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973401.0000Combination of genetically diverse Pseudomonas phages enhances the cocktail efficiency against bacteria. Phage treatment has been used as an alternative to antibiotics since the early 1900s. However, bacteria may acquire phage resistance quickly, limiting the use of phage treatment. The combination of genetically diverse phages displaying distinct replication machinery in phage cocktails has therefore become a novel strategy to improve therapeutic outcomes. Here, we isolated and studied lytic phages (SPA01 and SPA05) that infect a wide range of clinical Pseudomonas aeruginosa isolates. These relatively small myophages have around 93 kbp genomes with no undesirable genes, have a 30-min latent period, and reproduce a relatively high number of progenies, ranging from 218 to 240 PFU per infected cell. Even though both phages lyse their hosts within 4 h, phage-resistant bacteria emerge during the treatment. Considering SPA01-resistant bacteria cross-resist phage SPA05 and vice versa, combining SPA01 and SPA05 for a cocktail would be ineffective. According to the decreased adsorption rate of the phages in the resistant isolates, one of the anti-phage mechanisms may occur through modification of phage receptors on the target cells. All resistant isolates, however, are susceptible to nucleus-forming jumbophages (PhiKZ and PhiPA3), which are genetically distinct from phages SPA01 and SPA05, suggesting that the jumbophages recognize a different receptor during phage entry. The combination of these phages with the jumbophage PhiKZ outperforms other tested combinations in terms of bactericidal activity and effectively suppresses the emergence of phage resistance. This finding reveals the effectiveness of the diverse phage-composed cocktail for reducing bacterial growth and prolonging the evolution of phage resistance.202337264114
836910.9990Phage-resistant Pseudomonas aeruginosa against a novel lytic phage JJ01 exhibits hypersensitivity to colistin and reduces biofilm production. Pseudomonas aeruginosa, a major cause of nosocomial infections, has been categorized by World Health Organization as a critical pathogen urgently in need of effective therapies. Bacteriophages or phages, which are viruses that specifically kill bacteria, have been considered as alternative agents for the treatment of bacterial infections. Here, we discovered a lytic phage targeting P. aeruginosa, designated as JJ01, which was classified as a member of the Myoviridae family due to the presence of an icosahedral capsid and a contractile tail under TEM. Phage JJ01 requires at least 10 min for 90% of its particles to be adsorbed to the host cells and has a latent period of 30 min inside the host cell for its replication. JJ01 has a relatively large burst size, which releases approximately 109 particles/cell at the end of its lytic life cycle. The phage can withstand a wide range of pH values (3-10) and temperatures (4-60°C). Genome analysis showed that JJ01 possesses a complete genome of 66,346 base pairs with 55.7% of GC content, phylogenetically belonging to the genus Pbunavirus. Genome annotation further revealed that the genome encodes 92 open reading frames (ORFs) with 38 functionally predictable genes, and it contains neither tRNA nor toxin genes, such as drug-resistant or lysogenic-associated genes. Phage JJ01 is highly effective in suppressing bacterial cell growth for 12 h and eradicating biofilms established by the bacteria. Even though JJ01-resistant bacteria have emerged, the ability of phage resistance comes with the expense of the bacterial fitness cost. Some resistant strains were found to produce less biofilm and grow slower than the wild-type strain. Among the resistant isolates, the resistant strain W10 which notably loses its physiological fitness becomes eight times more susceptible to colistin and has its cell membrane compromised, compared to the wild type. Altogether, our data revealed the potential of phage JJ01 as a candidate for phage therapy against P. aeruginosa and further supports that even though the use of phages would subsequently lead to the emergence of phage-resistant bacteria, an evolutionary trade-off would make them more sensitive to antibiotics.202236274728
975520.9990Phages for treatment Pseudomonas aeruginosa infection. Pseudomonas aeruginosa is denoted as one of the highly threatening bacteria to the public health. It has acquired many virulent factors and resistant genes that make it difficult to control with conventional antibiotics. Thus, bacteriophage therapy (phage therapy) is a proposed alternative to antibiotics to fight against multidrug-resistant P. aeruginosa. Many phages have been isolated that exhibit a broad spectrum of activity against P. aeruginosa. In this chapter, the common virulent factors and the prevalence of antibiotic-resistance genes in P. aeruginosa were reported. In addition, recent efforts in the field of phage therapy against P. aeruginosa were highlighted, including wild-type phages, genetically modified phages, phage cocktails, and phage in combination with antibiotics against P. aeruginosa in the planktonic and biofilm forms. Recent regulations on phage therapy were also covered in this chapter.202337770166
975630.9989Genomewide identification of genetic determinants of antimicrobial drug resistance in Pseudomonas aeruginosa. The emergence of antimicrobial drug resistance is of enormous public concern due to the increased risk of delayed treatment of infections, the increased length of hospital stays, the substantial increase in the cost of care, and the high risk of fatal outcomes. A prerequisite for the development of effective therapy alternatives is a detailed understanding of the diversity of bacterial mechanisms that underlie drug resistance, especially for problematic gram-negative bacteria such as Pseudomonas aeruginosa. This pathogen has impressive chromosomally encoded mechanisms of intrinsic resistance, as well as the potential to mutate, gaining resistance to current antibiotics. In this study we have screened the comprehensive nonredundant Harvard PA14 library for P. aeruginosa mutants that exhibited either increased or decreased resistance against 19 antibiotics commonly used in the clinic. This approach identified several genes whose inactivation sensitized the bacteria to a broad spectrum of different antimicrobials and uncovered novel genetic determinants of resistance to various classes of antibiotics. Knowledge of the enhancement of bacterial susceptibility to existing antibiotics and of novel resistance markers or modifiers of resistance expression may lay the foundation for effective therapy alternatives and will be the basis for the development of new strategies in the control of problematic multiresistant gram-negative bacteria.200919332674
885740.9989Colistin-phage combinations decrease antibiotic resistance in Acinetobacter baumannii via changes in envelope architecture. Multidrug-resistant bacterial infections are becoming increasingly common, with only few last-resort antibiotics such as colistin available for clinical therapy. An alternative therapeutic strategy gaining momentum is phage therapy, which has the advantage of not being affected by bacterial resistance to antibiotics. However, a major challenge in phage therapy is the rapid emergence of phage-resistant bacteria. In this work, our main aim was to understand the mechanisms of phage-resistance used by the top priority pathogen Acinetobacter baumannii. We isolated the novel phage Phab24, capable of infecting colistin-sensitive and -resistant strains of A. baumannii. After co-incubating Phab24 with its hosts, we obtained phage-resistant mutants which were characterized on both genotypic and phenotypic levels. Using whole genome sequencing, we identified phage-resistant strains that displayed mutations in genes that alter the architecture of the bacterial envelope at two levels: the capsule and the outer membrane. Using an adsorption assay, we confirmed that phage Phab24 uses the bacterial capsule as its primary receptor, with the outer membrane possibly serving as the secondary receptor. Interestingly, the phage-resistant isolates were less virulent compared to the parental strains in a Galleria mellonella infection model. Most importantly, we observed that phage-resistant bacteria that evolved in the absence of antibiotics exhibited an increased sensitivity to colistin, even though the antibiotic resistance mechanism per se remained unaltered. This increase in antibiotic sensitivity is a direct consequence of the phage-resistance mechanism, and could potentially be exploited in the clinical setting.202134736365
884650.9989Phage Resistance Accompanies Reduced Fitness of Uropathogenic Escherichia coli in the Urinary Environment. Urinary tract infection (UTI) is among the most common infections treated worldwide each year and is caused primarily by uropathogenic Escherichia coli (UPEC). Rising rates of antibiotic resistance among uropathogens have spurred a consideration of alternative treatment strategies, such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, namely, HP3 and ES17, against clinical UPEC isolates using in vitro and in vivo models of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6 to 8 h of coincubation. Whole-genome sequencing revealed that UPEC strains resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. Phage-resistant strains displayed several in vitro phenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells and increased biofilm formation in some isolates. Interestingly, these phage-resistant UPEC isolates demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation and were more sensitive to several outer membrane-targeting antibiotics than parental strains. Additionally, phage-resistant UPEC isolates were attenuated in bladder colonization in a murine UTI model. In total, our findings suggest that while resistance to phages, such as HP3 and ES17, may arise readily in the urinary environment, phage resistance is accompanied by fitness costs which may render UPEC more susceptible to host immunity or antibiotics. IMPORTANCE UTI is one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control UTI unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest; however, much like with antibiotics, bacteria can readily become resistant to phages. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage resistance during bacterial infection. In our current study, we found that while phage-resistant bacteria quickly emerged in vitro, these bacteria were less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, of which each is evaded only at the cost of bacterial fitness.202235920561
886460.9989Resistance, mechanism, and fitness cost of specific bacteriophages for Pseudomonas aeruginosa. The bacteriophage is an effective adjunct to existing antibiotic therapy; however, in the course of bacteriophage therapy, host bacteria will develop resistance to bacteriophages, thus affecting the efficacy. Therefore, it is important to describe how bacteria evade bacteriophage attack and the consequences of the biological changes that accompany the development of bacteriophage resistance before the bacteriophage is applied. The specific bacteriophage vB3530 of Pseudomonas aeruginosa (P. aeruginosa) has stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. Ten bacteriophage-resistant strains (TL3780-R) were induced using the secondary infection approach, and the plaque assay showed that vB3530 was less sensitive to TL3780-R. Identification of bacteriophage adsorption receptors showed that the bacterial surface polysaccharide was probably the adsorption receptor of vB3530. In contrast to the TL3780 parental strain, TL3780-R is characterized by the absence of long lipopolysaccharide chains, which may be caused by base insertion of wzy or deletion of galU. It is also intriguing to observe that, in comparison to the parent strain, the bacteriophage-resistant strains TL3780-R mostly exhibited a large cost of fitness (growth rate, biofilm formation, motility, and ability to produce enhanced pyocyanin). In addition, TL3780-R9 showed increased susceptibility to aminoglycosides and chlorhexidine, which may be connected to the loss and down-regulation of mexX expression. Consequently, these findings fully depicted the resistance mechanism of P. aeruginosa to vB3530 and the fitness cost of bacteriophage resistance, laying a foundation for further application of bacteriophage therapy.IMPORTANCEThe bacteriophage is an effective adjunct to existing antibiotic therapy; However, bacteria also develop defensive mechanisms against bacteriophage attack. Thus, there is an urgent need to deeply understand the resistance mechanism of bacteria to bacteriophages and the fitness cost of bacteriophage resistance so as to lay the foundation for subsequent application of the phage. In this study, a specific bacteriophage vB3530 of P. aeruginosa had stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. In addition, we found that P. aeruginosa may lead to phage resistance due to the deletion of galU and the base insertion of wzy, involved in the synthesis of lipopolysaccharides. Simultaneously, we showed the association with the biological state of the bacteria after bacteria acquire bacteriophage resistance, which is extremely relevant to guide the future application of therapeutic bacteriophages.202438299825
973670.9988Coevolutionary phage training leads to greater bacterial suppression and delays the evolution of phage resistance. The evolution of antibiotic-resistant bacteria threatens to become the leading cause of worldwide mortality. This crisis has renewed interest in the practice of phage therapy. Yet, bacteria's capacity to evolve resistance may debilitate this therapy as well. To combat the evolution of phage resistance and improve treatment outcomes, many suggest leveraging phages' ability to counter resistance by evolving phages on target hosts before using them in therapy (phage training). We found that in vitro, λtrn, a phage trained for 28 d, suppressed bacteria ∼1,000-fold for three to eight times longer than its untrained ancestor. Prolonged suppression was due to a delay in the evolution of resistance caused by several factors. Mutations that confer resistance to λtrn are ∼100× less common, and while the target bacterium can evolve complete resistance to the untrained phage in a single step, multiple mutations are required to evolve complete resistance to λtrn. Mutations that confer resistance to λtrn are more costly than mutations for untrained phage resistance. Furthermore, when resistance does evolve, λtrn is better able to suppress these forms of resistance. One way that λtrn improved was through recombination with a gene in a defunct prophage in the host genome, which doubled phage fitness. This transfer of information from the host genome is an unexpected but highly efficient mode of training phage. Lastly, we found that many other independently trained λ phages were able to suppress bacterial populations, supporting the important role training could play during phage therapeutic development.202134083444
441080.9988Refuse in order to resist: metabolic bottlenecks reduce antibiotic susceptibility. The growth of pathogenic bacteria in the host is a prerequisite for infectious diseases. Antibiotic drugs are used to impair bacterial growth and thereby treat infections. In turn, growth of bacteria is underpinned by their primary metabolism. Thus, it has long been recognized that the activity of antibiotics is determined by the metabolic state of cells. However, only recently researchers have begun to systematically interrogate the links between metabolism and resistance (Jiang et al, ; Lopatkin et al, ; Pinheiro et al, ; Schrader et al, ; Zhao et al, 2021). In their recent study, Lubrano and colleagues (Lubrano et al, 2025) apply an elegant CRISPR-based approach to the model bacterium Escherichia coli to systematically screen the effect of 15,120 mutations in genes that encode for 346 proteins which are required for growth of E. coli (also referred to as ‘essential proteins’). The authors identified a multitude of mutations that reduce the susceptibility against two antibiotics related to two very distinct chemical classes; the β-lactam antibiotic carbenicillin and the aminoglycoside gentamicin. Strikingly, the majority of the identified mutations are directly linked to primary metabolism. The work highlights the importance of metabolism in order to understand antibiotic resistance mechanisms and the ecology and evolution of antibiotic resistance. In addition, the work provides leads to design metabolism-based intervention strategies to mitigate antibiotic resistance.202539966554
975790.9988Effects of different mechanisms on antimicrobial resistance in Pseudomonas aeruginosa: a strategic system for evaluating antibiotics against gram-negative bacteria. Our previous studies constructed a strategic system for testing antibiotics against specific resistance mechanisms using Klebsiella pneumoniae and Acinetobacter baumannii. However, it lacked resistance mechanisms specifically expressed only in Pseudomonas species. In this study, we constructed this system using Pseudomonas aeruginosa. In-frame deletion, site-directed mutagenesis, and plasmid transformation were used to generate genetically engineered strains with various resistance mechanisms from two fully susceptible P. aeruginosa strains. Antimicrobial susceptibility testing was used to test the efficacy of antibiotics against these strains in vitro. A total of 31 engineered strains with various antimicrobial resistance mechanisms from P. aeruginosa KPA888 and ATCC 27853 were constructed, and the same antibiotic resistance mechanism showed a similar effect on the MICs of the two strains. Compared to the parental strains, the engineered strains lacking porin OprD or lacking the regulator genes of efflux pumps all showed a ≥4-fold increase on the MICs of some of the 19 antibiotics tested. Mechanisms due to GyrA/ParC mutations and β-lactamases also contributed to their corresponding resistance as previously published. The strains constructed in this study possess well-defined resistance mechanisms and can be used to screen and evaluate the effectiveness of antibiotics against specific resistance mechanisms in P. aeruginosa. Building upon our previous studies on K. pneumoniae and A. baumannii, this strategic system, including a P. aeruginosa panel, has been expanded to cover almost all the important antibiotic resistance mechanisms of gram-negative bacteria that are in urgent need of new antibiotics.IMPORTANCEIn this study, an antibiotic assessment system for P. aeruginosa was developed, and the system can be expanded to include other key pathogens and resistance mechanisms. This system offers several benefits: (i) compound design: aid in the development of compounds that can bypass or counteract resistance mechanisms, leading to more effective treatments against specific resistant strains; (ii) combination therapies: facilitate the exploration of combination therapies, where multiple antibiotics may work synergistically to overcome resistance and enhance treatment efficacy; and (iii) targeted treatments: enable healthcare providers to prescribe more targeted treatments, reducing unnecessary antibiotic use and helping to slow the spread of antibiotic resistance. In summary, this system could streamline the development process, reduce costs, increase the success rate of new antibiotics, and help prevent and control antimicrobial resistance.202540042282
8935100.9988The Molecular and Genetic Basis of Repeatable Coevolution between Escherichia coli and Bacteriophage T3 in a Laboratory Microcosm. The objective of this study was to determine the genomic changes that underlie coevolution between Escherichia coli B and bacteriophage T3 when grown together in a laboratory microcosm. We also sought to evaluate the repeatability of their evolution by studying replicate coevolution experiments inoculated with the same ancestral strains. We performed the coevolution experiments by growing Escherichia coli B and the lytic bacteriophage T3 in seven parallel continuous culture devices (chemostats) for 30 days. In each of the chemostats, we observed three rounds of coevolution. First, bacteria evolved resistance to infection by the ancestral phage. Then, a new phage type evolved that was capable of infecting the resistant bacteria as well as the sensitive bacterial ancestor. Finally, we observed second-order resistant bacteria evolve that were resistant to infection by both phage types. To identify the genetic changes underlying coevolution, we isolated first- and second-order resistant bacteria as well as a host-range mutant phage from each chemostat and sequenced their genomes. We found that first-order resistant bacteria consistently evolved resistance to phage via mutations in the gene, waaG, which codes for a glucosyltransferase required for assembly of the bacterial lipopolysaccharide (LPS). Phage also showed repeatable evolution, with each chemostat producing host-range mutant phage with mutations in the phage tail fiber gene T3p48 which binds to the bacterial LPS during adsorption. Two second-order resistant bacteria evolved via mutations in different genes involved in the phage interaction. Although a wide range of mutations occurred in the bacterial waaG gene, mutations in the phage tail fiber were restricted to a single codon, and several phage showed convergent evolution at the nucleotide level. These results are consistent with previous studies in other systems that have documented repeatable evolution in bacteria at the level of pathways or genes and repeatable evolution in viruses at the nucleotide level. Our data are also consistent with the expectation that adaptation via loss-of-function mutations is less constrained than adaptation via gain-of-function mutations.201526114300
9759110.9988Rapid emergence of resistance to broad-spectrum direct antimicrobial activity of avibactam. Avibactam (AVI) is a diazabicyclooctane (DBO) β-lactamase inhibitor used clinically in combination with ceftazidime. At concentrations higher than those typically achieved in vivo, it also has broad-spectrum direct antibacterial activity against Enterobacterales strains, including metallo-β-lactamase-producing isolates, mediated by inhibition of penicillin-binding protein 2 (PBP2). This activity has some mechanistic similarities to that of more potent novel DBOs (zidebactam and nacubactam) in late clinical development. We found that resistance to AVI emerged readily, with a mutation frequency of 2 × 10(-6) to 8 × 10(-5). Whole-genome sequencing of resistant isolates revealed a heterogeneous mutational target that permitted bacterial survival and replication despite PBP2 inhibition, in line with prior studies of PBP2-targeting drugs. While such mutations are believed to act by upregulating the bacterial stringent response, we found a similarly high mutation frequency in bacteria deficient in components of the stringent response, although we observed a different set of mutations in these strains. Although avibactam-resistant strains had increased lag time, suggesting a fitness cost that might render them less problematic in clinical infections, there was no statistically significant difference in growth rates between susceptible and resistant strains. The finding of rapid emergence of resistance to avibactam as the result of a large and complex mutational target adds to our understanding of resistance to PBP2-targeting drugs and has potential implications for novel DBOs with potent direct antibacterial activity, which are being developed with the goal of expanding cell wall-active treatment options for multidrug-resistant gram-negative infections.IMPORTANCEAvibactam (AVI) is the first in a class of novel β-lactamase inhibitor antibiotics called diazabicyclooctanes (DBOs). In addition to its ability to inhibit bacterial β-lactamase enzymes that can destroy β-lactam antibiotics, we found that AVI had direct antibacterial activity, at concentrations higher than those used clinically, against even highly multidrug-resistant bacteria. This activity is the result of inhibition of the bacterial enzyme penicillin-binding protein 2 (PBP2). Resistance to other drugs that inhibit PBP2 occurs through mutations that involve upregulation of the bacterial "stringent response" to stress. We found that bacteria developed resistance to AVI at a high rate, as a result of mutations in stringent response genes. We also found that bacteria with impairments in the stringent response could still develop resistance to AVI through different mutations. Our findings indicate the importance of studying how resistance will emerge to newer, more potent DBOs in development and early clinical use.202540503840
9103120.9988Development of cannabidiol derivatives as potent broad-spectrum antibacterial agents with membrane-disruptive mechanism. The emergence of antibiotic resistance has brought a significant burden to public health. Here, we designed and synthesized a series of cannabidiol derivatives by biomimicking the structure and function of cationic antibacterial peptides. This is the first report on the design of cannabidiol derivatives as broad-spectrum antibacterial agents. Through the structure-activity relationship (SAR) study, we found a lead compound 23 that killed both Gram-negative and Gram-positive bacteria via a membrane-targeting mechanism of action with low resistance frequencies. Compound 23 also exhibited very weak hemolytic activity, low toxicity toward mammalian cells, and rapid bactericidal properties. To further validate the membrane action mechanism of compound 23, we performed transcriptomic analysis using RNA-seq, which revealed that treatment with compound 23 altered many cell wall/membrane/envelope biogenesis-related genes in Gram-positive and Gram-negative bacteria. More importantly, compound 23 showed potent in vivo antibacterial efficacy in murine corneal infection models caused by Staphylococcus aureus or Pseudomonas aeruginosa. These findings would provide a new design idea for the discovery of novel broad-spectrum antibacterial agents to overcome the antibiotic resistance crisis.202438266554
9748130.9988Resistance in antimicrobial photodynamic inactivation of bacteria. Antibiotics have increasingly lost their impact to kill bacteria efficiently during the last 10 years. The emergence and dissemination of superbugs with resistance to multiple antibiotic classes have occurred among Gram-positive and Gram-negative strains including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter strains. These six superbugs can "escape" more or less any single kind of antibiotic treatment. That means bacteria are very good at developing resistance against antibiotics in a short time. One new approach is called photodynamic antimicrobial chemotherapy (PACT) which already has demonstrated an efficient antimicrobial efficacy among multi-resistant bacteria. Until now it has been questionable if bacteria can develop resistance against PACT. This perspective summarises the current knowledge about the susceptibility of bacteria towards oxidative stress and sheds some light on possible strategies of the development of photodynamic inactivation of bacteria (PACT)-induced oxidative stress resistance by bacteria.201526098395
8855140.9988Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in Escherichia coli O157. Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene (sspA) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens.IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication.201830042196
8853150.9988Collateral sensitivity increases the efficacy of a rationally designed bacteriophage combination to control Salmonella enterica. The ability of virulent bacteriophages to lyse bacteria influences bacterial evolution, fitness, and population structure. Knowledge of both host susceptibility and resistance factors is crucial for the successful application of bacteriophages as biological control agents in clinical therapy, food processing, and agriculture. In this study, we isolated 12 bacteriophages termed SPLA phage which infect the foodborne pathogen Salmonella enterica. To determine phage host range, a diverse collection of Enterobacteriaceae and Salmonella enterica was used and genes involved in infection by six SPLA phages were identified using Salmonella Typhimurium strain ST4/74. Candidate host receptors included lipopolysaccharide (LPS), cellulose, and BtuB. Lipopolysaccharide was identified as a susceptibility factor for phage SPLA1a and mutations in LPS biosynthesis genes spontaneously emerged during culture with S. Typhimurium. Conversely, LPS was a resistance factor for phage SPLA5b which suggested that emergence of LPS mutations in culture with SPLA1a represented collateral sensitivity to SPLA5b. We show that bacteria-phage co-culture with SPLA1a and SPLA5b was more successful in limiting the emergence of phage resistance compared to single phage co-culture. Identification of host susceptibility and resistance genes and understanding infection dynamics are critical steps in the rationale design of phage cocktails against specific bacterial pathogens.IMPORTANCEAs antibiotic resistance continues to emerge in bacterial pathogens, bacterial viruses (phage) represent a potential alternative or adjunct to antibiotics. One challenge for their implementation is the predisposition of bacteria to rapidly acquire resistance to phages. We describe a functional genomics approach to identify mechanisms of susceptibility and resistance for newly isolated phages that infect and lyse Salmonella enterica and use this information to identify phage combinations that exploit collateral sensitivity, thus increasing efficacy. Collateral sensitivity is a phenomenon where resistance to one class of antibiotics increases sensitivity to a second class of antibiotics. We report a functional genomics approach to rationally design a phage combination with a collateral sensitivity dynamic which resulted in increased efficacy. Considering such evolutionary trade-offs has the potential to manipulate the outcome of phage therapy in favor of resolving infection without selecting for escape mutants and is applicable to other virus-host interactions.202438376991
4271160.9988Multi-step vs. single-step resistance evolution under different drugs, pharmacokinetics, and treatment regimens. The success of antimicrobial treatment is threatened by the evolution of drug resistance. Population genetic models are an important tool in mitigating that threat. However, most such models consider resistance emergence via a single mutational step. Here, we assembled experimental evidence that drug resistance evolution follows two patterns: (i) a single mutation, which provides a large resistance benefit, or (ii) multiple mutations, each conferring a small benefit, which combine to yield high-level resistance. Using stochastic modeling, we then investigated the consequences of these two patterns for treatment failure and population diversity under various treatments. We find that resistance evolution is substantially limited if more than two mutations are required and that the extent of this limitation depends on the combination of drug type and pharmacokinetic profile. Further, if multiple mutations are necessary, adaptive treatment, which only suppresses the bacterial population, delays treatment failure due to resistance for a longer time than aggressive treatment, which aims at eradication.202134001313
6280170.9988Genomic variation in Pseudomonas aeruginosa clinical respiratory isolates with de novo resistance to a bacteriophage cocktail. Pseudomonas aeruginosa is an opportunistic pathogen that can cause sinus infections and pneumonia in cystic fibrosis (CF) patients. Bacteriophage therapy is being investigated as a treatment for antibiotic-resistant P. aeruginosa infections. Although virulent bacteriophages have shown promise in treating P. aeruginosa infections, the development of bacteriophage-insensitive mutants (BIMs) in the presence of bacteriophages has been described. The aim of this study was to examine the genetic changes associated with the BIM phenotype. Biofilms of three genetically distinct P. aeruginosa strains, including PAO1 (ATCC 15692), and two clinical respiratory isolates (one CF and one non-CF) were grown for 7 days and treated with either a cocktail of four bacteriophages or a vehicle control for 7 consecutive days. BIMs isolated from the biofilms were detected by streak assays, and resistance to the phage cocktail was confirmed using spot test assays. Comparison of whole genome sequencing between the recovered BIMs and their respective vehicle control-treated phage-sensitive isolates revealed structural variants in two strains, and several small variants in all three strains. These variations involved a TonB-dependent outer membrane receptor in one strain, and mutations in lipopolysaccharide synthesis genes in two strains. Prophage deletion and induction were also noted in two strains, as well as mutations in several genes associated with virulence factors. Mutations in genes involved in susceptibility to conventional antibiotics were also identified in BIMs, with both decreased and increased antibiotic sensitivity to various antibiotics being observed. These findings may have implications for future applications of lytic phage therapy.IMPORTANCELytic bacteriophages are viruses that infect and kill bacteria and can be used to treat difficult-to-treat bacterial infections, including biofilm-associated infections and multidrug-resistant bacteria. Pseudomonas aeruginosa is a bacterium that can cause life-threatening infections. Lytic bacteriophage therapy has been trialed in the treatment of P. aeruginosa infections; however, sometimes bacteria develop resistance to the bacteriophages. This study sheds light on the genetic mechanisms of such resistance, and how this might be harnessed to restore the sensitivity of multidrug-resistant P. aeruginosa to conventional antibiotics.202540162801
8916180.9988Increased mutations in lipopolysaccharide biosynthetic genes cause time-dependent development of phage resistance in Salmonella. Understanding how bacteria evolve resistance to phages has implications for phage-based therapies and microbial evolution. In this study, the susceptibility of 335 Salmonella isolates to the wide host range Salmonella phage BPSELC-1 was tested. Potentially significant gene sets that could confer resistance were identified using bioinformatics approaches based on phage susceptibility phenotypes; more than 90 potential antiphage defense gene sets, including those involved in lipopolysaccharide (LPS) biosynthesis, DNA replication, secretion systems, and respiratory chain, were found. The evolutionary dynamics of Salmonella resistance to phage were assessed through laboratory evolution experiments, which showed that phage-resistant mutants rapidly developed and exhibited genetic heterogeneity. Most representative Salmonella hosts (58.1% of 62) rapidly developed phage resistance within 24 h. All phage-resistant mutant clones exhibited genetic heterogeneity and observed mutations in LPS-related genes (rfaJ and rfaK) as well as other genes such as cellular respiration, transport, and cell replication-related genes. The study also identified potential trade-offs, indicating that bacteria tend to escape fitness trade-offs through multi-site mutations, all tested mutants increased sensitivity to polymyxin B, but this does not always affect their relative fitness or biofilm-forming capacity. Furthermore, complementing the rfaJ mutant gene could partially restore the phage sensitivity of phage-resistant mutants. These results provide insight into the phage resistance mechanisms of Salmonella and the complexity of bacterial evolution resulting from phage predation, which can inform future strategies for phage-based therapies and microbial evolution.202438193669
8932190.9988Alternative Evolutionary Paths to Bacterial Antibiotic Resistance Cause Distinct Collateral Effects. When bacteria evolve resistance against a particular antibiotic, they may simultaneously gain increased sensitivity against a second one. Such collateral sensitivity may be exploited to develop novel, sustainable antibiotic treatment strategies aimed at containing the current, dramatic spread of drug resistance. To date, the presence and molecular basis of collateral sensitivity has only been studied in few bacterial species and is unknown for opportunistic human pathogens such as Pseudomonas aeruginosa. In the present study, we assessed patterns of collateral effects by experimentally evolving 160 independent populations of P. aeruginosa to high levels of resistance against eight commonly used antibiotics. The bacteria evolved resistance rapidly and expressed both collateral sensitivity and cross-resistance. The pattern of such collateral effects differed to those previously reported for other bacterial species, suggesting interspecific differences in the underlying evolutionary trade-offs. Intriguingly, we also identified contrasting patterns of collateral sensitivity and cross-resistance among the replicate populations adapted to the same drug. Whole-genome sequencing of 81 independently evolved populations revealed distinct evolutionary paths of resistance to the selective drug, which determined whether bacteria became cross-resistant or collaterally sensitive towards others. Based on genomic and functional genetic analysis, we demonstrate that collateral sensitivity can result from resistance mutations in regulatory genes such as nalC or mexZ, which mediate aminoglycoside sensitivity in β-lactam-adapted populations, or the two-component regulatory system gene pmrB, which enhances penicillin sensitivity in gentamicin-resistant populations. Our findings highlight substantial variation in the evolved collateral effects among replicates, which in turn determine their potential in antibiotic therapy.201728541480