The role of PemIK (PemK/PemI) type II TA system from Klebsiella pneumoniae clinical strains in lytic phage infection.

Since their discovery, toxin-antitoxin (TA) systems have captivated the attention of many scientists. Recent studies have demonstrated that TA systems play a key role in phage inhibition. The aim of the present study was to investigate the role of the PemIK (PemK/PemI) type II TA system in phage inhibition by its intrinsic expression in clinical strains of Klebsiella pneumoniae carrying the lncL plasmid, which harbours the carbapenemase OXA-48 and the PemK/PemI TA system. Furthermore, induced expression of the system in an IPTG-inducible plasmid in a reference strain of K. pneumoniae ATCC10031 was also studied. The results showed that induced expression of the whole TA system did not inhibit phage infection, whereas overexpression of the pemK toxin prevented early infection. To investigate the molecular mechanism involved in the PemK toxin-mediated inhibition of phage infection, assays measuring metabolic activity and viability were performed, revealing that overexpression of the PemK toxin led to dormancy of the bacteria. Thus, we demonstrate that the PemK/PemI TA system plays a role in phage infection and that the action of the free toxin induces a dormant state in the cells, resulting in inhibition of phage infections.

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.

Phenotypic and Genomic Comparison of Klebsiella pneumoniae Lytic Phages: vB_KpnM-VAC66 and vB_KpnM-VAC13.

Klebsiella pneumoniae is a human pathogen that worsens the prognosis of many immunocompromised patients. Here, we annotated and compared the genomes of two lytic phages that infect clinical strains of K. pneumoniae (vB_KpnM-VAC13 and vB_KpnM-VAC66) and phenotypically characterized vB_KpnM-VAC66 (time of adsorption of 12 min, burst size of 31.49 ± 0.61 PFU/infected cell, and a host range of 20.8% of the tested strains). Transmission electronic microscopy showed that vB_KpnM-VAC66 belongs to the Myoviridae family. The genomic analysis of the phage vB_KpnM-VAC66 revealed that its genome encoded 289 proteins. When compared to the genome of vB_KpnM-VAC13, they showed a nucleotide similarity of 97.56%, with a 93% of query cover, and the phylogenetic study performed with other Tevenvirinae phages showed a close common ancestor. However, there were 21 coding sequences which differed. Interestingly, the main differences were that vB_KpnM-VAC66 encoded 10 more homing endonucleases than vB_KpnM-VAC13, and that the nucleotidic and amino-acid sequences of the L-shaped tail fiber protein were highly dissimilar, leading to different three-dimensional protein predictions. Both phages differed significantly in their host range. These viruses may be useful in the development of alternative therapies to antibiotics or as a co-therapy increasing its antimicrobial potential, especially when addressing multidrug resistant (MDR) pathogens.

Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection.

To optimize phage therapy, we need to understand how bacteria evolve against phage attacks. One of the main problems of phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage-resistant strains that can be overcome by the analysis of metadata provided by whole-genome sequencing. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strains belonging to the ST-2 clonal complex during a decade (Ab2000 vs. 2010): 9 from 2000 to 9 from 2010. The presence of genes putatively associated with phage resistance was detected. Genes detected were associated with an abortive infection system, restriction-modification system, genes predicted to be associated with defense systems but with unknown function, and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands in the 2000 strains and 32% in the 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems. A moderately higher presence of these genes in the strains of 2010 in comparison with those of 2000 was found, especially those related to the restriction-modification system and CRISPR-Cas system. The presence of these genes in genomic islands at a higher rate in the strains of 2010 compared with those of 2000 was also detected. Whole-genome sequencing and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in possible phage therapy.

Temperate Bacteriophages (Prophages) in Pseudomonas aeruginosa Isolates Belonging to the International Cystic Fibrosis Clone (CC274).

Bacteriophages are important in bacterial ecology and evolution. Pseudomonas aeruginosa is the most prevalent bacterial pathogen in chronic bronchopulmonary infection in cystic fibrosis (CF). In this study, we used bioinformatics, microbiological and microscopy techniques to analyze the bacteriophages present in 24 P. aeruginosa isolates belonging to the international CF clone (ST274-CC274). Interestingly, we detected the presence of five members of the Inoviridae family of prophages (Pf1, Pf4, Pf5, Pf6, Pf7), which have previously been observed in P. aeruginosa. In addition, we identified a new filamentous prophage, designated Pf8, in the P. aeruginosa AUS411.500 isolate belonging to the international CF clone. We detected only one prophage, never previously described, from the family Siphoviridiae (with 66 proteins and displaying homology with PHAGE_Pseudo_phi297_NC_016762). This prophage was isolated from the P. aeruginosa AUS531 isolate carrying a new gene which is implicated in the phage infection ability, named Bacteriophage Control Infection (bci). We characterized the role of the Bci protein in bacteriophage infection and in regulating the host Quorum Sensing (QS) system, motility and biofilm and pyocyanin production in the P. aeruginosa isogenic mutant AUS531Δbci isolate. The findings may be relevant for the identification of targets in the development of new strategies to control P. aeruginosa infections, particularly in CF patients.

Viral Related Tools against SARS-CoV-2.

At the end of 2019, a new disease appeared and spread all over the world, the COVID-19, produced by the coronavirus SARS-CoV-2. As a consequence of this worldwide health crisis, the scientific community began to redirect their knowledge and resources to fight against it. Here we summarize the recent research on viruses employed as therapy and diagnostic of COVID-19: (i) viral-vector vaccines both in clinical trials and pre-clinical phases; (ii) the use of bacteriophages to find antibodies specific to this virus and some studies of how to use the bacteriophages themselves as a treatment against viral diseases; and finally, (iii) the use of CRISPR-Cas technology both to obtain a fast precise diagnose of the patient and also the possible use of this technology as a cure.

Mechanisms of Tolerance and Resistance to Chlorhexidine in Clinical Strains of Klebsiella pneumoniae Producers of Carbapenemase: Role of New Type II Toxin-Antitoxin System, PemIK.

Although the failure of antibiotic treatment is normally attributed to resistance, tolerance and persistence display a significant role in the lack of response to antibiotics. Due to the fact that several nosocomial pathogens show a high level of tolerance and/or resistance to chlorhexidine, in this study we analyzed the molecular mechanisms associated with chlorhexidine adaptation in two clinical strains of Klebsiella pneumoniae by phenotypic and transcriptomic studies. These two strains belong to ST258-KPC3 (high-risk clone carrying ß-lactamase KPC3) and ST846-OXA48 (low-risk clone carrying ß-lactamase OXA48). Our results showed that the K. pneumoniae ST258-KPC3CA and ST846-OXA48CA strains exhibited a different behavior under chlorhexidine (CHLX) pressure, adapting to this biocide through resistance and tolerance mechanisms, respectively. Furthermore, the appearance of cross-resistance to colistin was observed in the ST846-OXA48CA strain (tolerant to CHLX), using the broth microdilution method. Interestingly, this ST846-OXA48CA isolate contained a plasmid that encodes a novel type II toxin/antitoxin (TA) system, PemI/PemK. We characterized this PemI/PemK TA system by cloning both genes into the IPTG-inducible pCA24N plasmid, and found their role in persistence and biofilm formation. Accordingly, the ST846-OXA48CA strain showed a persistence biphasic curve in the presence of a chlorhexidine-imipenem combination, and these results were confirmed by the enzymatic assay (WST-1).

(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.

Genomic analysis of 40 prophages located in the genomes of 16 carbapenemase-producing clinical strains of Klebsiella pneumoniae.

Klebsiella pneumoniae is the clinically most important species within the genus Klebsiella and, as a result of the continuous emergence of multi-drug resistant (MDR) strains, the cause of severe nosocomial infections. The decline in the effectiveness of antibiotic treatments for infections caused by MDR bacteria has generated particular interest in the study of bacteriophages. In this study, we characterized a total of 40 temperate bacteriophages (prophages) with a genome range of 11.454-84.199 kb, predicted from 16 carbapenemase-producing clinical strains of K. pneumoniae belonging to different sequence types, previously identified by multilocus sequence typing. These prophages were grouped into the three families in the order Caudovirales (27 prophages belonging to the family Myoviridae, 10 prophages belonging to the family Siphoviridae and 3 prophages belonging to the family Podoviridae). Genomic comparison of the 40 prophage genomes led to the identification of four prophages isolated from different strains and of genome sizes of around 33.3, 36.1, 39.6 and 42.6 kb. These prophages showed sequence similarities (query cover >90 %, identity >99.9 %) with international Microbe Versus Phage (MVP) (http://mvp.medgenius.info/home) clusters 4762, 4901, 3499 and 4280, respectively. Phylogenetic analysis revealed the evolutionary proximity among the members of the four groups of the most frequently identified prophages in the bacterial genomes studied (33.3, 36.1, 39.6 and 42.6 kb), with bootstrap values of 100 %. This allowed the prophages to be classified into three clusters: A, B and C. Interestingly, these temperate bacteriophages did not infect the highest number of strains as indicated by a host-range assay, these results could be explained by the development of superinfection exclusion mechanisms. In addition, bioinformatic analysis of the 40 identified prophages revealed the presence of 2363 proteins. In total, 59.7 % of the proteins identified had a predicted function, mainly involving viral structure, transcription, replication and regulation (lysogenic/lysis). Interestingly, some proteins had putative functions associated with bacterial virulence (toxin expression and efflux pump regulators), phage defence profiles such as toxin-antitoxin modules, an anti-CRISPR/Cas9 protein, TerB protein (from terZABCDE operon) and methyltransferase proteins.

In vitro and in vivo efficacy of combinations of colistin and different endolysins against clinical strains of multi-drug resistant pathogens.

The emergence of multidrug resistant (MDR) pathogenic bacteria is jeopardizing the value of antimicrobials, which had previously changed the course of medical science. In this study, we identified endolysins ElyA1 and ElyA2 (GH108-PG3 family), present in the genome of bacteriophages Ab1051Φ and Ab1052Φ, respectively. The muralytic activity of these endolysins against MDR clinical isolates (Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae) was tested using the turbidity reduction assay. Minimal inhibitory concentrations (MICs) of endolysin, colistin and a combination of endolysin and colistin were determined, and the antimicrobial activity of each treatment was confirmed by time kill curves. Endolysin ElyA1 displayed activity against all 25 strains of A. baumannii and P. aeruginosa tested and against 13 out of 17 strains of K. pneumoniae. Endolysin ElyA2 did not display any such activity. The combined antimicrobial activity of colistin and ElyA1 yielded a reduction in the colistin MIC for all strains studied, except K. pneumoniae. These results were confirmed in vivo in G. mellonella survival assays and in murine skin and lung infection models. In conclusion, combining colistin (1/4 MIC) with the new endolysin ElyA1 (350 µg) enhanced the bactericidal activity of colistin in both in vitro and in vivo studies. This will potentially enable reduction of the dose of colistin used in clinical practice.

Strategies to Combat Multidrug-Resistant and Persistent Infectious Diseases.

Antibiotic failure is one of the most worrying health problems worldwide. We are currently facing an international crisis with several problematic facets: new antibiotics are no longer being discovered, resistance mechanisms are occurring in almost all clinical isolates of bacteria, and recurrent infections caused by persistent bacteria are hampering the successful treatment of infections. In this context, new anti-infectious strategies against multidrug-resistant (MDR) and persistent bacteria, as well as the rescue of Food and Drug Administration (FDA)-approved compounds (drug repurposing), are being explored. Among the highlighted new anti-infectious strategies, in this review, we focus on antimicrobial peptides, anti-virulence compounds, phage therapy, and new molecules. As drugs that are being repurposed, we highlight anti-inflammatory compounds, anti-psychotics, anti-helminthics, anti-cancerous drugs, and statins.

Combined Use of the Ab105-2φΔCI Lytic Mutant Phage and Different Antibiotics in Clinical Isolates of Multi-Resistant Acinetobacter baumannii.

Phage therapy is an abandoned antimicrobial therapy that has been resumed in recent years. In this study, we mutated a lysogenic phage from Acinetobacter baumannii into a lytic phage (Ab105-2phiΔCI) that displayed antimicrobial activity against A. baumannii clinical strain Ab177_GEIH-2000 (isolated in the GEIH-REIPI Spanish Multicenter A. baumannii Study II 2000/2010, Umbrella Genbank Bioproject PRJNA422585, and for which meropenem and imipenem MICs of respectively, 32 µg/mL, and 16 µg/mL were obtained). We observed an in vitro synergistic antimicrobial effect (reduction of 4 log-7 log CFU/mL) between meropenem and the lytic phage in all combinations analyzed (Ab105-2phiΔCI mutant at 0.1, 1 and 10 MOI and meropenem at 1/4 and 1/8 MIC). Moreover, bacterial growth was reduced by 8 log CFU/mL for the combination of imipenem at 1/4 MIC plus lytic phage (Ab105-2phiΔCI mutant) and by 4 log CFU/mL for the combination of imipenem at 1/8 MIC plus lytic phage (Ab105-2phiΔCI mutant) at both MOI 1 and 10. These results were confirmed in an in vivo model (G. mellonella), and the combination of imipenem and mutant Ab105-2phiΔCI was most effective (p < 0.05). This approach could help to reduce the emergence of phage resistant bacteria and restore sensitivity to antibiotics used to combat multi-resistant strains of Acinetobacter baumannii.

Quorum and Light Signals Modulate Acetoin/Butanediol Catabolism in Acinetobacter spp.

Acinetobacter spp. are found in all environments on Earth due to their extraordinary capacity to survive in the presence of physical and chemical stressors. In this study, we analyzed global gene expression in airborne Acinetobacter sp. strain 5-2Ac02 isolated from hospital environment in response to quorum network modulators and found that they induced the expression of genes of the acetoin/butanediol catabolism, volatile compounds shown to mediate interkingdom interactions. Interestingly, the acoN gene, annotated as a putative transcriptional regulator, was truncated in the downstream regulatory region of the induced acetoin/butanediol cluster in Acinetobacter sp. strain 5-2Ac02, and its functioning as a negative regulator of this cluster integrating quorum signals was confirmed in Acinetobacter baumannii ATCC 17978. Moreover, we show that the acetoin catabolism is also induced by light and provide insights into the light transduction mechanism by showing that the photoreceptor BlsA interacts with and antagonizes the functioning of AcoN in A. baumannii, integrating also a temperature signal. The data support a model in which BlsA interacts with and likely sequesters AcoN at this condition, relieving acetoin catabolic genes from repression, and leading to better growth under blue light. This photoregulation depends on temperature, occurring at 23°C but not at 30°C. BlsA is thus a dual regulator, modulating different transcriptional regulators in the dark but also under blue light, representing thus a novel concept. The overall data show that quorum modulators as well as light regulate the acetoin catabolic cluster, providing a better understanding of environmental as well as clinical bacteria.

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 Quorum Sensing and Secretion Systems.

Quorum sensing (QS) is a communication mechanism between bacteria that allows specific processes to be controlled, such as biofilm formation, virulence factor expression, production of secondary metabolites and stress adaptation mechanisms such as bacterial competition systems including secretion systems (SS). These SS have an important role in bacterial communication. SS are ubiquitous; they are present in both Gram-negative and Gram-positive bacteria and in Mycobacterium sp. To date, 8 types of SS have been described (T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, and T9SS). They have global functions such as the transport of proteases, lipases, adhesins, heme-binding proteins, and amidases, and specific functions such as the synthesis of proteins in host cells, adaptation to the environment, the secretion of effectors to establish an infectious niche, transfer, absorption and release of DNA, translocation of effector proteins or DNA and autotransporter secretion. All of these functions can contribute to virulence and pathogenesis. In this review, we describe the known types of SS and discuss the ones that have been shown to be regulated by QS. Due to the large amount of information about this topic in some pathogens, we focus mainly on Pseudomonas aeruginosa and Vibrio spp.

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.

Relationship Between the Quorum Network (Sensing/Quenching) and Clinical Features of Pneumonia and Bacteraemia Caused by A. baumannii.

Acinetobacter baumannii (Ab) is one of the most important pathogens associated with nosocomial infections, especially pneumonia. Interest in the Quorum network, i.e., Quorum Sensing (QS)/Quorum Quenching (QQ), in this pathogen has grown in recent years. The Quorum network plays an important role in regulating diverse virulence factors such as surface motility and bacterial competition through the type VI secretion system (T6SS), which is associated with bacterial invasiveness. In the present study, we investigated 30 clinical strains of A. baumannii isolated in the "II Spanish Study of A. baumannii GEIH-REIPI 2000-2010" (Genbank Umbrella Bioproject PRJNA422585), a multicentre study describing the relationship between the Quorum network in A. baumannii and the development of pneumonia and associated bacteraemia. Expression of the aidA gene (encoding the AidA protein, QQ enzyme) was lower (P < 0.001) in strains of A. baumannii isolated from patients with bacteraemic pneumonia than in strains isolated from patients with non-bacteraemic pneumonia. Moreover, aidA expression in the first type of strain was not regulated in the presence of environmental stress factors such as the 3-oxo-C12-HSL molecule (substrate of AidA protein, QQ activation) or H2O2 (inhibitor of AidA protein, QS activation). However, in the A. baumannii strains isolated from patients with non-bacteraemic pneumonia, aidA gene expression was regulated by stressors such as 3-oxo-C12-HSL and H2O2. In an in vivo Galleria mellonella model of A. baumannii infection, the A. baumannii ATCC 17978 strain was associated with higher mortality (100% at 24 h) than the mutant, abaI-deficient, strain (carrying a synthetase enzyme of Acyl homoserine lactone molecules) (70% at 24 h). These data suggest that the QS (abaR and abaI genes)/QQ (aidA gene) network affects the development of secondary bacteraemia in pneumonia patients and also the virulence of A. baumannii.