Phage-resistant Mutants Research Articles

The lytic bacteriophage vB_EfaH_EF1TV, a new member of the Herelleviridae family, disrupts biofilm produced by Enterococcus faecalis clinical strains

ObjectivesThe aim of this study is to characterize a new bacteriophage able to infect Enterococcus faecalis, and to evaluate its ability to disrupt biofilm. MethodsThe vB_EfaH_EF1TV (EF1TV) host-range was determined by spot test and efficiency of plating using a collection of 15E. faecalis clinical strains. The phage genome was sequenced with a next generation sequencing approach. Anti-biofilm activity was tested by crystal violet method and confocal laser scanning microscopy. Phage-resistant mutants were selected and sequenced to investigate receptors exploited by phage for infection. ResultsEF1TV is a newly discoveredE. faecalis phage which belongs to the Herelleviridae family. EF1TV, whose genome is 98% identical to φEF24C, is characterized by a linear dsDNA genome of 143,507 bp with direct terminal repeats of 1,911 bp. The phage is able to infect E. faecalis and shows also the ability to degrade biofilm produced by strains of this species. The results were confirmed by confocal laser scanning microscopy analyzing the biofilm reduction in the same optical field before and after phage infection. ConclusionsThe EF1TV phage shows promising features such as an obligatory lytic nature, an anti-biofilm activity and the absence of integration-related proteins, antibiotic resistance determinants and virulence factors, and therefore could be a promising tool for therapeutic applications.

Designing phagebodies.

A study reports the generation of synthetic ‘phagebodies’ with a broadened host range that were able to target naturally occurring phage-resistant bacterial mutants.

Identification of Brevibacterium flavum genes related to receptors involved in bacteriophage BFK20 adsorption

Phage infection of bacterial cells is a process requiring the interaction between phage receptor binding proteins and receptors on the bacterial cell surface. We prepared a Brevibacterium flavum CCM 251 EZ-Tn5 transposon insertional library and isolated phage-resistant mutants. Analysis of the DNA fragments produced by single-primer PCR was used to determine the EZ-Tn5 transposon insertion sites in the genomes of phage-resistant B. flavum mutants. Seven disrupted genes were identified in forty B. flavum mutants. The phage resistance of these mutants was demonstrated by cultivation analysis in the presence of BFK20, and the adsorption rate of BFK20 to these mutants was tested. B. flavum mutants displayed significantly reduced adsorption rates; the lowest rate was observed for mutants containing interrupted major facilitator superfamily (MFS) protein and glycosyltransferase genes. Uninterrupted forms of these genes were cloned into corynebacterial vector pJUP06 and used for in trans complementation of the corresponding B. flavum mutants. The growth of these complemented mutants when infected with BFK20 closely resembled that of wild-type B. flavum. These complemented mutants also exhibited similar BFK20 adsorption as the wild-type control. We infer that the disrupted MFS protein and glycosyltransferase genes are responsible for the phage-resistant phenotype of these B. flavum transposition mutants.

Phage resistance evolution in vitro is not reflective of in vivo outcome in a plant-bacteria-phage system.

The evolution of resistance to parasites is fundamentally important to disease ecology, yet we remain unable to predict when and how resistance will evolve. This is largely due to the context-dependent nature of host-parasite interactions, as the benefit of resistance will depend on the abiotic and biotic environment. Through experimental evolution of the plant pathogenic bacterium Pseudomonas syringae and two lytic bacteriophages across two different environments (high-nutrient media and the tomato leaf apoplast), we demonstrate that de novo evolution of resistance is negligible in planta despite high levels of resistance evolution in vitro. We find no evidence supporting the evolution of phage-selected resistance in planta despite multiple passaging experiments, multiple assays for resistance, and high multiplicities of infection. Additionally, we find that phage-resistant mutants (evolved in vitro) did not realize a fitness benefit over phage-sensitive cells when grown in planta in the presence of phage, despite reduced growth of sensitive cells, evidence of phage replication in planta, and a large fitness benefit in the presence of phage observed invitro. Thus, this context-dependent benefit of phage resistance led to different evolutionary outcomes across environments. These results underscore the importance of studying the evolution of parasite resistance in ecologically relevant environments.

Efficacy of a phage cocktail in controlling phage resistance development in multidrug resistant Acinetobacter baumannii

The control and treatment of multidrug resistant pathogens infections has become a grand challenge for clinicians worldwide. Virulent phage has long been considered as an effective bactericidal agent, which may be a potentially alternative to antibiotics. However, the rapid development of phage resistance seriously hinders the wide and continuous application of virulent phages. In this study, Acinetobacter baumannii phage vB_AbaS_D0 was isolated, characterized and used to control the phage resistance development in bacterial strains. Transmission electron microscopy analysis of vB_AbaS_D0 indicated it belonged to the Siphoviridae family with an icosahedral head. Its whole genome was 43, 051 bp in size, with a GC content of 45.48% and 55 putative open reading frames. The data showed that vB_AbaS_D0 was a virulent phage. Although vB_AbaS_D0 had a very weak bactericidal activity, a wide range of Acinetobacter baumannii strains were sensitive to it. The results suggested that the cocktail of vB_AbaS_D0 and another Acinetobacter baumannii phage vB_AbaP_D2 could improve the therapeutic efficacy in vivo and in vitro. The resistance mutation frequency of A. baumannii cells infected with D0 or phage cocktail was significantly lower than cells treated with D2 (P < 0.01). Phage therapy in the murine bacteremia model results showed that the percentage of phage resistant mutant occurrence in the phage D0 or cocktail treatment group was significantly lower than in phage D2 treatment group (P < 0.01).

Assessment of antibiotic resistance in bacteriophage-insensitive Klebsiella pneumoniae

This study was design to evaluate the physiological properties of bacteriophage-insensitive Klebsiella pneumoniae (BIKP) mutants in association with the antibiotic cross-resistance, β-lactamase activity, and gene expression. Klebsiella pneumoniae ATCC 23357(KPWT), ciprofloxacin-induced antibiotic-resistant K. pneumoniae ATCC 23357 (KPCIP), and clinically isolated antibiotic-resistant K. pneumoniae 10263 (KPCLI) were used to isolate BIKP mutants against KPB1, PBKP02, PBKP21, PBKP29, PBKP33, and PBKP35. PBKP35-induced mutants, including bacteriophage-insensitive K. pneumoniae ATCC 23357 (BIKPWT), ciprofloxacin-induced K. pneumoniae ATCC 23357 (BIKPCIP), and clinically isolated antibiotic-resistant K. pneumoniae CCARM 10263 (BIKPCLI). BIKPWT, BIKPCIP, and BIKPCLI were resistant to Klebsiella bacteriophages, KPB1, PBKP02, PBKP21, PBKP29, and PBKP33. The antibiotic cross-resistance to cefotaxime, cephalothin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, levofloxacin, and nalidixic acid was observed in BIKPWT. The relative expression levels of vagC was increased by more than 8-folds in BIKPWT, corresponding to the increased β-lactamase activity. The aac(6′)-Ib-cr was overexpressed in BIKP mutants, responsible for aminoglycoside and quinolone resistance. The phage-resistant mutants decreased the antibiotic susceptibilities in association with β-lactamase activity and antibiotic resistance-related gene expression. The results pointed out the cross-resistance of BIKP mutants to antibiotics, which might be considered when applying for the therapeutic use of bacteriophage.

Three Capsular Polysaccharide Synthesis-Related Glucosyltransferases, GT-1, GT-2 and WcaJ, Are Associated With Virulence and Phage Sensitivity of Klebsiella pneumoniae.

Klebsiella pneumoniae (K. pneumoniae) spp. are important nosocomial and community-acquired opportunistic pathogens, which cause various infections. We observed that K. pneumoniae strain K7 abruptly mutates to rough-type phage-resistant phenotype upon treatment with phage GH-K3. In the present study, the rough-type phage-resistant mutant named K7RR showed much lower virulence than K7. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis indicated that WcaJ and two undefined glycosyltransferases (GTs)- named GT-1, GT-2- were found to be down-regulated drastically in K7RR as compared to K7 strain. GT-1, GT-2, and wcaJ are all located in the gene cluster of capsular polysaccharide (CPS). Upon deletion, even of single component, of GT-1, GT-2, and wcaJ resulted clearly in significant decline of CPS synthesis with concomitant development of GH-K3 resistance and decline of virulence of K. pneumoniae, indicating that all these three GTs are more likely involved in maintenance of phage sensitivity and bacterial virulence. Additionally, K7RR and GT-deficient strains were found sensitive to endocytosis of macrophages. Mitogen-activated protein kinase (MAPK) signaling pathway of macrophages was significantly activated by K7RR and GT-deficient strains comparing with that of K7. Interestingly, in the presence of macromolecular CPS residues (>250 KD), K7(ΔGT-1) and K7(ΔwcaJ) could still be bounded by GH-K3, though with a modest adsorption efficiency, and showed minor virulence, suggesting that the CPS residues accumulated upon deletion of GT-1 or wcaJ did retain phage binding sites as well maintain mild virulence. In brief, our study defines, for the first time, the potential roles of GT-1, GT-2, and WcaJ in K. pneumoniae in bacterial virulence and generation of rough-type mutation under the pressure of bacteriophage.

Efficiency of Single Phage Suspensions and Phage Cocktail in the Inactivation of Escherichia coli and Salmonella Typhimurium: An In Vitro Preliminary Study.

Enterobacteriaceae Escherichia coli and Salmonella enterica serovar Typhimurium strains are among the main pathogens responsible for moderate and serious infections at hospital and community environments, in part because they frequently present resistance to antibiotics. As the treatment of Enterobacteriaceae infections is empiric, using the same antibiotics to treat E. coli and Salmonella infections, the same concept can be applied with phages. The use of different phages combined in cocktails, frequently used to circumvent the development of phage-resistant mutants, also allows for the treatment of multiple pathogens, broadening the phages’ action spectrum. As such, the aim of this study was to evaluate the efficiency of a cocktail of two phages (ELY-1, produced on E. coli and phSE-5, produced on S. Typhimurium) to control E. coli and S. Typhimurium. Phages ELY-1 and phSE-5 were effective against E. coli (maximum reductions of 4.5 and 3.8 log CFU/mL, respectively), S. Typhimurium (maximum reductions of 2.2 and 2.6 log CFU/mL, respectively), and the mixture of both bacteria (maximum reductions of 2.2 and 2.0 log CFU/mL, respectively). The cocktail ELY-1/phSE-5 was more effective against S. Typhimurium and the mixture of both bacteria (maximum reduction of 3.2 log CFU/mL for both) than the single phage suspensions and as effective against E. coli as its specific phage ELY-1 (maximum reductions of 4.5 log CFU/mL). The use of both the phage cocktails, as well as the single-phage suspensions, however, did not prevent the occurrence of phage-resistant mutants. Overall, the results indicate that the application of the phages in the form of a cocktail show their potential to be used presumptively, that is, prior to the identification of the pathogens, paving its use to control E. coli or S. Typhimurium.

High-throughput LPS profiling as a tool for revealing of bacteriophage infection strategies

O-antigens of Gram-negative bacteria modulate the interactions of bacterial cells with diverse external factors, including the components of the immune system and bacteriophages. Some phages need to acquire specific adhesins to overcome the O-antigen layer. For other phages, O-antigen is required for phage infection. In this case, interaction of phage receptor binding proteins coupled with enzymatic degradation or modification of the O-antigen is followed by phage infection. Identification of the strategies used by newly isolated phages may be of importance in their consideration for various applications. Here we describe an approach based on screening for host LPS alterations caused by selection by bacteriophages. We describe an optimized LPS profiling procedure that is simple, rapid and suitable for mass screening of mutants. We demonstrate that the phage infection strategies identified using a set of engineered E. coli 4 s mutants with impaired or altered LPS synthesis are in good agreement with the results of simpler tests based on LPS profiling of phage-resistant spontaneous mutants.

Phage Reduce Stability for Regaining Infectivity during Antagonistic Coevolution with Host Bacterium.

The coevolution between phage and host bacterium is an important force that drives the evolution of the microbial community, yet the coevolution mechanisms have still not been well analyzed. Here, by analyzing the interaction between a Bacillus phage vB_BthS_BMBphi and its host bacterium, the coevolution mechanisms of the first-generation phage-resistant bacterial mutants and regained-infectivity phage mutants were studied. The phage-resistant bacterial mutants showed several conserved mutations as a potential reason for acquiring phage resistance, including the mutation in flagellum synthesis protein FlhA and cell wall polysaccharide synthesis protein DltC. All the phage-resistant bacterial mutants showed a deleted first transmembrane domain of the flagellum synthesis protein FlhA. Meanwhile, the regain-infectivity phage mutants all contained mutations in three baseplate-associated phage tail proteins by one nucleotide, respectively. A polymorphism analysis of the three mutant nucleotides in the wild-type phage revealed that the mutations existed before the interaction of the phage and the bacterium, while the wild-type phage could not infect the phage-resistant bacterial mutants, which might be because the synchronized mutations of the three nucleotides were essential for regaining infectivity. This study for the first time revealed that the synergism mutation of three phage baseplate-associated proteins were essential for the phages’ regained infectivity. Although the phage mutants regained infectivity, their storage stability was decreased and the infectivity against the phage-resistant bacterial mutants was reduced, suggesting the phage realized the continuation of the species by way of “dying to survive”.

Isolation and characterization of spontaneous phage-resistant mutants of Lactobacillus paracasei

Spontaneous phage-resistant mutants were isolated from Lactobacillus paracasei LPC by agar plate (AP) and secondary-culture (SC) methods. They were characterized by cell and colony morphologies, carbohydrate fermentation patterns, phage resistance stability, efficiency of plaquing (EOP), acidifying and milk acidification kinetics. Only 98 out of 175 isolates (56%) proved to be true phage-resistant mutants and the SC method was more efficient than the AP method. Although the phage resistance stability varied among the mutants isolated, the EOP values were mostly very high (<10−11). Three phage-resistant strains were selected based on their extreme resistance capacity to the phage ΦT25 (EOP<10−10) and their nonlysogenic property. Acidifying activity did not differ between phage-sensitive strains and their three respective phage-resistant derivatives. Most of the mutants were completely or partially unable to adsorb phage particles. Restriction-modification type systems were not detected in all phage-resistant derivatives. All three selected mutants were identical to the corresponding parent strain of L. paracasei LPC in the cell and colony morphologies. The comparison of random amplified polymorphic DNA profiles obtained with four arbitrary primers also revealed the highest similarity coefficient (87%) among the parent strain and the three mutants, indicating that each mutant has been derived from this parent strain. A good performance during milk fermentation and the subsequent refrigerated storage were obtained when these mutants were employed. These phage-resistant derivatives could be used as improved strains or for strain rotation programs when commercial strains become sensitive to the phages present in industrial environments.

Sequential Combined Effect of Phages and Antibiotics on the Inactivation of Escherichia coli.

The emergence of antibiotic resistance in bacteria is a global concern. The use of bacteriophages (or phages) alone or combined with antibiotics is consolidating itself as an alternative approach to inactivate antibiotic-resistant bacteria. However, phage-resistant mutants have been considered as a major threat when phage treatment is employed. Escherichia coli is one of the main responsible pathogens for moderate and serious infections in hospital and community environments, being involved in the rapid evolution of fluoroquinolones and third-generation cephalosporin resistance. The aim of this study was to evaluate the effect of combined treatments of phages and antibiotics in the inactivation of E. coli. For this, ciprofloxacin at lethal and sublethal concentrations was added at different times (0, 6, 12 and 18 h) and was tested in combination with the phage ELY-1 to inactivate E. coli. The efficacy of the combined treatment varied with the antibiotic concentration and with the time of antibiotic addition. The combined treatment prevented bacterial regrowth when the antibiotic was used at minimum inhibitory concentration (MIC) and added after 6 h of phage addition, causing less bacterial resistance than phage and antibiotic applied alone (4.0 × 10−7 for the combined treatment, 3.9 × 10−6 and 3.4 × 10−5 for the antibiotics and the phages alone, respectively). Combined treatment with phage and antibiotic can be effective in reducing the bacterial density and it can also prevent the emergence of resistant variants. However, the antibiotic concentration and the time of antibiotic application are essential factors that need to be considered in the combined treatment.

Reviving Phage Therapy for the Treatment of Cholera.

Cholera remains a major risk in developing countries, particularly after natural or man-made disasters. Vibrio cholerae El Tor is the most important cause of these outbreaks, and is becoming increasingly resistant to antibiotics, so alternative therapies are urgently needed. In this study, a single bacteriophage, Phi_1, was used to control cholera prophylactically and therapeutically in an infant rabbit model. In both cases, phage-treated animals showed no clinical signs of disease, compared with 69% of untreated control animals. Bacterial counts in the intestines of phage-treated animals were reduced by up to 4 log10 colony-forming units/g. There was evidence of phage multiplication only in animals that received a V. cholerae challenge. No phage-resistant bacterial mutants were isolated from the animals, despite extensive searching. This is the first evidence that a single phage could be effective in the treatment of cholera, without detectable levels of resistance. Clinical trials in human patients should be considered.

Efficacy of φkm18p phage therapy in a murine model of extensively drug-resistant Acinetobacter baumannii infection.

PurposeFew effective antibiotics are available for treating extensively drug-resistant Acinetobacter baumannii (XDRAB) sepsis. Phage therapy may show potential in treating XDRAB infections.Materials and methodsWe studied φkm18p phage therapy in BALB/c and C57BL/6 mice models of XDRAB bacteremia.ResultsWe observed survival rates of nearly 100% in groups given phage therapy concurrent with XDRAB at different multiplicities of infection. In mice that received phage therapy after a 1-hour delay, the survival rate decreased to about 50%. The bacterial load in the blood decreased from 108 to 102 and 103 colony-forming units (CFU)/mL in the concurrent treatment group. In the phage therapy group, the levels of the cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), were low at 3 hours after infection. Although some phage-resistant mutants were isolated after phage therapy, a cytotoxicity study showed that they had reduced fitness.ConclusionPhage therapy in XDRAB bacteremia increased the animal survival rates, decreased the bacteremia loads, and decreased the levels of inflammatory markers TNF-α and IL-6. However, the reduced therapeutic effect with delayed administrations may be a concern in developing a successful phage therapy for treating acute infections of multidrug-resistant pathogens.

Bacteriophage-resistant industrial fermentation strains: from the cradle to CRISPR/Cas9.

Bacteriophage contamination and cell lysis have been recurring issues with some actinomycetes used in the pharmaceutical fermentation industry since the commercialization of streptomycin in the 1940s. In the early years, spontaneous phage-resistant mutants or lysogens were isolated to address the problem. In some cases, multiple phages were isolated from different contaminated fermentors, so strains resistant to multiple phages were isolated to stabilize the fermentation processes. With the advent of recombinant DNA technology, the early scaleup of the Escherichia coli fermentation process for the production of human insulin A and B chains encountered contamination with multiple coliphages. A genetic engineering solution was to clone and express a potent restriction/modification system in the production strains. Very recently, an E. coli fermentation of 1,3-propanediol was contaminated by a coliphage related to T1. CRISPR/Cas9 technology was applied to block future contamination by targeting seven different phage genes for double-strand cleavage. These approaches employing spontaneous mutation, genetic engineering, and synthetic biology can be applied to many current and future microorganisms used in the biotechnology industry.

High Throughput Manufacturing of Bacteriophages Using Continuous Stirred Tank Bioreactors Connected in Series to Ensure Optimum Host Bacteria Physiology for Phage Production.

Future industrial demand for large quantities of bacteriophages e.g., for phage therapy, necessitates the development of scalable Good Manufacturing Practice compliant (cGMP) production platforms. The continuous production of high titres of E coli T3 phages (1011 PFU mL−1) was achieved using two continuous stirred tank bioreactors connected in series, and a third bioreactor was used as a final holding tank operated in semi-batch mode to finish the infection process. The first bioreactor allowed the steady-state propagation of host bacteria using a fully synthetic medium with glucose as the limiting substrate. Host bacterial growth was decoupled from the phage production reactor downstream of it to suppress the production of phage-resistant mutants, thereby allowing stable operation over a period of several days. The novelty of this process is that the manipulation of the host reactor dilution rates (range 0.1–0.6 hr−1) allows control over the physiological state of the bacterial population. This results in bacteria with considerably higher intracellular phage production capability whilst operating at high dilution rates yielding significantly higher overall phage process productivity. Using a pilot-scale chemostat system allowed optimisation of the upstream phage amplification conditions conducive for high intracellular phage production in the host bacteria. The effect of the host reactor dilution rates on the phage burst size, lag time, and adsorption rate were evaluated. The host bacterium physiology was found to influence phage burst size, thereby affecting the productivity of the overall process. Mathematical modelling of the dynamics of the process allowed parameter sensitivity evaluation and provided valuable insights into the factors affecting the phage production process. The approach presented here may be used at an industrial scale to significantly improve process control, increase productivity via process intensification, and reduce process manufacturing costs through process footprint reduction.

More than Rotating Flagella: Lipopolysaccharide as a Secondary Receptor for Flagellotropic Phage 7-7-1.

Bacteriophage 7-7-1, a member of the family Myoviridae, infects the soil bacterium Agrobacterium sp. strain H13-3. Infection requires attachment to actively rotating bacterial flagellar filaments, with flagellar number, length, and rotation speed being important determinants for infection efficiency. To identify the secondary receptor(s) on the cell surface, we isolated motile, phage-resistant Agrobacterium sp. H13-3 transposon mutants. Transposon insertion sites were pinpointed using arbitrary primed PCR and bioinformatics analyses. Three genes were recognized, whose corresponding proteins had the following computationally predicted functions: AGROH133_07337, a glycosyltransferase; AGROH133_13050, a UDP-glucose 4-epimerase; and AGROH133_08824, an integral cytoplasmic membrane protein. The first two gene products are part of the lipopolysaccharide (LPS) synthesis pathway, while the last is predicted to be a relatively small (13.4-kDa) cytosolic membrane protein with up to four transmembrane helices. The phenotypes of the transposon mutants were verified by complementation and site-directed mutagenesis. Additional characterization of motile, phage-resistant mutants is also described. Given these findings, we propose a model for Agrobacterium sp. H13-3 infection by bacteriophage 7-7-1 where the phage initially attaches to the flagellar filament and is propelled down toward the cell surface by clockwise flagellar rotation. The phage then attaches to and degrades the LPS to reach the outer membrane and ejects its DNA into the host using its syringe-like contractile tail. We hypothesize that the integral membrane protein plays an important role in events following viral DNA ejection or in LPS processing and/or deployment. The proposed two-step attachment mechanism may be conserved among other flagellotropic phages infecting Gram-negative bacteria.IMPORTANCE Flagellotropic bacteriophages belong to the tailed-phage order Caudovirales, the most abundant phages in the virome. While it is known that these viruses adhere to the bacterial flagellum and use flagellar rotation to reach the cell surface, their infection mechanisms are poorly understood. Characterizing flagellotropic-phage-host interactions is crucial to understanding how microbial communities are shaped. Using a transposon mutagenesis approach combined with a screen for motile, phage-resistant mutants, we identified lipopolysaccharides as the secondary cell surface receptor for phage 7-7-1. This is the first cell surface receptor identified for flagellotropic phages. One hypothetical membrane protein was also recognized as essential for infection. These new findings, together with previous results, culminated in an infection model for phage 7-7-1.

New insights on phage efficacy to control Aeromonas salmonicida in aquaculture systems: An in vitro preliminary study

A major source of financial loss for the fish-farming industry is the occurrence of bacterial infections. Phage therapy can be a useful alternative tool to conventional treatments to control bacterial infections in aquaculture. The promising results obtained with phage AS-A to control the agent of furunculosis, Aeromonas salmonicida, led us to isolate two new phages and evaluate their dynamics, in cocktails and individually, to control this pathogenic bacterium. Moreover, considering that in outdoor facilities aquaculture water is exposed to the natural variability of physical and chemical parameters, the influence of pH, salinity, temperature, solar radiation and UV radiation on phage AS-D stability was also evaluated in this study, in order to develop an effective phage therapy protocol. Phages were assigned to the family Myoviridae and revealed identical morphological characteristics. Phage AS-A presents a higher burst size and a shorter latent period, decreasing the concentration of A. salmonicida sooner than phages AS-D and AS-E. However, phage AS-D presented higher rate of bacterial reduction, inducing also less bacterial resistance. Bacterial control with the cocktails was higher, namely when phage AS-A was combined with one of the two new phages or with both, but the main difference in the bacterial control was in the treatment time. Phage cocktails decrease the concentration of A. salmonicida sooner than single suspensions. The use of phage cocktails, in general, decreased phage-resistant mutants. The survival of AS-D phage was mostly affected by sunlight exposure (decrease of 3 log PFU/mL after 12 h) and high temperatures (decrease of 3 PFU/mL after 21 days and of 7 log PFU/mL after 49 days at 37 °C, but no decrease after 21 and 49 days at 25 °C and decrease of only 1 and 2 log PFU/mL after 21 and 49 days, respectively, at ambient temperature). The high bacterial control and low development of phage-resistant bacterial clones suggest that these phages can be used to control the furunculosis in aquaculture. Nonetheless, the stability of the phages was affected by solar radiation, this can be overcome by the application of phages at the end of the day or at night.

Adaptation of Pseudomonas aeruginosa to Phage PaP1 Predation via O-Antigen Polymerase Mutation.

Adaptation of bacteria to phage predation poses a major obstacle for phage therapy. Bacteria adopt multiple mechanisms, such as inhibition of phage adsorption and CRISPR/Cas systems, to resist phage infection. Here, a phage-resistant mutant of Pseudomonas aeruginosa strain PA1 under the infection of lytic phage PaP1 was selected for further study. The PaP1-resistant variant, termed PA1RG, showed decreased adsorption to PaP1 and was devoid of long chain O-antigen on its cell envelope. Whole genome sequencing and comparative analysis revealed a single nucleotide mutation in the gene PA1S_08510, which encodes the O-antigen polymerase Wzy that is involved in lipopolysaccharide (LPS) biosynthesis. PA1_Wzy was classified into the O6 serotype based on sequence homology analysis and adopts a transmembrane topology similar to that seem with P. aeruginosa strain PAO1. Complementation of gene wzy in trans enabled the mutant PA1RG to produce the normal LPS pattern with long chain O-antigen and restored the susceptibility of PA1RG to phage PaP1 infection. While wzy mutation did not affect bacterial growth, mutant PA1RG exhibited decreased biofilm production, suggesting a fitness cost of PA1 associated with resistance of phage PaP1 predation. This study uncovered the mechanism responsible for PA1RG resistance to phage PaP1 via wzy mutation and revealed the role of phages in regulating bacterial behavior.

Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment.

The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells.