Systematic interrogation of CRISPR antimicrobials in Klebsiella pneumoniae reveals nuclease-, guide- and strain-dependent features influencing antimicrobial activity.

CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials using multidrug-resistant and hypervirulent strains of Klebsiella pneumoniae as models. Comparing different Cas nucleases, DNA-targeting nucleases outperformed RNA-targeting nucleases based on the tested targets. Focusing on AsCas12a that exhibited robust targeting across different strains, we found that the elucidated modes of escape varied widely, restraining opportunities to enhance killing. We also encountered individual guide RNAs yielding different extents of clearance across strains, which were linked to an interplay between improper gRNA folding and strain-specific DNA repair and survival. To explore features that could improve targeting across strains, we performed a genome-wide screen in different K. pneumoniae strains that yielded guide design rules and trained an algorithm for predicting guide efficiency. Finally, we showed that Cas12a antimicrobials can be exploited to eliminate K. pneumoniae when encoded in phagemids delivered by T7-like phages. Altogether, our results highlight the importance of evaluating antimicrobial activity of CRISPR antimicrobials across relevant strains and define critical parameters for efficient CRISPR-based targeting.

Extending the Host Range of Bacteriophage Particles for DNA Transduction.

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.

Highly active CRISPR-adaptation proteins revealed by a robust enrichment technology.

Natural prokaryotic defense via the CRISPR-Cas system requires spacer integration into the CRISPR array in a process called adaptation. To search for adaptation proteins with enhanced capabilities, we established a robust perpetual DNA packaging and transfer (PeDPaT) system that uses a strain of T7 phage to package plasmids and transfer them without killing the host, and then uses a different strain of T7 phage to repeat the cycle. We used PeDPaT to identify better adaptation proteins-Cas1 and Cas2-by enriching mutants that provide higher adaptation efficiency. We identified two mutant Cas1 proteins that show up to 10-fold enhanced adaptation in vivo. In vitro, one mutant has higher integration and DNA binding activities, and another has a higher disintegration activity compared to the wild-type Cas1. Lastly, we showed that their specificity for selecting a protospacer adjacent motif is decreased. The PeDPaT technology may be used for many robust screens requiring efficient and effortless DNA transduction.

A phage mechanism for selective nicking of dUMP-containing DNA.

Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.

An efficient, scarless, selection-free technology for phage engineering.

Most recently developed phage engineering technologies are based on the CRISPR-Cas system. Here, we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harbouring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, which yields 1-14% of desired recombinants, allows low-throughput screening for the desired mutant. We have demonstrated the use of this technology for single-base substitutions, for deletions of 50-201 bases, for insertions of 20 bases, and for four different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.[Figure: see text].

CRISPR adaptation biases explain preference for acquisition of foreign DNA.

CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.

Superhydrophobic polypyrene films to prevent Staphylococcus aureus and Pseudomonas aeruginosa biofilm adhesion on surfaces: high efficiency deciphered by fluorescence microscopy.

A blue luminescent and superhydrophobic coating based on an electropolymerized fluorinated-pyrene monomer and its planktonic bacteria and biofilm repellent properties are reported. Two different pathogenic bacterial strains (Gram-positive and Gram-negative) at two different incubation times (2 h planktonic bacterial and 24 h biofilm adhesion) were studied and monitored (analyzed) using multicolor scanning confocal fluorescence microscopy. The coating was proved to reduce bacterial adhesion by 65%. It is highly effective against biofilm attachment, with 90% reduction of bacteria surface coverage. This blue fluorescent surface provides a facile method to characterize the coating, observe the bacterial distribution and quantify the bacterial coverage rate by fluorescence imaging of different colors. Furthermore, the film does not show significant bacterial toxicity during the working incubation times.

DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins constitute a recently identified prokaryotic defense system against invading nucleic acids. DNA segments, termed protospacers, are integrated into the CRISPR array in a process called adaptation. Here, we establish a PCR-based assay that enables evaluating the adaptation efficiency of specific spacers into the type I-E Escherichia coli CRISPR array. Using this assay, we provide direct evidence that the protospacer adjacent motif along with the first base of the protospacer (5'-AAG) partially affect the efficiency of spacer acquisition. Remarkably, we identified a unique dinucleotide, 5'-AA, positioned at the 3' end of the spacer, that enhances efficiency of the spacer's acquisition. Insertion of this dinucleotide increased acquisition efficiency of two different spacers. DNA sequencing of newly adapted CRISPR arrays revealed that the position of the newly identified motif with respect to the 5'-AAG is important for affecting acquisition efficiency. Analysis of approximately 1 million spacers showed that this motif is overrepresented in frequently acquired spacers compared with those acquired rarely. Our results represent an example of a short nonprotospacer adjacent motif sequence that affects acquisition efficiency and suggest that other as yet unknown motifs affect acquisition efficiency in other CRISPR systems as well.

Safety assessment of a novel active ingredient, acetyl aspartic acid, according to the EU Cosmetics Regulation and the Scientific Committee on Consumer Safety guidelines.

OBJECTIVE: Acetyl aspartic acid (A-A-A) was proposed as a new novel active ingredient for use in cosmetics. The safety of A-A-A was assessed by following an in-house-developed 'New Ingredient Testing Strategy', which was designed in accordance with the Scientific Committee on Consumer Safety (SCCS) notes of guidance and the requirements of Annex I of the EU Cosmetics Regulation. The aim of the project was to determine whether A-A-A was safe for use in cosmetics and to determine a maximum permitted safe level in the formulations. METHODS: A literature review was conducted, consulting over 40 different information sources. This highlighted a number of gaps which required testing data. A-A-A was tested for phototoxicity according to OECD test guideline 432, skin irritation according to OECD test guideline 439 and eye irritation according to OECD test guideline 437. Dermal absorption of A-A-A was measured according to OECD test guideline 428 and was used to calculate the margin of safety (MoS). Finally, A-A-A was tested in a human repeat insult patch test (HRIPT) and a 14-day in-use tolerance study. RESULTS: A-A-A was non-phototoxic and was non-irritating to skin and eyes in in vitro testing. Dermal absorption was calculated to be 5%. The MoS for A-A-A was 351, at a level of 5%, for all cosmetic product types, indicating no systemic safety toxicity concern. A-A-A at 5% under occlusive patch on a panel of 50 adult volunteers induced no skin irritation or allergic reaction in the HRIPT study. Finally, repeated application of A-A-A to the periocular area, twice per day for 14 days, in 21 female volunteers, demonstrated that 1% A-A-A was well tolerated following dermatological and ophthalmological assessment in a cosmetic formulation. CONCLUSION: A-A-A was assessed as safe by the cosmetic safety assessor for use in cosmetics at a level of 5% in all cosmetic product types, in line with the requirements of the EU Cosmetics Regulation and in accordance with the SCCS notes of guidance.

Novel concepts in inflammatory bowel disease.

INTRODUCTION: Clinical management in inflammatory bowel disease (IBD) is constantly changing. Although improvement in symptoms is of paramount importance, using this as the only surrogate marker of disease activity might underestimate disease burden. SOURCES OF DATA: New data from randomized clinical trials are now available. Treatment paradigms are constantly changing leading to an evolution in the therapeutic approach in routine IBD practice. AREAS OF AGREEMENT: Patients with an aggressive disease phenotype should be identified at the onset and treated more intensely in order to achieve long-lasting mucosal remission. AREAS OF CONTROVERSY: Patients who have mild and indolent disease need to be identified and not over treated. GROWING POINTS: The primary endpoint in IBD management should ideally be mucosal healing. Ample data are now available that correlates mucosal healing with surgical-free outcomes with minimal intestinal damage and patient disability. However, the exact degree of mucosal healing that will lead to improved long-term remission, decreased hospital and surgical rates remains unknown. AREAS TIMELY FOR DEVELOPING RESEARCH: Clinical translational work is needed to identify novel pathways in IBD pathogenesis that sub-select patients who would benefit by specific-cytokine pathway modulation.

Fatigue, psychological and cognitive impairment following transient ischaemic attack and minor stroke: a systematic review.

Transient ischaemic attack (TIA) and minor stroke are characterized by short-lasting symptoms; however, anecdotal and empirical evidence suggests that these patients experience ongoing cognitive/psychological impairment for which they are not routinely treated. The aims were (i) to investigate the prevalence and time course of fatigue, anxiety, depression, post-traumatic stress disorder(PTSD) and cognitive impairment following TIA/minor stroke; (ii) to explore the impact on quality of life (QoL), change in emotions and return to work; and (iii) to identify where further research is required and potentially inform an intervention study. A systematic review of MEDLINE, EMBASE, PSYCINFO, CINAHL, the Cochrane libraries and the grey literature between January 1993 and April 2013 was undertaken. Literature was screened and data were extracted by two independent reviewers. Studies were included of adult TIA/minor stroke participants with any of the outcomes of interest: fatigue, anxiety, depression, PTSD, cognitive impairment, QoL, change in emotions and return to work. Random-effects meta-analysis pooled outcomes by measurement tool. Searches identified 5976 records, 289 were assessed for eligibility and 31 studies were included. Results suggest high levels of cognitive impairment and depression post-TIA/minor stroke which decreased over time. However, frequencies varied between studies. Limited information was available on anxiety, PTSD and fatigue. Meta-analysis revealed that the measurement tool administered influenced the prevalence of cognitive impairment: Mini-Mental State Examination 17% [95% confidence interval (CI) 7, 26]; neuropsychological test battery 39% (95% CI 28, 50); Montreal Cognitive Assessment 54% (95% CI 43, 66). There is evidence to suggest that TIA/minor stroke patients may experience residual impairments; however, results should be interpreted with caution because of the few high quality studies. Notwithstanding, it is important to raise awareness of potential subtle but meaningful residual impairments.

Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.

High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system.

Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.

CRISPR adaptation in Escherichia coli subtypeI-E system.

The CRISPRs (clustered regularly interspaced short palindromic repeats) and their associated Cas (CRISPR-associated) proteins are a prokaryotic adaptive defence system against foreign nucleic acids. The CRISPR array comprises short repeats flanking short segments, called 'spacers', which are derived from foreign nucleic acids. The process of spacer insertion into the CRISPR array is termed 'adaptation'. Adaptation allows the system to rapidly evolve against emerging threats. In the present article, we review the most recent studies on the adaptation process, and focus primarily on the subtype I-E CRISPR-Cas system of Escherichia coli.

Clinical differences in a multidisciplinary pediatric pain unit between primary and secondary chronic pain.

INTRODUCTION: Chronic pain affects an important part of the pediatric population in developed countries. secondary chronic pain (SCP) can have a well-defined medical cause, but primary chronic pain (PCP) can have an unknown etiology. In Spain, there is as yet no information on the clinical differences between patients treated in multidisciplinary units. METHODS: Retrospective analysis of the clinical records of patients seen in 2018 at the Children's Chronic Pain Unit in University La Paz Hospital. RESULTS: A total of 92 patients were included, (age between 3 and 19 years), with a mean age of 12.4 (SD = 4.1) years, mostly female (55%), with a mean duration of pain of 11.3 (SD = 10.4) months. A comparison of patients with PCP (n = 31) and SCP (n = 61) showed that both groups, on average, presented intense pain (X = 5.9; SD = 2.2; range = 0-10), with similar duration and functional repercussions, although PCP was less likely to be associated with neuropathic descriptors than SCP (p = 0.040), and was more extensive (p < 0.001). Both groups received similar treatment, based on rehabilitation, psychotherapy, invasive techniques and analgesic medication, although patients in the PCP group received less analgesic medication (gabapentinoids and opioids) than the SCP (p = 0.011). CONCLUSION: Patients treated in a multidisciplinary Child Pain Unit for PCP or SCP present a very similar clinical profile, though with differences in the number and type of analgesic drugs used. This shows the importance of etiologic diagnosis for adequate pharmacological treatment.

Inhibition of host cell division by T5 protein 008 (Hdi).

IMPORTANCE: We have identified a novel phage-encoded inhibitor of the major cytoskeletal protein in bacterial division, FtsZ. The inhibition is shown to confer T5 bacteriophage with a growth advantage in dividing hosts. Our studies demonstrate a strategy in bacteriophages to maximize their progeny number by inhibiting escape of one of the daughter cells of an infected bacterium. They further emphasize that FtsZ is a natural target for bacterial growth inhibition.

The association of antimicrobial resistance with cure and quality of life among women with acute uncomplicated cystitis.

BACKGROUND: The association of in vitro resistance with bacteriologic, clinical, and health-related quality of life (HRQoL) outcomes for acute uncomplicated cystitis is unclear. METHODS: We conducted a prospective study of women aged 18-40 years with acute uncomplicated cystitis symptoms for ≤7 days who subsequently grew an Enterobacteriaceae sp. and initially received trimethoprim/sulfamethoxazole (TMP/SMX) and phenazopyridine. We conducted telephone follow-up evaluating clinical cure at 1-3 days and in-person follow-up evaluating clinical, bacteriologic, and HRQoL outcomes at 3-7 days and 4-6 weeks post-treatment. RESULTS: An Enterobacteriaceae sp. was isolated in 139 (96.5%) patients (25.2% TMP/SMX-resistant). At 1-3 days post-treatment, clinical cure occurred in 56/81 (69.1%) and 14/31 (45.2%) of cases with susceptible and resistant strains, respectively (difference 23.9%; 95% confidence interval [CI], 1.5-46.4%). At 3-7 days post-treatment, bacteriologic cure occurred in 70/73 (95.9%) and 15/25 (60%) of cases with susceptible and resistant strains, respectively (difference 35.9%; 95% CI, 13.5-58.3%). Sustained clinical cure rates at 3-7 days and 4-6 weeks post-treatment were 65.4 and 56.8% with susceptible strains, and 45.2 and 45.2% with resistant strains, respectively. The HRQoL scale assessing role limitations due to physical health problems was lower in TMP/SMX-resistant versus TMP/SMX-susceptible infections, with twice as many hours of missed activities reported (mean, 18.4 vs. 9.1 h). Differences in HRQoL appeared to be largely related to differences in clinical cure rates. CONCLUSIONS: Among women treated for acute uncomplicated cystitis with TMP/SMX, in vitro TMP/SMX resistance was associated with lower bacteriologic and clinical cure rates, and had greater impact on the time lost from daily activities compared to those with TMP/SMX-susceptible infections.

Transfer of carbapenem-resistant plasmid from Klebsiella pneumoniae ST258 to Escherichia coli in patient.

Klebsiella pneumoniae carbapenemase (KPC) 3-producing Escherichia coli was isolated from a carrier of KPC-3-producing K. pneumoniae. The KPC-3 plasmid was identical in isolates of both species. The patient's gut flora contained a carbapenem-susceptible E. coli strain isogenic with the KPC-3-producing isolate, which suggests horizontal interspecies plasmid transfer.

Carbapenem-resistant KPC-2-producing Escherichia coli in a Tel Aviv Medical Center, 2005 to 2008.

All of the carbapenem-resistant Escherichia coli (CREC) isolates identified in our hospital from 2005 to 2008 (n = 10) were studied. CREC isolates were multidrug resistant, all carried bla(KPC-2), and six of them were also extended-spectrum beta-lactamase producers. Pulsed-field gel electrophoresis indicated six genetic clones; within the same clone, similar transferable bla(KPC-2)-containing plasmids were found whereas plasmids differed between clones. Tn4401 elements were identified in all of these plasmids.