Computational Design of Pore-Forming Peptides with Potent Antimicrobial and Anticancer Activities.

Peptides that form transmembrane barrel-stave pores are potential alternative therapeutics for bacterial infections and cancer. However, their optimization for clinical translation is hampered by a lack of sequence-function understanding. Recently, we have de novo designed the first synthetic barrel-stave pore-forming antimicrobial peptide with an identified function of all residues. Here, we systematically mutate the peptide to improve pore-forming ability in anticipation of enhanced activity. Using computer simulations, supported by liposome leakage and atomic force microscopy experiments, we find that pore-forming ability, while critical, is not the limiting factor for improving activity in the submicromolar range. Affinity for bacterial and cancer cell membranes needs to be optimized simultaneously. Optimized peptides more effectively killed antibiotic-resistant ESKAPEE bacteria at submicromolar concentrations, showing low cytotoxicity to human cells and skin model. Peptides showed systemic anti-infective activity in a preclinical mouse model of Acinetobacter baumannii infection. We also demonstrate peptide optimization for pH-dependent antimicrobial and anticancer activity.

Prevalence and factors associated with suicidal ideation, cannabis, and alcohol use during the COVID-19 pandemic in Saskatchewan: findings from a joint-effect modeling.

BACKGROUND: Generally, pandemics such as COVID-19 take an enormous toll on people's lives. As the pandemic now turns to an endemic state, growing attention has been paid to the multiple adverse mental health and behavioral issues, such as suicidal ideation and substance use. However, the interplay of suicidality and substance misuse during the pandemic has been limited. We aimed to investigate the prevalence of co-occurrence of suicide ideation, alcohol and cannabis misuse, and the factors that are associated with these co-occurrences in the province of Saskatchewan during the COVID-19 pandemic. METHODS: We performed a multivariable trivariate probit regression on a sample of 666 Saskatchewan adolescents and adults (16 years or older), drawn from the cycle 10 data collection (March 2022) of the Mental Health Commission of Canada, and Canadian Centre on Substance Use and Addiction (MHCC-CCSA) dataset. RESULTS: The prevalence of suicidal ideation was higher among respondents who reported both problematic cannabis and alcohol use (25.8%) than single users of alcohol (23.2%) and cannabis (18.7%). Younger respondents (16-34 years) and those who reported recent changes in other substance use were independent factors that were associated with the common experience of suicide ideation, problematic cannabis, and alcohol use. Having a diagnosis of mental health disorders either before or during the pandemic, and the perceived inability to bounce back after the pandemic (low resilience) are strong correlates of suicidal ideation. Those who lived alone, between 35 and 55 years of age were more likely to report problematic alcohol use. Those who reported changes in alternative activities, who reported pandemic stress, and declared a LGBTQIA2S + identity had higher probability of problematic cannabis use. CONCLUSIONS: As the pandemic persists, improving access to suicide and substance use interventions for the vulnerable groups identified in this study may be impactful.

Harm Reduction Strategies for Severe Alcohol Use Disorder in the Context of Homelessness: A Rapid Review.

Severe alcohol use disorder (AUD) in the context of housing instability remains one of the most complex health and social issues. Homelessness is related to increased vulnerability to stigma, marginalization and harmful ways of alcohol consumption, including non-beverage alcohol use (NBA). As a result, severe intoxication, alcohol poisoning, injury and death are common occurrences. Although harm minimization strategies have been readily proposed and examined in the context of drug use, applying the same principles to severe AUD remains controversial within the research and treatment community. This article summarizes the emerging research on managed alcohol programs to increase awareness about alcohol-related strategies that address severe AUD and provide other wrap-around supports such as housing, health and social services to mitigate various harms, including COVID-19.

Clostridioides difficile infections were predominantly driven by fluoroquinolone-resistant Clostridioides difficile ribotypes 176 and 001 in Slovakia in 2018-2019.

AIM: To investigate the epidemiology of Clostridioides difficile infection (CDI) in Slovakian hospitals after the emergence of ribotype 176 (027-like) in 2016. METHODS: Between 2018 and 2019, European Centre for Disease Control and Prevention CDI surveillance protocol v2.3 was applied to 14 hospitals, with additional data collected on recent antimicrobial use and the characterization of C. difficile isolates. RESULTS: The mean hospital incidence of CDI was 4.1 cases per 10,000 patient bed-days. One hundred and five (27.6%) in-hospital deaths were reported among the 381 cases. Antimicrobial treatment within the previous 4 weeks was recorded in 90.5% (333/368) of cases. Ribotype (RT)176 was detected in 50% (n=185/370, 14 hospitals) and RT001 was detected in 34.6% (n=128/370,13/14 hospitals) of cases with RT data. Overall, 86% (n=318/370) of isolates were resistant to moxifloxacin by Thr82Ile in GyrA (99.7%). Multi-locus variable tandem repeat analysis showed clonal relatedness of predominant RTs within and between hospitals. Seven of 14 sequenced RT176 isolates and five of 13 RT001 isolates showed between zero and three allelic differences by whole-genome multi-locus sequence typing. The majority of sequenced isolates (24/27) carried the erm(B) gene and 16/27 also carried the aac(6')-aph(2'') gene with the corresponding antimicrobial susceptibility phenotypes. Nine RT176 strains carried the cfr(E)gene and one RT001 strain carried the cfr(C) gene, but without linezolid resistance. CONCLUSIONS: The newly-predominant RT176 and endemic RT001 are driving the epidemiology of CDI in Slovakia. In addition to fluoroquinolones, the use of macrolide-lincosamide-streptogramin B antibiotics can represent another driving force for the spread of these epidemic lineages. In C. difficile, linezolid resistance should be confirmed phenotypically in strains with detected cfr gene(s).

Role of membrane proteins in bacterial resistance to antimicrobial peptides.

Several natural antimicrobial peptides (AMPs), including the novel semisynthetic lipoglycopeptide antibiotics telavancin, dalbavancin, and oritavancin, have been approved for clinical use to address the growing problem of multiple antibiotic-resistant Gram-positive bacterial infections. Nevertheless, the efficacy of these antibiotics has already been compromised. The SARS-CoV-2 pandemic led to the increased clinical use of all antibiotics, further promoting the development of bacterial resistance. Therefore, it is critical to gain a deeper understanding of the role of resistance mechanisms to minimize the consequential risks of long-term antibiotic use and misuse. Here, we summarize for the first time the current knowledge of resistance mechanisms that have been shown to cause resistance to clinically used AMPs, with particular focus on membrane proteins that have been reported to interfere with the activity of AMPs by affecting the binding of AMPs to bacteria.

Two Novel Semisynthetic Lipoglycopeptides Active against Staphylococcus aureus Biofilms and Cells in Late Stationary Growth Phase.

The increase in antibiotic resistance among Gram-positive bacteria underscores the urgent need to develop new antibiotics. New antibiotics should target actively growing susceptible bacteria that are resistant to clinically accepted antibiotics including bacteria that are not growing or are protected in a biofilm environment. In this paper, we compare the in vitro activities of two new semisynthetic glycopeptide antibiotics, MA79 and ERJ390, with two clinically used glycopeptide antibiotics-vancomycin and teicoplanin. The new antibiotics effectively killed not only exponentially growing cells of Staphylococcus aureus, but also cells in the stationary growth phase and biofilm.

Beyond Self-Resistance: ABCF ATPase LmrC Is a Signal-Transducing Component of an Antibiotic-Driven Signaling Cascade Accelerating the Onset of Lincomycin Biosynthesis.

In natural environments, antibiotics are important means of interspecies competition. At subinhibitory concentrations, they act as cues or signals inducing antibiotic production; however, our knowledge of well-documented antibiotic-based sensing systems is limited. Here, for the soil actinobacterium Streptomyces lincolnensis, we describe a fundamentally new ribosome-mediated signaling cascade that accelerates the onset of lincomycin production in response to an external ribosome-targeting antibiotic to synchronize antibiotic production within the population. The entire cascade is encoded in the lincomycin biosynthetic gene cluster (BGC) and consists of three lincomycin resistance proteins in addition to the transcriptional regulator LmbU: a lincomycin transporter (LmrA), a 23S rRNA methyltransferase (LmrB), both of which confer high resistance, and an ATP-binding cassette family F (ABCF) ATPase, LmrC, which confers only moderate resistance but is essential for antibiotic-induced signal transduction. Specifically, antibiotic sensing occurs via ribosome-mediated attenuation, which activates LmrC production in response to lincosamide, streptogramin A, or pleuromutilin antibiotics. Then, ATPase activity of the ribosome-associated LmrC triggers the transcription of lmbU and consequently the expression of lincomycin BGC. Finally, the production of LmrC is downregulated by LmrA and LmrB, which reduces the amount of ribosome-bound antibiotic and thus fine-tunes the cascade. We propose that analogous ABCF-mediated signaling systems are relatively common because many ribosome-targeting antibiotic BGCs encode an ABCF protein accompanied by additional resistance protein(s) and transcriptional regulators. Moreover, we revealed that three of the eight coproduced ABCF proteins of S. lincolnensis are clindamycin responsive, suggesting that the ABCF-mediated antibiotic signaling may be a widely utilized tool for chemical communication. IMPORTANCE Resistance proteins are perceived as mechanisms protecting bacteria from the inhibitory effect of their produced antibiotics or antibiotics from competitors. Here, we report that antibiotic resistance proteins regulate lincomycin biosynthesis in response to subinhibitory concentrations of antibiotics. In particular, we show the dual character of the ABCF ATPase LmrC, which confers antibiotic resistance and simultaneously transduces a signal from ribosome-bound antibiotics to gene expression, where the 5' untranslated sequence upstream of its encoding gene functions as a primary antibiotic sensor. ABCF-mediated antibiotic signaling can in principle function not only in the induction of antibiotic biosynthesis but also in selective gene expression in response to any small molecules targeting the 50S ribosomal subunit, including clinically important antibiotics, to mediate intercellular antibiotic signaling and stress response induction. Moreover, the resistance-regulatory function of LmrC presented here for the first time unifies functionally inconsistent ABCF family members involving antibiotic resistance proteins and translational regulators.

R/G Value-A Numeric Index of Individual Periodontal Health and Oral Microbiome Dynamics.

The dysbiosis of oral microbiome (OM) precedes the clinical signs of periodontal disease. Its simple measure thus could indicate individuals at risk of periodontitis development; however, such a tool is still missing. Up to now, numerous microbial taxa were associated with periodontal health or periodontitis. The outputs of most studies could, nevertheless, be slightly biased from following two reasons: First, the healthy group is often characterized only by the absence of the disease, but the individuals could already suffer from dysbiosis without any visible signs. Second, the healthy/diseased OM characteristics are frequently determined based on average data obtained for whole groups of periodontally healthy persons versus patients. Especially in smaller sets of tested individuals the typical individual variability can thus complicate the unambiguous assignment of oral taxa to respective state of health. In this work the taxonomic composition of OM was evaluated for 20 periodontally healthy individuals and 15 patients with chronic periodontitis. The narrowed selection set of the most diseased patients (confirmed by clinical parameters) and the most distant group of healthy individuals with the lowest probability of dysbiosis was determined by clustering analysis and used for identification of marker taxa. Based on their representation in each individual oral cavity we proposed the numeric index of periodontal health called R/G value. Its diagnostic potential was further confirmed using independent set of 20 periodontally healthy individuals and 20 patients with periodontitis with 95 percent of samples assigned correctly. We also assessed the individual temporal OM dynamics in periodontal health and we compared it to periodontitis. We revealed that the taxonomic composition of the system changes dynamically but generally it ranges within values typical for periodontal health or transient state, but far from values typical for periodontitis. R/G value tool, formulated from individually evaluated data, allowed us to arrange individual OMs into a continuous series, instead of two distinct groups, thus mimicking the gradual transformation of a virtual person from periodontal health to disease. The application of R/G value index thus represents a very promising diagnostic tool for early prediction of persons at risk of developing periodontal disease.

Ribosome-Mediated Attenuation of vga(A) Expression Is Shaped by the Antibiotic Resistance Specificity of Vga(A) Protein Variants.

Vga(A) protein variants confer different levels of resistance to lincosamides, streptogramin A, and pleuromutilins (LSAP) by displacing antibiotics from the ribosome. Here, we show that expression of vga(A) variants from Staphylococcus haemolyticus is regulated by cis-regulatory RNA in response to the LSAP antibiotics by the mechanism of ribosome-mediated attenuation. The specificity of induction depends on Vga(A)-mediated resistance rather than on the sequence of the riboregulator. Fine tuning between Vga(A) activity and its expression in response to the antibiotics may contribute to the selection of more potent Vga(A) variants because newly acquired mutation can be immediately phenotypically manifested.

VanZ Reduces the Binding of Lipoglycopeptide Antibiotics to Staphylococcus aureus and Streptococcus pneumoniae Cells.

vanZ, a member of the VanA glycopeptide resistance gene cluster, confers resistance to lipoglycopeptide antibiotics independent of cell wall precursor modification by the vanHAX genes. Orthologs of vanZ are present in the genomes of many clinically relevant bacteria, including Enterococcus faecium and Streptococcus pneumoniae; however, vanZ genes are absent in Staphylococcus aureus. Here, we show that the expression of enterococcal vanZ paralogs in S. aureus increases the minimal inhibitory concentrations of lipoglycopeptide antibiotics teicoplanin, dalbavancin, oritavancin and new teicoplanin pseudoaglycone derivatives. The reduction in the binding of fluorescently labeled teicoplanin to the cells suggests the mechanism of VanZ-mediated resistance. In addition, using a genomic vanZ gene knockout mutant of S. pneumoniae, we have shown that the ability of VanZ proteins to compromise the activity of lipoglycopeptide antibiotics by reducing their binding is a more general feature of VanZ-superfamily proteins.

Draft genome sequences of three clinical isolates of teicoplanin-resistant Staphylococcus epidermidis from patients without prior exposure to glycopeptide antibiotics.

OBJECTIVES: The aim of this study was to analyse the DNA sequences of three teicoplanin-resistant Staphylococcus epidermidis isolates collected from patients not previously treated with glycopeptide antibiotics. METHODS: The minimum inhibitory concentrations (MICs) of 12 antibiotics, including teicoplanin and vancomycin, were determined by the broth microdilution method. Genomic DNA was isolated, was sequenced by HiSeqX paired-end sequencing and was assembled into draft genome sequences using MyPro pipeline. RESULTS: Analysis of the draft genome sequences demonstrated that the teicoplanin-resistant S. epidermidis isolates belonged to multilocus sequence typing (MLST) sequence types ST5 and ST87 and encoded multiple antimicrobial resistance genes, including the methicillin resistance gene mecA. CONCLUSIONS: This report highlights the risk of dissemination of S. epidermidis strains resistant to a wide range of clinically important antibiotics.

Fluorescence assay to predict activity of the glycopeptide antibiotics.

Here, we describe a fluorescent assay developed to study competitive binding of the glycopeptide antibiotics to live bacteria cells. This assay demonstrated that the mechanism of action of the lipoglycopeptide antibiotics strongly depends on the hydrophobicity of the substitutes, with the best antibacterial activity of the glycopeptide antibiotics equally sharing properties of binding to D-Ala-D-Ala residues of the nascent peptidoglycan and to the membrane.

Teicoplanin resistance in Staphylococcus haemolyticus is associated with mutations in histidine kinases VraS and WalK.

We investigated the genetic basis of glycopeptide resistance in laboratory-derived strains of S. haemolyticus with emphasis on differences between vancomycin and teicoplanin. The genomes of two stable teicoplanin-resistant laboratory mutants selected on vancomycin or teicoplanin were sequenced and compared to parental S. haemolyticus strain W2/124. Only the two non-synonymous mutations, VraS Q289K and WalK V550L were identified. No other mutations or genome rearrangements were detected. Increased cell wall thickness, resistance to lysostaphin-induced lysis and adaptation of cell growth rates specifically to teicoplanin were phenotypes observed in a sequenced strain with the VraS Q289K mutation. Neither of the VraS Q289K and WalK V550L mutations was present in the genomes of 121S. haemolyticus clinical isolates. However, all but two of the teicoplanin resistant strains carried non-synonymous SNPs in vraSRTU and walKR-YycHIJ operons pointing to their importance for the glycopeptide resistance.

Assessment and Treatment for Persons with Coexisting Ability and Substance Use Issues: A Review and Analysis of the Literature.

Persons with ability issues are at considerably higher risk to develop substance use problems when compared to the general population. Yet, little is known about the current status of substance use treatment for this population. A comprehensive search of the literature revealed a need for (a) population-specific instruments for screening and assessment of the use of alcohol and drugs, including the misuse of prescription medication; (b) tailored treatment methods and individualized treatment plans that meet diverse literacy or cognitive needs;

In Vivo Characterization of the Activation and Interaction of the VanR-VanS Two-Component Regulatory System Controlling Glycopeptide Antibiotic Resistance in Two Related Streptomyces Species.

The VanR-VanS two-component system is responsible for inducing resistance to glycopeptide antibiotics in various bacteria. We have performed a comparative study of the VanR-VanS systems from two streptomyces strains, Streptomyces coelicolor and Streptomyces toyocaensis, to characterize how the two proteins cooperate to signal the presence of antibiotics and to define the functional nature of each protein in each strain background. The results indicate that the glycopeptide antibiotic inducer specificity is determined solely by the differences between the amino acid sequences of the VanR-VanS two-component systems present in each strain rather than by any inherent differences in general cell properties, including cell wall structure and biosynthesis. VanR of S. coelicolor (VanRsc) functioned with either sensor kinase partner, while VanR of S. toyocaensis (VanRst) functioned only with its cognate partner, S. toyocaensis VanS (VanSst). In contrast to VanRsc, which is known to be capable of phosphorylation by acetylphosphate, VanRst could not be activated in vivo independently of a VanS sensor kinase. A series of amino acid sequence modifications changing residues in the N-terminal receiver (REC) domain of VanRst to the corresponding residues present in VanRsc failed to create a protein capable of being activated by VanS of S. coelicolor (VanSsc), which suggests that interaction of the response regulator with its cognate sensor kinase may require a region more extended than the REC domain. A T69S amino acid substitution in the REC domain of VanRst produced a strain exhibiting weak constitutive resistance, indicating that this particular amino acid may play a key role for VanS-independent phosphorylation in the response regulator protein.

ClpP-independent function of ClpX interferes with telithromycin resistance conferred by Msr(A) in Staphylococcus aureus.

The ABCF family protein Msr(A) confers high resistance to macrolides but only low resistance to ketolides in staphylococci. Mutations in conserved functional regions of ClpX as well as deletion of clpX significantly increased Msr(A)-mediated resistance to the ketolide antibiotic telithromycin. ClpX is the chaperone component of the ClpXP two-component proteolytic system. Nevertheless, no changes in resistance were observed in a clpP knockout strain expressing msr(A), demonstrating that ClpX affects Msr(A) independently of ClpP.

Detailed mutational analysis of Vga(A) interdomain linker: implication for antibiotic resistance specificity and mechanism.

Detailed mutational analysis examines the roles of individual residues of the Vga(A) linker in determining the antibiotic resistance phenotype. It defines a narrowed region of residues 212 to 220 whose composition determines the resistance specificity to lincosamides, pleuromutilins, and/or streptogramins A. From the analogy with the recently described function of the homologous ABC-F protein EttA as a translational factor, we infer that the Vga(A) linker interacts with the ribosome and directly or indirectly affects the binding of the respective antibiotic.

Does the redesign of a psychiatric inpatient unit change the treatment process and outcomes?

OBJECTIVE: This study investigated whether ward atmosphere mediated the associations between the physical and therapeutic characteristics of an inpatient ward and patient outcomes. METHODS: Individuals (N=290) receiving inpatient care for mood and anxiety disorders before and after an extensive renovation project were surveyed about ward atmosphere, quality of life, and treatment satisfaction. Global functioning at admission and discharge and other clinical characteristics were obtained from patients' charts. RESULTS: After the redesign, participants perceived improved ward atmosphere, and the improvement was associated with greater treatment satisfaction and quality of life. Change in global functioning was independent of ward atmosphere. CONCLUSIONS: Efforts to improve the inpatient environment by supporting patient autonomy, peer support, and practical skill development may be expected to meet with improved outcomes, at least for quality of life and satisfaction with treatment. These findings are consistent with patient-centered design as well as with broader perspectives on recovery-oriented services.