Adaptation of clinical isolates of Klebsiella pneumoniae to the combination of niclosamide with the efflux pump inhibitor phenyl-arginine-ß-naphthylamide (PaßN): co-resistance to antimicrobials.

OBJECTIVES: To search for new means of combatting carbapenemase-producing strains of Klebsiella pneumoniae by repurposing the anti-helminth drug niclosamide as an antimicrobial agent and combining it with the efflux pump inhibitor (EPI) phenyl-arginine-ß-naphthylamide (PaßN). METHODS: Niclosamide and PaßN MICs were determined for six clinical K. pneumoniae isolates harbouring different carbapenemases by broth microdilution and chequerboard assays. Time-kill curves in the presence of each drug alone and in combination were conducted. The viability of bacterial cells in the presence of repetitive exposures at 8 h to the treatment at the same concentration of niclosamide and/or PaßN (adapted isolates) was determined. The acrAB-tolC genes and their regulators were sequenced and quantitative RT-PCR was performed to assess whether the acrA gene was overexpressed in adapted isolates compared with non-adapted isolates. Finally, the MICs of several antimicrobials were determined for the adapted isolates. RESULTS: Niclosamide and PaßN had synergistic effects on the six isolates in vitro, but adaptation appeared when the treatment was applied to the medium every 8 h, with an increase of 6- to 12-fold in the MIC of PaßN. Sequencing revealed different mutations in the regulators of the tripartite AcrAB-TolC efflux pump (ramR and acrR) that may be responsible for the overexpression of the efflux pump and the adaptation to this combination. Co-resistance to different antimicrobials confirmed the overexpression of the AcrAB-TolC efflux pump. CONCLUSIONS: Despite the synergistic effect that preliminary in vitro stages may suggest, the combinations of drugs and EPI may generate adapted phenotypes associated with antimicrobial resistance that must be taken into consideration.

Executive functions in deaf and hearing children: The mediating role of language skills in inhibitory control.

This study aimed to analyze one of the critical components of inhibitory control-the ability to suppress interference-in deaf and hearing children and to investigate the mediating role of language skills in this central component of executive functions. To this end, a cross-sectional study was carried out with 40 deaf children with and without cochlear implants (CIs) and 21 hearing children age 7-10 years. The ability to suppress interference was assessed with the children's version of the Attentional Network Test (child-ANT), and language skills were assessed with a computerized version of the Carolina Picture Vocabulary Test (CPVT), a receptive vocabulary test. As a measure of control of nonverbal cognitive abilities, we used the Test of Nonverbal Intelligence (TONI-2). The results showed that deaf children had lower nonverbal IQ than hearing children. In addition, deaf children, compared with hearing children and regardless of whether they used CIs, showed a lower range of receptive vocabulary and a poorer ability to suppress the interference of distractors in the child-ANT. Linear regression mediation analyses revealed that this more significant interference effect was mediated by receptive vocabulary level and not by hearing deprivation. These results are consistent with the hypothesis that language is one of the critical factors in the development of executive functions.

CRISPR-Cas Controls Cryptic Prophages.

The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, Escherichia coli K-12. We show here that the E. coli CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., cryptic) prophages. Specifically, compared to the wild-type strain, reducing the amounts of specific interfering RNAs (crRNA) decreases growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA rather than through cleavage of cryptic prophage DNA, i.e., self-targeting. Consistently, four of the 13 E. coli spacers contain complementary regions to the mRNA sequences of seven cryptic prophages, and inactivation of CRISPR-Cas increases the level of mRNA for lysis protein YdfD of cryptic prophage Qin and lysis protein RzoD of cryptic prophage DLP-12. In addition, lysis is clearly seen via transmission electron microscopy when the whole CRISPR-Cas array is deleted, and eliminating spacer #12, which encodes crRNA with complementary regions for DLP-12 (including rzoD), Rac, Qin (including ydfD), and CP4-57 cryptic prophages, also results in growth inhibition and cell lysis. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages, likely through RNAi, i.e., unlike with active lysogens, active CRISPR-Cas and cryptic prophages may stably co-exist.

Enhanced Antibacterial Activity of Repurposed Mitomycin C and Imipenem in Combination with the Lytic Phage vB_KpnM-VAC13 against Clinical Isolates of Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that employs different strategies (resistance and persistence) to counteract antibiotic treatments. This study aimed to search for new means of combatting imipenem-resistant and persister strains of K. pneumoniae by repurposing the anticancer drug mitomycin C as an antimicrobial agent and by combining the drug and the conventional antibiotic imipenem with the lytic phage vB_KpnM-VAC13. Several clinical K. pneumoniae isolates were characterized, and an imipenem-resistant isolate (harboring OXA-245 ß-lactamase) and a persister isolate were selected for study. The mitomycin C and imipenem MICs for both isolates were determined by the broth microdilution method. Time-kill curve data were obtained by optical density at 600 nm (OD600) measurement and CFU enumeration in the presence of each drug alone and with the phage. The frequency of occurrence of mutants resistant to each drug and the combinations was also calculated, and the efficacy of the combination treatments was evaluated using an in vivo infection model (Galleria mellonella). The lytic phage vB_KpnM-VAC13 and mitomycin C had synergistic effects on imipenem-resistant and persister isolates, both in vitro and in vivo. The phage-imipenem combination successfully killed the persisters but not the imipenem-resistant isolate harboring OXA-245 ß-lactamase. Interestingly, the combinations decreased the emergence of in vitro resistant mutants of both isolates. Combinations of the lytic phage vB_KpnM-VAC13 with mitomycin C and imipenem were effective against the persister K. pneumoniae isolate. The lytic phage-mitomycin C combination was also effective against imipenem-resistant K. pneumoniae strains harboring OXA-245 ß-lactamase.

Heavy metal solubilisation during the hydrothermal treatment of sludge.

The present study analyses the combined effects of temperature (from 160 to 200 °C), and, for the first time ever, type of atmosphere (oxidising or inert) and pressure (from 60 to 100 bar) on the solubilisation of heavy metals during the hydrothermal treatment of wet no-diluted sewage sludge. Results revealed that Cd, Pb, Al and Fe were hardly affected by neither the atmosphere nor temperature, remained almost completely in the solid phase during all the hydrothermal treatments tested; while Cr, Ni, Cu and Zn were partially solubilised, being this solubilisation favoured by the presence of an oxidising atmosphere. In contrast, initial dissolved Hg was partially precipitated under both types of atmosphere, although it was re-dissolved after 30 min under an inert atmosphere. Regarding the working conditions, the highest temperature (200 °C) caused the greatest metal immobilisation, whereas the range of pressures tested barely had any effect on it. Concerning the reaction time, the lowest concentration of metals in the liquid fraction of the hydrolysed sludge was obtained during the initial 30 min of treatment.

(p)ppGpp and Its Role in Bacterial Persistence: New Challenges.

Antibiotic failure not only is due to the development of resistance by pathogens but can also often be explained by persistence and tolerance. Persistence and tolerance can be included in the "persistent phenotype," with high relevance for clinics. Two of the most important molecular mechanisms involved in tolerance and persistence are toxin-antitoxin (TA) modules and signaling via guanosine pentaphosphate/tetraphosphate [(p)ppGpp], also known as "magic spot." (p)ppGpp is a very important stress alarmone which orchestrates the stringent response in bacteria; hence, (p)ppGpp is produced during amino acid or fatty acid starvation by proteins belonging to the RelA/SpoT homolog family (RSH). However, (p)ppGpp levels can also accumulate in response to a wide range of signals, including oxygen variation, pH downshift, osmotic shock, temperature shift, or even exposure to darkness. Furthermore, the stringent response is not only involved in responses to environmental stresses (starvation for carbon sources, fatty acids, and phosphates or heat shock), but it is also used in bacterial pathogenesis, host invasion, and antibiotic tolerance and persistence. Given the exhaustive and contradictory literature surrounding the role of (p)ppGpp in bacterial persistence, and with the aim of summarizing what is known so far about the magic spot in this bacterial stage, this review provides new insights into the link between the stringent response and persistence. Moreover, we review some of the innovative treatments that have (p)ppGpp as a target, which are in the spotlight of the scientific community as candidates for effective antipersistence agents.

Multiplex Real-Time PCR-shortTUB Assay for Detection of the Mycobacterium tuberculosis Complex in Smear-Negative Clinical Samples with Low Mycobacterial Loads.

Tuberculosis (TB) remains a major health problem worldwide. Control of TB requires rapid, accurate diagnosis of active disease. However, extrapulmonary TB is very difficult to diagnose because the clinical specimens have very low bacterial loads. Several molecular methods involving direct detection of the Mycobacterium tuberculosis complex (MTBC) have emerged in recent years. Real-time PCR amplification simultaneously combines the amplification and detection of candidate sequences by using fluorescent probes (mainly TaqMan or Molecular Beacons) in automated systems. The multiplex real-time PCR-short assay is performed using locked nucleic acid (LNA) probes (length, 8 to 9 nucleotides) in combination with CodUNG to detect multiple pathogens in clinical samples. In this study, we evaluated the performance of this novel multiplex assay for detecting the MTBC in comparison with that of the conventional culture-based method. The multiplex real-time PCR-shortTUB assay targets two genes, whiB3 (redox-responsive transcriptional regulator) and pstS1 (phosphate-specific transporter), yielding limits of detection (LOD) of 10 copies and 100 copies, respectively, and amplification efficiencies of 92% and 99.7%, respectively. A total of 94 extrapulmonary samples and pulmonary samples with low mycobacterial loads (all smear negative; 75 MTBC culture positive) were analyzed using the test, yielding an overall sensitivity of 88% and a specificity of 95%. For pleural fluid and tissues/biopsy specimens, the sensitivity was 83% and 85%, respectively. In summary, this technique could be implemented in routine clinical microbiology testing to reduce the overall turnaround time for MTBC detection and may therefore be a useful tool for the diagnosis of extrapulmonary tuberculosis and diagnosis using pulmonary samples with low mycobacterial loads.

Relationship between Tolerance and Persistence Mechanisms in Acinetobacter baumannii Strains with AbkAB Toxin-Antitoxin System.

The molecular mechanisms of tolerance and persistence associated with several compounds in Acinetobacter baumannii clinical isolates are unknown. Using transcriptomic and phenotypic studies, we found a link between mechanisms of bacterial tolerance to chlorhexidine and the development of persistence in the presence of imipenem in an A. baumannii strain belonging to clinical clone ST-2 (OXA-24 ß-lactamase and AbkAB toxin-antitoxin [TA] system carried in a plasmid). Interestingly, the strain A. baumannii ATCC 17978 (AbkAB TA system from plasmid) showed persistence in the presence of imipenem and chlorhexidine.

Toxin/antitoxin systems induce persistence and work in concert with restriction/modification systems to inhibit phage.

IMPORTANCE: To date, there are no reports of phage infection-inducing persistence. Therefore, our results are important since we show for the first time that a phage-defense system, the MqsRAC toxin/antitoxin system, allows the host to survive infection by forming persister cells, rather than inducing cell suicide. Moreover, we demonstrate that the MqsRAC system works in concert with restriction/modification systems. These results imply that if phage therapy is to be successful, anti-persister compounds need to be administered along with phages.

Development of full-body rhythmic synchronization in middle childhood.

Rhythmic entrainment is a fundamental aspect of musical behavior, but the skills required to accurately synchronize movement to the beat seem to develop over many years. Motion capture studies of corporeal synchronization have shown immature abilities to lock in to the beat in children before age 5, and reliable synchronization ability in adults without musical training; yet there is a lack of data on full-body synchronization skills between early childhood and adulthood. To document typical rhythmic synchronization during middle childhood, we used a wireless motion capture device to measure period- and phase-locking of full body movement to rhythm and metronome stimuli in 6 to 11 year-old children in comparison with adult data. Results show a gradual improvement with age; however children's performance did not reach adult levels by age 12, suggesting that these skills continue to develop during adolescence. Our results suggest that in the absence of specific music training, full-body rhythmic entrainment skills improve gradually during middle childhood, and provide metrics for examining the continued maturation of these skills during adolescence.

Regulation of anti-phage defense mechanisms by using cinnamaldehyde as a quorum sensing inhibitor.

Background: Multidrug-resistant bacteria and the shortage of new antibiotics constitute a serious health problem. This problem has led to increased interest in the use of bacteriophages, which have great potential as antimicrobial agents but also carry the risk of inducing resistance. The objective of the present study was to minimize the development of phage resistance in Klebsiella pneumoniae strains by inhibiting quorum sensing (QS) and thus demonstrate the role of QS in regulating defense mechanisms. Results: Cinnamaldehyde (CAD) was added to K. pneumoniae cultures to inhibit QS and thus demonstrate the role of the signaling system in regulating the anti-phage defense mechanism. The QS inhibitory activity of CAD in K. pneumoniae was confirmed by a reduction in the quantitative expression of the lsrB gene (AI-2 pathway) and by proteomic analysis. The infection assays showed that the phage was able to infect a previously resistant K. pneumoniae strain in the cultures to which CAD was added. The results were confirmed using proteomic analysis. Thus, anti-phage defense-related proteins from different systems, such as cyclic oligonucleotide-based bacterial anti-phage signaling systems (CBASS), restriction-modification (R-M) systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system, and bacteriophage control infection (BCI), were present in the cultures with phage but not in the cultures with phage and CAD. When the QS and anti-phage defense systems were inhibited by the combined treatment, proteins related to phage infection and proliferation, such as the tail fiber protein, the cell division protein DamX, and the outer membrane channel protein TolC, were detected. Conclusion: Inhibition of QS reduces phage resistance in K. pneumoniae, resulting in the infection of a previously resistant strain by phage, with a significant increase in phage proliferation and a significant reduction in bacterial growth. QS inhibitors could be considered for therapeutic application by including them in phage cocktails or in phage-antibiotic combinations to enhance synergistic effects and reduce the emergence of antimicrobial resistance.

Single-cell analysis reveals that cryptic prophage protease LfgB protects Escherichia coli during oxidative stress by cleaving antitoxin MqsA.

Although toxin/antitoxin (TA) systems are ubiquitous, beyond phage inhibition and mobile element stabilization, their role in host metabolism is obscure. One of the best-characterized TA systems is MqsR/MqsA of Escherichia coli, which has been linked previously to protecting gastrointestinal species during the stress it encounters from the bile salt deoxycholate as it colonizes humans. However, some recent whole-population studies have challenged the role of toxins such as MqsR in bacterial physiology since the mqsRA locus is induced over a hundred-fold during stress, but a phenotype was not found upon its deletion. Here, we investigate further the role of MqsR/MqsA by utilizing single cells and demonstrate that upon oxidative stress, the TA system MqsR/MqsA has a heterogeneous effect on the transcriptome of single cells. Furthermore, we discovered that MqsR activation leads to induction of the poorly characterized yfjXY ypjJ yfjZF operon of cryptic prophage CP4-57. Moreover, deletion of yfjY makes the cells sensitive to H2O2, acid, and heat stress, and this phenotype was complemented. Hence, we recommend yfjY be renamed to lfgB (less fatality gene B). Critically, MqsA represses lfgB by binding the operon promoter, and LfgB is a protease that degrades MqsA to derepress rpoS and facilitate the stress response. Therefore, the MqsR/MqsA TA system facilitates the stress response through cryptic phage protease LfgB.IMPORTANCEThe roles of toxin/antitoxin systems in cell physiology are few and include phage inhibition and stabilization of genetic elements; yet, to date, there are no single-transcriptome studies for toxin/antitoxin systems and few insights for prokaryotes from this novel technique. Therefore, our results with this technique are important since we discover and characterize a cryptic prophage protease that is regulated by the MqsR/MqsA toxin/antitoxin system in order to regulate the host response to oxidative stress.

Improving phage therapy by evasion of phage resistance mechanisms.

Antibiotic failure is one of the most worrisome threats to global health. Among the new therapeutic efforts that are being explored, the use of bacteriophages (viruses that kill bacteria), also known as 'phages', is being extensively studied as a strategy to target bacterial pathogens. However, one of the main drawbacks of phage therapy is the plethora of defence mechanisms that bacteria use to defend themselves against phages. This review aims to summarize the therapeutic approaches that are being evaluated to overcome the bacterial defence systems, including the most innovative therapeutic approaches applied: circumvention of phage receptor mutations; modification of prophages; targeting of CRISPR-Cas systems and the biofilm matrix; engineering of safer and more efficacious phages; and inhibition of the anti-persister strategies used by bacteria.

Phage-Defense Systems Are Unlikely to Cause Cell Suicide.

As new phage-defense systems (PDs) are discovered, the overlap between their mechanisms and those of toxin/antitoxin systems (TAs) is becoming clear in that both use similar means to reduce cellular metabolism; for example, both systems have members that deplete energetic compounds (e.g., NAD+, ATP) and deplete nucleic acids, and both have members that inflict membrane damage. Moreover, both TAs and PDs are similar in that rather than altruistically killing the host to limit phage propagation (commonly known as abortive infection), both reduce host metabolism since phages propagate less in slow-growing cells, and slow growth facilitates the interaction of multiple phage-defense systems.

Ribosome inactivation by Escherichia coli GTPase RsgA inhibits T4 phage.

Introduction: Bacteria must combat phages, and myriad bacterial anti-phage systems have been discovered that reduce host metabolism, for example, by depleting energetic compounds like ATP and NAD+. Hence, these systems indirectly inhibit protein production. Surprisingly, direct reduction of ribosome activity has not been demonstrated to thwart phage. Methods: Here, by producing each of the 4,287 Escherichia coli proteins and selecting for anti-phage activity that leads to enhanced growth, we investigated the role of host proteins in phage inhibition. Results and discussion: We identified that E. coli GTPase RsgA inhibits lytic phage T4 by inactivating ribosomes.

Effectiveness of rhythmic training on linguistics skill development in deaf children and adolescents with cochlear implants: A systematic review.

OBJECTIVE: This review compiles the scientific evidence to date on the effectiveness of musical/rhythmic training for improving and/or enhancing the development of language skills in deaf children aged 6-16 years with cochlear implants. METHODS: PubMed, ScienceDirect, and Web of Science were used for the research following the PRISMA protocol. RESULTS: The reviewed studies indicate that rhythmic training can improve language skills (perception, production, and comprehension) in this population, as well as in other cognitive skills. CONCLUSION: Although further research is still needed, the current evidence can help identify new and more effective early intervention methods for deaf children.

Proteomic Study of the Interactions between Phages and the Bacterial Host Klebsiella pneumoniae.

Phages and bacteria have acquired resistance mechanisms for protection. In this context, the aims of the present study were to analyze the proteins isolated from 21 novel lytic phages of Klebsiella pneumoniae in search of defense mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defense mechanisms of two clinical isolates of K. pneumoniae infected by phages. For this purpose, the 21 lytic phages were sequenced and de novo assembled. The host range was determined in a collection of 47 clinical isolates of K. pneumoniae, revealing the variable infective capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the order Caudovirales. Phage sequence analysis revealed that the proteins were organized in functional modules within the genome. Although most of the proteins have unknown functions, multiple proteins were associated with defense mechanisms against bacteria, including the restriction-modification system, the toxin-antitoxin system, evasion of DNA degradation, blocking of host restriction and modification, the orphan CRISPR-Cas system, and the anti-CRISPR system. Proteomic study of the phage-host interactions (i.e., between isolates K3574 and K3320, which have intact CRISPR-Cas systems, and phages vB_KpnS-VAC35 and vB_KpnM-VAC36, respectively) revealed the presence of several defense mechanisms against phage infection (prophage, defense/virulence/resistance, oxidative stress and plasmid proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages. IMPORTANCE Researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about their defense mechanisms. In this study, we analyzed the molecular mechanisms of viral and bacterial defense in phages infecting clinical isolates of K. pneumoniae. Viral defense mechanisms included restriction-modification system evasion, the toxin-antitoxin (TA) system, DNA degradation evasion, blocking of host restriction and modification, and resistance to the abortive infection system, anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defense mechanisms, proteomic analysis revealed expression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defense/virulence/resistance (porins, efflux pumps, lipopolysaccharide, pilus elements, quorum network proteins, TA systems, and methyltransferases), oxidative stress mechanisms, and Acr candidates (anti-CRISPR protein). The findings reveal some important molecular mechanisms involved in the phage-host bacterial interactions; however, further study in this field is required to improve the efficacy of phage therapy.

Study of 32 new phage tail-like bacteriocins (pyocins) from a clinical collection of Pseudomonas aeruginosa and of their potential use as typing markers and antimicrobial agents.

Phage tail-like bacteriocins (PTLBs) are large proteomic structures similar to the tail phages. These structures function in bacterial competition by making pores in the membrane of their competitors. The PTLBs identified in Pseudomonas aeruginosa are known as R-type and F-type pyocins, which have a narrow spectrum of action. Their specificity is determined by the tail fiber and is closely related to the lipopolysaccharide type of the target competitor strain. In this study, the genome sequences of 32 clinical of P. aeruginosa clinical isolates were analysed to investigate the presence of R-type and F-type pyocins, and one was detected in all strains tested. The pyocins were classified into 4 groups on the basis of the tail fiber and also the homology, phylogeny and structure of the cluster components. A relationship was established between these groups and the sequence type and serotype of the strain of origin and finally the killing spectrum of the representative pyocins was determined showing a variable range of activity between 0 and 37.5%. The findings showed that these pyocins could potentially be used for typing of P. aeruginosa clinical isolates, on the basis of their genomic sequence and cluster structure, and also as antimicrobial agents.