naturally avirulent C. difficile strain.

Clostridioides difficile (C. difficile) strains belonging to the epidemic BI/NAP1/027 (RT027) group have been associated with increased transmissibility and disease severity. In addition to the major toxin A and toxin B virulence factors, RT027 strains also encode the CDT binary toxin. Our lab previously identified a toxigenic RT027 isolate, ST1-75, that is avirulent in mice despite densely colonizing the colon. Here, we show that coinfecting mice with the avirulent ST1-75 and virulent R20291 strains protects mice from colitis due to rapid clearance of the virulent strain and persistence of the avirulent strain. Although avirulence of ST1-75 is due to a mutation in the cdtR gene, which encodes a response regulator that modulates the production of all three C. difficile toxins, the ability of ST1-75 to protect against acute colitis is not directly attributable to the cdtR mutation. Metabolomic analyses indicate that the ST1-75 strain depletes amino acids more rapidly than the R20291 strain and supplementation with amino acids ablates ST1-75's competitive advantage, suggesting that the ST1-75 strain limits the growth of virulent R20291 bacteria by amino acid depletion. Since the germination kinetics and sensitivity to the co-germinant glycine are similar for the ST1-75 and R20291 strains, our results identify the rapidity of in vivo nutrient depletion as a mechanism providing strain-specific, virulence-independent competitive advantages to different BI/NAP1/027 strains. They also suggest that the ST1-75 strain may, as a biotherapeutic agent, enhance resistance to CDI in high-risk patients.

The long and sinuous road to phage-based therapy of Clostridioides difficile infections.

With the antibiotic crisis and the rise in antimicrobial resistance worldwide, new therapeutic alternatives are urgently needed. Phage therapy represents one of the most promising alternatives but for some pathogens, such as Clostridioides difficile, important challenges are being faced. The perspective of phage therapy to treat C. difficile infections is complicated by the fact that no strictly lytic phages have been identified so far, and current temperate phages generally have a narrow host range. C. difficile also harbors multiple antiphage mechanisms, and the bacterial genome is often a host of one or multiple prophages that can interfere with lytic phage infection. Nevertheless, due to recent advances in phage host receptor recognition and improvements in genetic tools to manipulate phage genomes, it is now conceivable to genetically engineer C. difficile phages to make them suitable for phage therapy. Other phage-based alternatives such as phage endolysins and phage tail-like bacteriocins (avidocins) are also being investigated but these approaches also have their own limitations and challenges. Last but not least, C. difficile produces spores that are resistant to phage attacks and all current antibiotics, and this complicates therapeutic interventions. This mini-review gives a brief historical overview of phage work that has been carried out in C. difficile, presents recent advances in the field, and addresses the most important challenges that are being faced, with potential solutions.

Assessment of antibacterial properties and skin irritation potential of anodized aluminum impregnated with various quaternary ammonium.

The importance of the inert environment in the transmission of pathogens has been reassessed in recent years. To reduce cross-contamination, new biocidal materials used in high touch surfaces (e.g., stair railings, door handles) have been developed. However, their impact on skin remains poorly described. The present study aimed to evaluate the antibacterial properties and the risk of skin irritation of two materials based on hard-anodized aluminum (AA) impregnated with quaternary ammonium compound solutions (QAC#1 or QAC#2). The QAC#1 or QAC#2 solutions vary in composition, QAC#2 being free of dioctyl dimethyl ammonium chloride (Dio-DAC) and octyl decyl dimethyl ammonium chloride (ODDAC). Unlike AA used as a control, both AA-QAC#1 and AA-QAC#2 had excellent and rapid antibacterial efficacy, killing 99.9 % of Staphylococcus aureus and Escherichia coli bacteria, in 15 s and 1 min, respectively. The impregnation solutions (QAC#1 and QAC#2) did not show any skin sensitizing effect on transformed human keratinocytes. Nevertheless, these solutions as well as the materials (AA-QAC#1, AA-QAC#2), and the liquid extracts derived from them, induced a very rapid cytotoxicity on L929 murine fibroblasts (>70 % after 1 h of contact) as shown by LDH, MTS and neutral red assays. This cytotoxicity can be explained by the fast QACs release occurring when AA-QAC#1 and AA-QAC#2 were immersed in aqueous medium. To overcome the limitation of assays based on liquid condition, an in vitro skin irritation assay on reconstructed human epidermis (RHE) was developed. The effect of the materials upon their direct contact with the epidermis grown at the liquid-air interface was determined by evaluating tissue viability and quantifying interleukin-1 alpha (IL-1α) which is released in skin during injury or infection. AA-QAC#1 induced a significant decrease in RHE viability, close to OECD and ISO 10993-10 acceptability thresholds and enhanced the pro-inflammatory IL-1α secretion compared with AA-QAC#2. Finally, these results were corroborated by in vivo assays on mice using erythema and edema visual scores, histological observations, and epidermal thickness measurement. AA had no effect on the skin, while a stronger irritation was induced by AA-QAC#1 compared with AA-QAC#2. Hence, these materials were classified as moderate and slight irritants, respectively. In summary, this study revealed that AA-QAC#2 without Dio-DAC and ODDAC could be a great candidate for high touch surface applications, showing an extremely effective and rapid bactericidal activity, without inducing adverse effects for skin tissue.

Clostridioides difficile S-Layer Protein A (SlpA) Serves as a General Phage Receptor.

Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against Clostridioides difficile infections. To be efficient, phages should have a wide host range, buthe lack of knowledge about the cell receptor used by C. difficile phages hampers the rational design of phage cocktails. Recent reports suggested that the C. difficile surface layer protein A (SlpA) is an important phage receptor, but available data are still limited. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by ϕCD38-2, ϕCD111, and ϕCD146, which normally infect the parental strain. Complementation with 12 different S-layer cassette types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by ϕCD111 and SLCT-11 enabled infection by ϕCD38-2 and ϕCD146. Of note, the expression of SLCT-1, -6, -8, -9, -10, or -12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain. Also, deletion of the D2 domain within the low-molecular-weight fragment of SlpA was found to abolish infection by ϕCD38-2 and ϕCD146 but not ϕCD111. Altogether, our data suggest that many phages use SlpA as their receptor and, most importantly, that both siphophages and myophages target SlpA despite major differences in their tail structures. Our study therefore represents an important step in understanding the interactions between C. difficile and its phages. IMPORTANCE Phage therapy represents an interesting alternative to treat Clostridioides difficile infections because, contrary to antibiotics, most phages are highly species specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against C. difficile have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage receptor-binding protein binds to a specific SLCT, the identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.

Interleukin 15 in murine models of colitis.

Inflammatory bowel diseases (IBDs) are characterized by abnormal, non-antigen specific chronic inflammation of unknown etiology. Genome-wide association studies show that many IBD genetic susceptibility loci map to immune function genes and compelling evidence indicate that environmental factors play a critical role in IBD pathogenesis. Clinical and experimental evidence implicate the pro-inflammatory cytokine IL-15 in the pathogenesis of IBD. IL-15 and IL-15α expression is increased in the inflamed mucosa of IBD patients. IL-15 contributes to the maintenance of different cell subsets in the intestinal mucosa. However, very few studies have addressed the role of IL-15 in pre-clinical models of colitis. In this study, we use three well-characterized models of experimental colitis to determine the contribution of IL-15 to pathological intestinal inflammation.