11 research outputs found
Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection
[Abstract] 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.This study was funded by grants PI16/01163 and PI19/00878 awarded to MT within the State Plan for R + D + I 2013–2016 (National Plan for Scientific Research, Technological Development and Innovation 2008–2011) and co-financed by the ISCIII-Deputy General Directorate of evaluation and Promotion of Research-European Regional Development Fund “A way of Making Europe” and Instituto de Salud Carlos III FEDER. MT was financially supported by the Miguel Servet Research Programme (SERGAS and ISCIII
Molecular studies of phages-Klebsiella pneumoniae in mucoid environment: innovative use of mucolytic agents prior to the administration of lytic phages
Mucins are important glycoproteins that form a protective layer throughout the gastrointestinal and respiratory tracts. There is scientific evidence of increase in phage-resistance in the presence of mucin for some bacterial pathogens. Manipulation in mucin composition may ultimately influence the effectiveness of phage therapy. In this work, two clinical strains of K. pneumoniae (K3574 and K3325), were exposed to the lytic bacteriophage vB_KpnS-VAC35 in the presence and absence of mucin on a long-term co-evolution assay, in an attempt to mimic in vitro the exposure to mucins that bacteria and their phages face in vivo. Enumerations of the bacterial and phage counts at regular time intervals were conducted, and extraction of the genomic DNA of co-evolved bacteria to the phage, the mucin and both was performed. We determined the frequency of phage-resistant mutants in the presence and absence of mucin and including a mucolytic agent (N-acetyl L-cysteine, NAC), and sequenced them using Nanopore. We phenotypically demonstrated that the presence of mucin induces the emergence of bacterial resistance against lytic phages, effectively decreased in the presence of NAC. In addition, the genomic analysis revealed some of the genes relevant to the development of phage resistance in long-term co-evolution, with a special focus on the mucoid environment. Genes involved in the metabolism of carbohydrates were mutated in the presence of mucin. In conclusion, the use of mucolytic agents prior to the administration of lytic phages could be an interesting therapeutic option when addressing K. pneumoniae infections in environments where mucin is overproduced.The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study has been funded by Instituto de Salud Carlos III (ISCIII) through the projects PI19/00878 and PI22/00323 and co-funded by the European Union, and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC). (SEIMC, http://www.seimc.org/). This research was also supported by CIBERINFEC (CIBER21/13/00095) and by Personalized and precision medicine grant from the Instituto de Salud Carlos III (MePRAM Project, PMP22/00092). MT was financially supported by the Miguel Servet Research Programme (SERGAS and ISCIII). OP, LF-G, and ML were financially supported by the grants IN606A-2020/035, IN606B-2021/013, and IN606C-2022/002, respectively (GAIN, Xunta de Galicia). IB was financially supported by the pFIS program (ISCIII, FI20/00302). Finally, to thank to PIRASOA laboratory which is the reference laboratory for molecular typing of nosocomial pathogens and detection of mechanisms of resistance to antimicrobials of health interest in Andalusia, Virgen Macarena Hospital, Seville, to send us the clinical isolates. Thanks to Alvaro Pascual and Luis Martínez-Martínez from Virgen Macarena Hospital, Seville and Reina Sofia Hospital, Cordoba.S
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.This study was funded by grant PI19/00878 and PI22/00323 awarded to M.T. within the State Plan for R1D1I 2013-2016 (National Plan for Scientific Research, Technological Development, and Innovation 2008-2011) and cofinanced by the ISCIII-Deputy General Directorate for Evaluation and Promotion of Research/European Regional Development Fund “A Way of Making Europe” and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI; RD16/0016/0006 and RD16/0016/0008), CIBERINFEC (CIBER21/13/00012, CB21/13/00049, CIBER21/13/00084, and CIBER21/13/00095), and Personalized Medicine Project (MePRAM; PMP/00092) and also by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC; http://www.seimc.org/). M.T. was financially supported by the Miguel Servet Research Program (SERGAS and ISCIII). I.B. was financially supported by pFIS program (ISCIII, FI20/00302). O.P., L.F.-G., and M.L. were financially supported by grants IN606A-2020/035, IN606B-2021/013, and IN606C-2022/002, respectively (GAIN; Xunta de Galicia). The authors acknowledge CESGA (www.cesga.es) in Santiago de Compostela, Spain, for providing access to computing facilities and the RIAIDT-USC analytical facilities. Finally, We thank researchers from the Spanish Network of Bacteriophages and Transducer Elements (FAGOMA) for contributing the lytic phages. I.B., L.B., O.P., and L.F.-G. developed the experiments, analyzed the results, and wrote the original manuscript. M.L., C.O.C. and A.B.P. helped to prepare the visual presentation of the results. F.F.C., Á.P., L.M.-M., and J.O.-I. rewrote the manuscript. M.T. financed and directed the experiments and supervised the writing of the originalmanuscript. We declare that there are no conflicts of interest.S
Phenotypic and Genomic Comparison of Klebsiella pneumoniae Lytic Phages: vB_KpnM-VAC66 and vB_KpnM-VAC13
[Abstract] 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.This study was funded by grants PI19/00878 awarded to M. Tomás within the State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological Development and Innovation 2008–2011) and co-financed by the ISCIII-Deputy General Directorate for Evaluation and Promotion of Research—European Regional Development Fund “A way of Making Europe” and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI, RD16/0016/0001, RD16/0016/0006 and RD16/CIII/0004/0002) and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC, http://www.seimc.org/ accessed on 1 February 2021) and project PID2020-112835RA-I00 funded by MCIN/AEI /10.13039/501100011033, and project SEJIGENT/2021/014 funded by Conselleria d’Innovació, Universitats, Ciència i Societat Digital (Generalitat Valenciana). M. Tomás was financially supported by the Miguel Servet Research Programme (SERGAS and ISCIII). O. Pacios, L. Fernández-García and M. López were financially supported by the grants IN606A-2020/035, IN606B-2021/013 and IN606B-2018/008, respectively (GAIN, Xunta de Galicia). I. Bleriot was financially supported by pFIS program (ISCIII, FI20/00302). P. Domingo-Calap was financially supported by a Ramón y Cajal contract RYC2019-028015-I funded by MCIN/AEI /10.13039/501100011033, ESF Invest in your futureGeneralitat Valenciana; SEJIGENT/2021/014Xunta de Galicia; IN606A-2020/035Xunta de Galicia; IN606B-2021/013Xunta de Galicia; IN606B-2018/00
Adaptation of clinical isolates of Klebsiella pneumoniae to the combination of niclosamide with the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PaβN): 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-β-naphthylamide (PaβN).
Methods: Niclosamide and PaβN 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 PaβN (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 PaβN 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 PaβN. 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
The Role of PemIK (PemK/PemI) Type II TA System from Klebsiella pneumoniae Clinical Strains in Lytic Phage Infection
[Abstract] 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.This study was funded by grant PI19/00878 awarded to M. Tomás within the State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological Development and Innovation 2008-2011) and co-financed by the ISCIII-Deputy General Directorate for Evaluation and Promotion of Research—European Regional Development Fund "A way of Making Europe" and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI, RD16/0016/0001, RD16/0016/0006 and RD16/CIII/0004/0002) and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC, http://www.seimc.org/). M. Tomás was financially supported by the Miguel Servet Research Programme (SERGAS and ISCIII). I. Bleriot was financially supported by pFIS program (ISCIII, FI20/00302). O. Pacios and M. López was financially supported by a grant IN606A-2020/035 and IN606B-2018/008, respectively (GAIN, Xunta de Galicia) and M. Gonzalez-Bardanca was financially supported by the Rio Hortega program (ISCIII, CM20/00198)Xunta de Galicia; IN606A-2020/035Xunta de Galicia; IN606B-2018/00
Reverse Transcription-Loop-Mediated Isothermal Amplification- CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2
At the end of 2019, a new coronavirus, severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in
more than 6.2 million deaths. In the last couple of years, researchers have made great
efforts to develop a diagnostic technique that maintains high levels of sensitivity and
specificity, since an accurate and early diagnosis is required to minimize the prevalence
of SARS-CoV-2 infection. In this context, CRISPR-Cas systems are proposed as promising
tools for development as diagnostic techniques due to their high specificity, highlighting
that Cas13 endonuclease discriminates single nucleotide changes and displays collateral
activity against single-stranded RNA molecules. With the aim of improving the sensitivity
of diagnosis, this technology is usually combined with isothermal preamplification reactions
(SHERLOCK, DETECTR). Based on this, we developed a reverse transcription-loopmediated
isothermal amplification (RT-LAMP)-CRISPR-Cas13a method for SARS-CoV-2
virus detection in nasopharyngeal samples without using RNA extraction that exhibits
100% specificity and 83% sensitivity, as well as a positive predictive value (PPV) of 100%
and negative predictive values (NPVs) of 100%, 81%, 79.1%, and 66.7% for cycle threshold
(CT) values of ,20, 20 to 30, .30 and overall, respectively
Prophage identification and molecular analysis in the genomes of Pseudomonas aeruginosa strains isolated from critical care patients
Prophages are bacteriophages integrated into the bacterial host’s chromosome. This research aims to analyze and characterize the existing prophages within a collection of 53 Pseudomonas aeruginosa strains from intensive care units (ICUs) in Portugal and Spain. A total of 113 prophages were localized in the collection, with 18 of them being present in more than one strain simultaneously. After annotation, five of them were discarded as incomplete, and the 13 remaining prophages were characterized. Of 13, 10 belonged to the siphovirus tail morphology group, 2 to the podovirus tail morphology group, and 1 to the myovirus tail morphology group. All prophages had a length ranging from 20,199 to 63,401 bp and a GC% between 56.2%
and 63.6%. The number of open reading frames (ORFs) oscillated between 32 and 88, and in 3/13 prophages, more than 50% of the ORFs had an unknown function. With our findings, we show that prophages are present in the majority of the P. aeruginosa strains isolated from Portuguese and Spanish critically ill patients, many of them found in more than one circulating strain at the same time and following a similar clonal distribution pattern. Although a great sum of ORFs had an unknown function, number of proteins in relation to viral defense (anti-CRISPR proteins, toxin/antitoxin modules, proteins against restriction-modification systems) as well as to prophage interference into their host’s quorum sensing system and regulatory cascades were found. This supports the idea that prophages have an influence in bacterial pathogenesis and anti-phage defense
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
Data_Sheet_1_Molecular studies of phages-Klebsiella pneumoniae in mucoid environment: innovative use of mucolytic agents prior to the administration of lytic phages.PDF
Mucins are important glycoproteins that form a protective layer throughout the gastrointestinal and respiratory tracts. There is scientific evidence of increase in phage-resistance in the presence of mucin for some bacterial pathogens. Manipulation in mucin composition may ultimately influence the effectiveness of phage therapy. In this work, two clinical strains of K. pneumoniae (K3574 and K3325), were exposed to the lytic bacteriophage vB_KpnS-VAC35 in the presence and absence of mucin on a long-term co-evolution assay, in an attempt to mimic in vitro the exposure to mucins that bacteria and their phages face in vivo. Enumerations of the bacterial and phage counts at regular time intervals were conducted, and extraction of the genomic DNA of co-evolved bacteria to the phage, the mucin and both was performed. We determined the frequency of phage-resistant mutants in the presence and absence of mucin and including a mucolytic agent (N-acetyl L-cysteine, NAC), and sequenced them using Nanopore. We phenotypically demonstrated that the presence of mucin induces the emergence of bacterial resistance against lytic phages, effectively decreased in the presence of NAC. In addition, the genomic analysis revealed some of the genes relevant to the development of phage resistance in long-term co-evolution, with a special focus on the mucoid environment. Genes involved in the metabolism of carbohydrates were mutated in the presence of mucin. In conclusion, the use of mucolytic agents prior to the administration of lytic phages could be an interesting therapeutic option when addressing K. pneumoniae infections in environments where mucin is overproduced.</p