A genotypic analysis of five P. aeruginosa strains after biofilm infection by phages targeting different cell surface receptors

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] Antibiotic resistance constitutes currently one of the most serious threats to the global public health and it urgently requires new and effective solutions. Bacteriophages are bacterial viruses increasingly recognized as being good alternatives to the traditional antibiotic therapies [1]. In the present study, the efficacy of phages against Pseudomonas aeruginosa PAO1 biofilm and planktonic cell cultures was evaluated over the course of 48 hours. Although significant reductions in the number of viable cells were achieved for both cases, the high level of adaptability of the bacteria in response to the selective pressure caused by phage treatment resulted in the emergence of phage-resistant variants. However, very few studies have explored this phenomenon. [...]info:eu-repo/semantics/publishedVersio

Fast emergence of phage-resistant Pseudomonas aeruginosa biofilm cells in response to the pressure exerted by bacteriophage treatment

Antibiotic resistance constitutes currently one of the most serious threats to the global public health and it urgently requires new and effective solutions. Bacteriophages are bacterial viruses increasingly recognized as an attractive alternative to the conventional antibiotic therapies. In the present study, the efficacy of phages against Pseudomonas aeruginosa PAO1 biofilm and planktonic cell cultures was evaluated over the course of 48 hours. Although significant reductions in the number of viable cells were achieved for both cases, the high adaptation capability of bacteria in response to the selective pressure caused by phage treatment, resulted in the inevitable arising of phage-resistant variants. In most cases, those variants appeared later in planktonic cultures than in biofilms. Given the interest in further understanding their genetic makeup and possible mutations accumulated, some were selected for further phenotypic and genotypic characterization. The complete genomes of five P. aeruginosa PAO1 phage-resistant variants were sequenced and all revealed to carry mutations in the galU gene, which is involved in lipopolysaccharide core biosynthesis, as well as in one pil gene, which is involved in type IV pilus synthesis. Three of the P. aeruginosa PAO1 variants further revealed large deletions (> 200 kbp) in their genomes. Overall the results of this study reveal that the selective pressure caused by phages while targeting biofilms results in a faster emergence of resistance compared to planktonic cultures, probably due to the high genetic diversity of cells within biofilms. Furthermore phage-resistant variants seem to be quite adapted to the biofilm phenotype

Understanding the complex phage-host interactions in biofilm communities

Bacteriophages and bacterial biofilms are widely present in natural environments, a fact that has accelerated the evolution of phages and their bacterial hosts in these particular niches. Phage-host interactions in biofilm communities are rather complex, where phages are not always merely predators but also can establish symbiotic relationships that induce and strengthen biofilms. In this review we provide an overview of the main features affecting phage-biofilm interactions as well as the currently available methods of studying these interactions. In addition, we address the applications of phages for biofilm control in different contexts.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of project PTDC/BIA-MIC/2312/2020 and the strategic funding of unit UIDB/04469/2020.info:eu-repo/semantics/publishedVersio

Phages for the biocontrol of bacterial canker of kiwifruit

Bacterial canker of kiwifruit (Actinidia spp.) caused by Pseudomonas syringae pv. actinidiae (Psa), causes significant yield and financial losses. The use of copper-based products and antibiotics are the current techniques for Psa control. These compounds are phytotoxic and also promote copper and antibiotic resistance. The isolation and characterization of (bacterio)phages for the control of Psa was the focus of this research, motivated by the demand for safe and effective biocontrol techniques against this disease. A Portuguese collection of Psa strains was characterized by molecular and phenotypic tests. Phages were isolated from branches, buds, leaves and flowers of kiwifruit plants in the North of Portugal. Phages were isolated by the enrichment procedure with Psa strains CFBP 7286 and P84 as possible hosts, and the lytic spectra of 6 selected ones were tested against the Psa collection. The two phages displaying broader host ranges (between 71% and 84% of efficacy among Psa strains) were stable between -20ºC and 50ºC, pH range of 3 to 11 and UV light at 366 nm. Transmission Electron Microscopy was used to characterize phages morphology. In vitro efficacy studies revealed that, with MOI=1, phage 177T decreased the number of CFUs after 4 hours of inoculation while maintaining a low bacterial load for up to 24 hours. Over 24 hours, phage VC3 maintained the bacterial growth stable. Preliminary ex vivo and in vivo assays on kiwifruit leaf discs and directly applied to the plant, showed differences between the phage application and the control after 12 days of inoculation. One phage has been sequenced and confirmed to be lysogenic, data that corresponded to the ex vivo results. Even so, this lysogenic phage showed potential to be used as a biological control agent against Psa. This work was supported by the project GesPSAKiwi - Ferramenta Operacional para gestão sustentável do cancro bacteriano (Psa) da Actinídea.info:eu-repo/semantics/publishedVersio

Challenging mono and dual-species biofilms with phages and antibiotics

Pseudomonas aeruginosa is a relevant opportunistic pathogen, and worryingly it frequently shows a low antibiotic susceptibility. This bacterium is responsible for 65% of mortality in hospitals all over the world. One of its virulence factors is associated with the ability to adhere to surfaces and form virulent biofilms. This work describes the results from several years of investigation using P. aeruginosa phages alone and combined with antibiotics or other phages against single and mixedspecies biofilms. The mixed species biofilms of P. aeruginosa reported herein were formed with fungi (Candida albicans) since these two microorganisms co-inhabit a wide variety of environments and the interactions between them can result in huge medical and economic impacts; and with Acinetobacter baumannii also an opportunistic pathogen associated with nosocomial infections. In both mixed species biofilms there was observed an inhibitory effect of P. aeruginosa since the levels of C. albicans and A. baumannii were highly reduced in the presence of P. aeruginosa. In P. aeruginosa - C. albicans biofilms, the Pseudomonas phages were able to attack their host population; however, as soon as the phages had killed P. aeruginosa, the numbers of viable C. albicans cells increased rapidly. Furthermore, C. albicans’ morphology and virulence were significantly affected in the presence of P. aeruginosa. In P. aeruginosa – A. baumannii biofilms, phages applied only to one of the hosts decreased, as expected, that specific population already after 6 h. Nevertheless, while after treatment of the mixed species biofilms with P. aeruginosa phages we observed a growth of A. baumannii, the same did not occur when the biofilms were only treated with the A. baumannii phage. The use of both phages was effective and reduced significantly the numbers of viable cells of the mixed population biofilm. Despite the potential of the phages used in this work as antimicrobial agents, it is well known that bacteria can quickly adapt and create new survival strategies and the emergence of phage-resistant phenotypes is inevitable. Indeed, we observed the rapid appearance of Pseudomonas aeruginosa resistant phenotypes following 24h of biofilm contact with phages and those phenotypes exhibited altered LPS structures. Thus, the combination therapy of phages and 4 commonly used antibiotics in the treatment of P. aeruginosa infections was also evaluated. The results obtained proved that certain antibiotics and phages have potentially more benefits and even act synergistically compared to just phages or antibiotics alone

The ability of C. albicans to form biofilm in the presence of sensitive and phage-resistant P. aeruginosa phenotypes

P. aeruginosa and C. albicans are human opportunistic pathogens frequently associated with nosocomial infections. Pathogenic interactions between them have already been identified and it has been reported that C. albicans’ morphology and virulence are significantly affected by the presence of P. aeruginosa. In this work, the interaction between these 2 microorganisms in mixed biofilms was studied. The behavior of C. albicans cells after applying P. aeruginosa-specific phages, which lyse P. aeruginosa biofilm cells in the mixed biofilms, was analyzed as well as the ability of C. albicans biofilm formation in the presence of P. aeruginosa phage-resistant phenotypes. Biofilms were formed in 24-well microplates, containing 1 ml of YPD medium and 10 µl of each cellular suspension (OD600nm = 1), during 24 and 48 h. Phages (phiIBB-PAA2 and phiIBB-PAP21) were applied in 24 h old mixed biofilms and samples were taken after 2, 6 and 24 h of phage biofilm infection for CFU counts. The results revealed that C. albicans proliferation was clearly inhibited by the presence of wildtype P. aeruginosa ATCC 10145 and PAO1 strains. Conversely, the proliferation of P. aeruginosa was not influenced by the presence of C. albicans. After the infection, it was observed that both phages were effective in depleting Pseudomonas biofilm cells from the mixed biofilms. This reduction resulted in an increase in the amount of C. albicans cells during phage infection. However, at 24 h post-infection of mixed biofilms, an increase of P. aeruginosa biofilm cells was also observed as compared to the numbers at 6 h post-infection. This suggests that P. aeruginosa cells acquired resistance to the phages between 6 and 24 h of infection. Surprisingly, this increase in P. aeruginosa at 24 h post-infection did not interfere with C. albicans biofilm growth and accordingly, an increase in C. albicans cells was observed. These results suggest that the surviving P. aeruginosa cells after phage attack have changes in their phenotype resulting in a diminished ability to inhibit C. albicans biofilm growth. To evaluate if the regrowth of C. albicans cells in the infected biofilms was caused by the emergence of phage-resistant P. aeruginosa phenotypes, which did not inhibit C. albicans biofilm growth, several P. aeruginosa colonies were isolated after 24 h of mixed biofilm infection with phage phiIBB-PAA2 and tested for their susceptibility to the phages used in this study. Most of these phenotypes showed resistance to the phage phiIBB-PAA2 and 1 of these strains was also resistant to phage phiIBB-PAP21. Mixed biofilms with C. albicans were once again performed with these P. aeruginosa ATCC 10145-phenotype variants and revealed that most of them could co-inhabit better with C. albicans than their wildtype strain

Can bacteriofages be effective in controlling harmful biofilms?

(Bacterio)phages are viruses that specifically infect bacteria, causing cell lysis and therefore can be considered a valuable strategy for bacterial control. Recent studies have demonstrated the potential of using phages to control bacterial biofilms. Phages are able to penetrate the extracellular matrnc and can cause up to 90% of biofilm mass reduction even in old biofilms. However phage action can be impaired by components of the biofilm matrix, the slow growth of biofilm bacteria and the fast emergence of phage resistant phenotypes. We have conducted several studies of phage biofilm interaction and based on our experimental data, we have hypothesized that the general mechanisms of a virulent phage-biofilm infection, in a very simplistic model, can occur in four stages: 1) Transport of the phage particles through the biofilm matrix (by diffusion or convection mechanisms); 2) Settlement and/or attachment of phages onto bacterial cells embedded in the biofilm matrix, followed by adsorption and phage replicatiOn inside host cells; 3) Release of phage progeny to planktonic and biofilm phases, through host cell lysis and infection of neighbourhood biofilm cells resulting in biofilm biomomass reduct1on; 4) Detachment of biofilm portions and phages into the planktomc phase. Nevertheless, the interaction between phage and biofilms is a rather complex process. Theoretically, a biofilm should be rapidly infected because cells are more close to each other and this fact can enhance phage replication, when compared to the less accessible bacteria of planktonic cultures. On the other hand, the structure and compositiOn of the biofilm as well the physiology of the biofilm cells may impose some limitations to biofilm infection. Indeed, phage·biofilm interaction is greatly inFluenced by the biofilm age, biofilm structure, biofilm mode of growth and most importantly the host and phage characteristics. This work is a summary of all phage/biofilm interaction studies conducted by our team involving different phage types and host species

The influence of P. fluorescens cell morphology on the lytic performance and production of phage ϕIBB-PF7A

This study aims at assessing the influence of Pseudomonas fluorescence cell morphology on the effectiveness and production of the lytic bacteriophage /IBBPF7A. P. fluorescens were cultured as rods or as elongated cells by varying the temperature and rotary agitation conditions. Cells presented rod shape when grown at temperatures up to 25C and also at 30C under static conditions, and elongated morphology only at 30C when cultures were grown under agitation. Elongated cells were 0.4 up to 27.9 lm longer than rod cells. Rod-shaped hosts were best infected by phages at 25C which resulted in an 82% cell density reduction. Phage infection of elongated cells was successful, and the cell density reductions achieved was statistically similar (P[0.05) to those obtained at the optimum growth temperature of P. fluorescens. Phage burst size varied with the cell growth conditions and was approximately 58 and 153 PFU per infected rod and elongated cells, grown at 160 rpm, at 25C (the optimal temperature) and 30C, respectively. Phage adsorption was faster to elongated cells, most likely due to the longer length of the host. The surface composition of rod and elongated cells is similar in terms of outer membrane proteins and lipopolysaccharide profiles. The results of this study suggest that the change of rod cells to an elongated morphology does not prevent cells from being attacked by phages and also does not impair the phage infection.This work was supported by a grant (SFRH/BD/18485/2004) from the Portuguese Foundation for Science and Technology (FCT)

Adaptive evolution of phages towards Pseudomonas aeruginosa biofilm control

Pseudomonas aeruginosa is the leading cause of chronic lung infection in patients with Cystic Fibrosis (CF). The main reason for the persistence of P. aeruginosa in CF lung environment is its biofilm mode of growth, associated with increased tolerance to antibiotics and host immune defenses. Phage therapy is a promising approach to treat biofilm-related infections such as CF. However, the complete eradication of biofilms is almost impossible. Given the natural ability of (bacterio)phages to evolve and counterattack the bacterial defense mechanisms, the aim here was to improve the anti-biofilm activity of phages through adaptive evolution. The phage evolution was stimulated for 8 days in 24h-old biofilms formed by a P. aeruginosa clinical isolate recovered from a CF patient. The biofilms were treated with phage PE1, a Pseudomonas PB1-like phage. After 24h of infection, the phages were recovered from the wells and added to a fresh 24h-biofilm. This procedure was repeated daily in 24-well plates and the final biofilm-adapted phages were recovered for phage production and characterization. The evolution process resulted in an increased anti-biofilm activity of the adapted phages compared to the wildtype phage, leading to a greater biofilm reduction. The two adapted-phages with the best anti-biofilm activity revealed an increased efficiency-of-plating against several P. aeruginosa clinical strains and P. aeruginosa colonies isolated from the biofilm. When comparing the phage genomes, it was possible to identify two SNPs in genes encoding a tail-fiber and a baseplate. The emergence of mutations in genes involved in bacterial recognition and binding, together with the increased efficiency-of-plating, indicate that the biofilm evolution process improves phage host range and infectivity efficiency. Given the common heterogenicity of biofilms, the enhancement of bacterial recognition may be the key for the increased anti-biofilm activity of the evolved phages.info:eu-repo/semantics/publishedVersio