Biology subjects

Fernandez-Garcia, L.

Publications and source records attributed to Fernandez-Garcia, L..

8 recordsLinked to original sources

bioRxiv2023

Phages Produce Persisters

Bacteria primarily encounter stress, and, arguably, their greatest threats are phages. It is often assumed that those bacteria that escape phage attack have mutated; however, another possibility is that a subpopulation forms the dormant persister state, in a manner similar to that demonstrated for bacterial cells undergoing nutritive, oxidative, and antibiotic stress. Persister cells do not undergo mutation and survive lethal conditions by ceasing growth transiently. Slower growth and dormancy play a key physiological role as they allow host phage defense systems more time to clear the phage infection. Here we investigated how bacteria survive lytic phage infection by isolating surviving cells from the plaques of T2, T4, and lambda (cI mutant) virulent phages. We found that bacteria in plaques can escape phage attack both by mutation (i.e., become resistant) and without mutation (i.e., become persistent). Specifically, whereas T4-resistant and lambda-resistant bacteria with over a 100,000-fold less sensitivity were isolated from plaques with obvious genetic mutations (e.g., causing mucoidy), cells were also found after T2 infection that undergo no significant mutation, retain wild-type phage sensitivity, and survive lethal doses of antibiotics. Corroborating this, adding T2 phage to persister cells resulted in 137,000-fold more survival compared to that of addition to exponentially-growing cells. Phage treatments with Klebsiella pneumonia and Pseudomonas aeruginosa also generated persister cells. Hence, along with resistant strains, bacteria also form persister cells during phage infection.

microbiology↗

bioRxiv2023

Single-Cell Analysis Reveals 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 this 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 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.

molecular biology↗

bioRxiv2023

Molecular Studies of Phages-Klebsiella pneumoniae in a 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 these conditions 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.

microbiology↗

bioRxiv2023

In vitro and in vivo combination of lytic phages and octapeptin OPX10053 against B-lactamase-producing clinical isolates of Klebsiella pneumoniae

Backgroundnovel approaches to treat Klebsiella pneumoniae infections are desperately needed, such as the use of rationally designed combination therapies. Objectivesto evaluate the in vitro and in vivo therapeutic potential of lytic phages against K. pneumoniae in combination with octapeptin, a promising class of lipopeptides with broad spectrum Gram-negative activity. Methodswe determined the MICs to twenty-two lipopeptide compounds and chose one octapeptin (OPX10053) for evaluation of potential synergism in combination with lytic phages using checkerboard assays, optical density growth curves and time-kill (CFU enumeration). Toxicity and efficacy in vivo assays were conducted on Galleria mellonella larvae. Resultsthis study reports the synergy found in vitro between the octapeptin OPX10053 and two lytic phages previously characterized by our research group (vB_KpnM-VAC13 and vB_KpnM-VAC66) against clinical isolates of K. pneumoniae. This synergy was validated by the FIC index, OD growth curves and time-kill assay when OPX10053 was added following 4 hours of phage exposure. Preliminary evaluation of toxicity revealed that OPX10053, even at subinhibitory concentrations and in phage combinations, exerts a toxic effect on larvae, which requires further investigation. ConclusionsThe in vitro application of lytic phages in combination with octapeptin OPX10053 showed synergistic activity. Exposure of G. mellonella to the lytic phages was well tolerated, whereas combination treatment with subinhibitory concentrations of OPX10053 did not attenuate toxicity. Even so, this innovative approach of combining lytic phages could open the door to some interesting associations between chemically synthesized drugs and biological entities. Sequential or simultaneous application alongside time, dosing and stewardship warrants further research.

microbiology↗

bioRxiv2023

Toxin/Antitoxin Systems Induce Persistence and Work in Concert with Restriction/Modification Systems to Inhibit Phage

Myriad bacterial anti-phage systems have been described and often the mechanism of programmed cell death is invoked for phage inhibition. However, there is little evidence of suicide under physiological conditions for these systems. Instead of death to stop phage propagation, we show here that persister cells, i.e., transiently-tolerant, dormant, antibiotic-insensitive cells, are formed and survive using the Escherichia coli C496_10 tripartite toxin/antitoxin system MqsR/MqsA/MqsC to inhibit T2 phage. Specifically, MqsR/MqsA/MqsC inhibited T2 phage by one million-fold and reduced T2 titers by 500-fold. During T2 phage attack, in the presence of MqsR/MqsA/MqsC, evidence of persistence include the single-cell physiological change of reduced metabolism (via flow cytometry), increased spherical morphology (via transmission electron microscopy), and heterogeneous resuscitation. Critically, we found restriction-modification systems (primarily EcoK McrBC) work in concert with the toxin/antitoxin system to inactivate phage, likely while the cells are in the persister state. Phage attack also induces persistence in Klebsiella and Pseudomonas spp. Hence, phage attack invokes a stress response similar to antibiotics, starvation, and oxidation, which leads to persistence, and this dormant state likely allows restriction/modification systems to clear phage DNA.

molecular biology↗

bioRxiv2022

Molecular analysis of the interactions between phages and the bacterial host Klebsiella pneumoniae

Lytic phages are currently considered among the best options for treating infections caused by multi-drug resistant pathogens. Phages have some advantages over conventional antibiotics. For example, phages acquire modifications in accordance with their environment, and thus with the bacteria present, which has led to the co-evolution of both types of organism. Therefore, both 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 twenty-one novel lytic phages of Klebsiella pneumoniae in search of defence mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defence mechanisms of two clinical isolates of Klebsiella pneumoniae infected by phages. For this purpose, the twenty-one lytic phages were sequenced and de novo assembled using the Illumina-Miseq system and Spades V.3.15.2 respectively. Gene annotation was performed with Patric, Blast, Hhmer and Hhpred tools. The evolutionary relationships between phages were determined by RaxML. The host-range was determined in a collection of forty-seven clinical isolates of K. pneumoniae, revealing the variable infectivity capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the family Caudovirales. The size and GC content of the phages ranged from 39,371 to 178,532 bp and from 41.72 % to 53.76 %, respectively. 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 defence mechanisms against bacteria, including the restriction-modification (RM) system, the toxin-antitoxin (TA) system, evasion of DNA degradation, blocking of host RM, 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 defence mechanisms against phage infection (prophage, plasmid, defence/virulence/resistance and oxidative stress proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages. IMPORTANCEPhages, viral parasites of bacteria, have long protected the Earths biosphere against bacterial overgrowth and could now help in the fight against antimicrobial resistance. However, researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about the defence mechanisms in both viruses and bacteria. In this study, we analyzed the molecular mechanisms of viral and bacterial defence in phages infecting clinical isolates of Klebsiella pneumoniae. Viral defence mechanisms included RM system evasion, the Toxin-Antitoxin system, DNA degradation evasion, blocking of host RM and resistance to the abortive infection system (Abi), anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defence mechanisms, proteomic analysis revealed overexpression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defence/virulence/resistance (porins, efflux pumps, LPS, pili 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.

microbiology↗

bioRxiv2022

Application of RT-LAMP-CRISPR-Cas13a technology to the detection of OXA-48 producing Klebsiella pneumoniae

Carbapenem-resistant pathogens have been recognized as a health concern because of their ability to cause severe infections and because they are difficult to detect in laboratories. Researchers are making great efforts to develop a diagnostic technique with high levels of sensitivity and specificity, as accurate, early diagnosis is required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic techniques due to their high specificity: Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. This technology is usually combined with isothermal pre-amplification reactions in order to increase the sensitivity of diagnosis. We have developed an RT-LAMP-CRISPR-Cas13a-based assay for the detection of Klebsiella pneumoniae OXA-48 producer strains in clinical samples without the need for RNA extraction. The assay exhibited 100 % specificity, sensitivity, positive predictive value and negative predictive value.

microbiology↗

bioRxiv2022

Molecular characteristics of phages located in Carbapenemase-Producing Escherichia coli clinical isolates: New Phage-Like Plasmids

Escherichia coli normally inhabits the gastrointestinal tract of humans and animals. Most E. coli bacteria do not cause problems, but the acquisition of different resistance and virulence genes encoded by mobile plasmids or phages by different bacterial isolates has been associated with the appearance of successful high-risk clones of multidrug-resistant (MDR) E. coli such as ST131 or ST405. In the present study, 50 temperate bacteriophages present in 21 clinical isolates of carbapenemase-producing E. coli of sequence types (STs) ST38, ST131, ST167, ST405 and ST410 were analysed. These phages were classified in the three families of the order Caudovirales: 24 within the family Siphoviridae, 23 in Myoviridae and 3 in Podoviridae. The size of the phages studied ranged from 11 to 95 Kb. Phylogenetic analysis of the terminase large subunit allowed us to classify these phages into different groups showing similarity with the phage sequences deposited in the Microbe Versus Phage (MVP) database and which belonged to clusters 229, 604, 2503 and 2725. On the other hand, bioinformatic study revealed that most of the identified proteins exerted a structural function (26.73%) but also functions involved in lysis/lysogeny (6.70%) or regulation (5.20%) among others. In addition, the ParA-ParB partitioning system and the type II toxin-antitoxin Phd-Doc system were also found in two of the phages studied, which could indicate the presence of plasmid-prophages. Host range testing revealed that two isolates were more susceptible to infection than the other isolates. IMPORTANCEEscherichia coli is one of the pathogens that causes most problems in human health, as it presents multiple resistances to different antibiotics. The study of bacteriophages located in different isolates of this species is important for the development of new anti-infective therapies. Currently, antibiotic resistance is a major problem, but more and more studies are pointing to experimental treatments with bacteriophages as a possible solution.

bioinformatics↗