Corinda Taylor

A new macrocyclic antibiotic, fidaxomicin (OPT- 80), causes less alteration to the bowel microbiota of Clostridium difficile-infected patients than does vancomycin

Clostridium difficile infection (CDI) is the most common identifiable cause of diarrhoea in hospitalized patients. Current therapies rely on the administration of metronidazole or vancomycin, which reduce vegetative populations of C. difficile in the bowel. Recurrence of the disease when treatment with these antibiotics ceases indicates that metronidazole and vancomycin affect not only C. difficile but also commensal populations that normally mediate competitive exclusion. Fidaxomicin is a new antibiotic that inhibits C. difficile. Our study shows that fidaxomicin had little effect on the composition of the faecal microbiota in terms of its major phylogenetic clusters. Notably, clostridial clusters XIVa and IV, and Bifidobacterium, were much less affected by fidaxomicin compared to vancomycin treatment. These findings help to explain the substantially reduced rates of relapse following treatment of CDI with fidaxomicin in recent clinical trials.

Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.

In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.

Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species.

CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids1,2. They are present in approximately half of all sequenced prokaryotes3 and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function4,5. We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti-CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems.

Comprehensive analysis of the bacterial content of stool from patients with chronic pouchitis, normal pouches, or familial adenomatous polyposis pouches.

Background: Chronic pouchitis is an important long‐term complication following ileal pouch–anal anastomosis for ulcerative colitis. Antibiotic administration reduces symptoms of pouchitis, indicating that bacteria have a role in pathogenesis. The aim of the research was to investigate the bacterial content of pouches using nucleic acid‐based methods. Methods: Stool microbiota of 17 patients with normal pouches (NP), 17 patients with pouchitis (CP) utilizing samples collected from each patient when antibiotic‐treated (CP‐on, asymptomatic) and when untreated (CP‐off, symptomatic), and 14 familial adenomatous polyposis (FAP) patients were analyzed by high‐throughput sequencing, fluorescence in situ hybridization technologies, and quantitative polymerase chain reaction (qPCR). Results: Fluorescence in situ hybridization analysis revealed an expanded phylogenetic gap in NP and CP‐off patients relative to FAP. Antibiotic treatment reduced the gap in CP stool. The phylogenetic gap of CP‐off patients was due to members of the bacterial families Caulobacteriaceae, Sphingomonadaceae, Comamonadaceae, Peptostreptococcaceae, and Clostridiaceae. There was a greater diversity of phylotypes of Clostridiaceae in CP‐off subjects. The phylogenetic gap of NP stool was enriched by Ruminococcaceae and Bifidobacteriaceae. CP stool microbiota had reduced diversity relative to NP and FAP stool due largely to a reduction in Lachnospiraceae/Insertae Sedis XIV/clostridial cluster IV groups. Conclusions: Bacterial groups within the expanded phylogenetic gap of pouch patients may have roles in the pathogenesis of pouchitis. Further research concerning the physiology of cultured members of these groups will be necessary to explain their specific roles. Members of the Lachnospiraceae, Incertae Sedis XIV, and clostridial cluster IV could be useful biomarkers of pouch health. (Inflamm Bowel Dis 2011;)

The immune response to autologous bacteroides in ankylosing spondylitis is characterized by reduced interleukin 10 production.

Objective. Ileocolitis is a recognized feature of ankylosing spondylitis (AS) and is likely to play a role in the pathogenesis of AS, in conjunction with the normal intestinal microbiota. In order to investigate the host immune response in AS, we measured cytokines in tissue culture following exposure of peripheral blood mononuclear cells (PBMC) to autologous colonic bacteria. Methods. Twenty-one patients with AS and 21 matched controls were recruited. Subjects in the AS group were assessed clinically. Bacteroides species belonging to the B. fragilis group were selectively cultured from stool samples and paired with blood samples from each participant. Ten cultures of autologous Bacteroides were randomly selected from cultures grown from the fecal specimens of each of the 21 patients with AS and 21 controls. These were then tested for reactivity with PBMC and the cytokines produced by proliferating lymphocytes [interleukin 10 (IL-10), IL-17, interferon-γ, tumor necrosis factor-α] were measured in cell culture supernatants. Differences between groups were analyzed using censored normal regression analysis. Results. The patients with AS had severe active AS with Bath AS Disease Activity Index 5.5 (± 1.6) and C-reactive protein (mg/l) 13.8 (± 12.2) (mean ± standard deviation). IL-10 concentrations in ex vivo assay supernatants were lower in the AS group compared with controls (p = 0.047). There were no statistically significant differences between the groups for other cytokines. Conclusion. In AS, reduced IL-10 production in response to stimulation with autologous Bacteroides cultures may represent a mechanism by which intestinal inflammation develops and persists, a situation analogous to inflammatory bowel disease.

Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems

Summary Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

ABSTRACT Pseudomonas syringae pv. actinidiae is a reemerging pathogen which causes bacterial canker of kiwifruit (Actinidia sp.). Since 2008, a global outbreak of P. syringae pv. actinidiae has occurred, and in 2010 this pathogen was detected in New Zealand. The economic impact and the development of resistance in P. syringae pv. actinidiae and other pathovars against antibiotics and copper sprays have led to a search for alternative management strategies. We isolated 275 phages, 258 of which were active against P. syringae pv. actinidiae. Extensive host range testing on P. syringae pv. actinidiae, other pseudomonads, and bacteria isolated from kiwifruit orchards showed that most phages have a narrow host range. Twenty-four were analyzed by electron microscopy, pulse-field gel electrophoresis, and restriction digestion. Their suitability for biocontrol was tested by assessing stability and the absence of lysogeny and transduction. A detailed host range was performed, phage-resistant bacteria were isolated, and resistance to other phages was examined. The phages belonged to the Caudovirales and were analyzed based on morphology and genome size, which showed them to be representatives of Myoviridae, Podoviridae, and Siphoviridae. Twenty-one Myoviridae members have similar morphologies and genome sizes yet differ in restriction patterns, host range, and resistance, indicating a closely related group. Nine of these Myoviridae members were sequenced, and each was unique. The most closely related sequenced phages were a group infecting Pseudomonas aeruginosa and characterized by phages JG004 and PAK_P1. In summary, this study reports the isolation and characterization of P. syringae pv. actinidiae phages and provides a framework for the intelligent formulation of phage biocontrol agents against kiwifruit bacterial canker.

Regulation of the Type I-F CRISPR-Cas system by CRP-cAMP and GalM controls spacer acquisition and interference

The CRISPR-Cas prokaryotic ‘adaptive immune systems’ represent a sophisticated defence strategy providing bacteria and archaea with protection from invading genetic elements, such as bacteriophages or plasmids. Despite intensive research into their mechanism and application, how CRISPR-Cas systems are regulated is less clear, and nothing is known about the regulation of Type I-F systems. We used Pectobacterium atrosepticum, a Gram-negative phytopathogen, to study CRISPR-Cas regulation, since it contains a single Type I-F system. The CRP-cAMP complex activated the cas operon, increasing the expression of the adaptation genes cas1 and cas2–3 in addition to the genes encoding the Csy surveillance complex. Mutation of crp or cyaA (encoding adenylate cyclase) resulted in reductions in both primed spacer acquisition and interference. Furthermore, we identified a galactose mutarotase, GalM, which reduced cas operon expression in a CRP- and CyaA-dependent manner. We propose that the Type I-F system senses metabolic changes, such as sugar availability, and regulates cas genes to initiate an appropriate defence response. Indeed, elevated glucose levels reduced cas expression in a CRP- and CyaA-dependent manner. Taken together, these findings highlight that a metabolite-sensing regulatory pathway controls expression of the Type I-F CRISPR-Cas system to modulate levels of adaptation and interference.

Testing probiotic strain Escherichia coli Nissle 1917 (Mutaflor) for its ability to reduce carriage of multidrug-resistant E. coli by elderly residents in long-term care facilities

A high carriage rate of multidrug-resistant Escherichia coli (MDREC) was observed in elderly residents in long-term care facilities. A double-blinded, placebo-controlled trial was carried out to determine whether the probiotic product E. coli strain Nissle 1917 (Mutaflor) would compete with MDREC in the bowel and thereby reduce the prevalence of the multiresistant bacteria in faeces and urine. Sixty-nine patients excreting norfloxacin-resistant E. coli were randomized to probiotic or placebo groups and administered capsules twice daily. The daily dose of probiotic was 5×10(9)-5×10(10) bacteria. Faecal and urine samples were cultured at baseline and during and after the treatment period. A reduction in baseline carriage was not influenced by probiotic administration. The probiotic strain was detected in faecal specimens collected during the treatment period of only two out of 12 probiotic group subjects that were tested. Genotyping of norfloxacin-resistant E. coli isolates showed that 32 strains were prevalent among the patients. Thus, E. coli Nissle 1917 does not have the capacity to compete effectively with MDREC in the bowel of elderly patients.