Molecular characterization of Pseudomonas aeruginosa from diabetic foot infections in Tunisia.

Background. Pseudomonas aeruginosa is an invasive organism that frequently causes severe tissue damage in diabetic foot ulcers.Gap statement. The characterisation of P. aeruginosa strains isolated from diabetic foot infections has not been carried out in Tunisia.Purpose. The aim was to determine the prevalence of P. aeruginosa isolated from patients with diabetic foot infections (DFIs) in Tunisia and to characterize their resistance, virulence and molecular typing.Methods. Patients with DFIs admitted to the diabetes department of the International Hospital Centre of Tunisia, from September 2019 to April 2021, were included in this prospective study. P. aeruginosa were obtained from the wound swabs, aspiration and soft tissue biopsies during routine clinical care and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing, serotyping, integron and OprD characterization, virulence, biofilm production, pigment quantification, elastase activity and molecular typing were analysed in all recovered P. aeruginosa isolates by phenotypic tests, specific PCRs, sequencing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.Results. Sixteen P. aeruginosa isolates (16.3 %) were recovered from 98 samples of 78 diabetic patients and were classified into 6 serotypes (O:11 the most frequent), 11 different PFGE patterns and 10 sequence types (three of them new ones). The high-risk clone ST235 was found in two isolates. The highest resistance percentages were observed to netilmicin (69 %) and cefepime (43.8 %). Four multidrug-resistant (MDR) isolates (25 %) were detected, three of them being carbapenem-resistant. The ST235-MDR strain harboured the In51 class 1 integron (intI1 +aadA6+orfD+qacED1-sul1). According to the detection of 14 genes involved in virulence or quorum sensing, 5 virulotypes were observed, including 5 exoU-positive, 9 exoS-positive and 2 exoU/exoS-positive strains. The lasR gene was truncated by ISPpu21 insertion sequence in one isolate, and a deletion of 64 bp in the rhlR gene was detected in the ST235-MDR strain. Low biofilm, pyoverdine and elastase production were detected in all P. aeruginosa; however, the lasR-truncated strain showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity, high production of phenazines and high biofilm formation.Conclusions. Our study demonstrated for the first time the prevalence and the molecular characterization of P. aeruginosa strains from DFIs in Tunisia, showing a high genetic diversity, moderate antimicrobial resistance, but a high number of virulence-related traits, highlighting their pathological importance.

Evolution and host-specific adaptation of Pseudomonas aeruginosa.

The major human bacterial pathogen Pseudomonas aeruginosa causes multidrug-resistant infections in people with underlying immunodeficiencies or structural lung diseases such as cystic fibrosis (CF). We show that a few environmental isolates, driven by horizontal gene acquisition, have become dominant epidemic clones that have sequentially emerged and spread through global transmission networks over the past 200 years. These clones demonstrate varying intrinsic propensities for infecting CF or non-CF individuals (linked to specific transcriptional changes enabling survival within macrophages); have undergone multiple rounds of convergent, host-specific adaptation; and have eventually lost their ability to transmit between different patient groups. Our findings thus explain the pathogenic evolution of P. aeruginosa and highlight the importance of global surveillance and cross-infection prevention in averting the emergence of future epidemic clones.

Modeling Host-Pathogen Interactions in C. elegans: Lessons Learned from Pseudomonas aeruginosa Infection.

Infections, such as that by the multiresistant opportunistic bacterial pathogen Pseudomonas aeruginosa, may pose a serious health risk, especially on vulnerable patient populations. The nematode Caenorhabditis elegans provides a simple organismal model to investigate both pathogenic mechanisms and the emerging role of innate immunity in host protection. Here, we review the virulence and infection strategies of P. aeruginosa and host defenses of C. elegans. We summarize the recognition mechanisms of patterns of pathogenesis, including novel pathogen-associated molecular patterns and surveillance immunity of translation, mitochondria, and lysosome-related organelles. We also review the regulation of antimicrobial and behavioral defenses by the worm's neuroendocrine system. We focus on how discoveries in this rich field align with well-characterized evolutionary conserved protective pathways, as well as on potential crossovers to human pathogenesis and innate immune responses.

Genomic Comparative of Pseudomonas aeruginosa Small Colony Variant, Mucoid and Non-mucoid Phenotypes Obtained from a Patient with Cystic Fibrosis During Respiratory Exacerbations.

Pseudomonas aeruginosa, the most prevalent opportunistic pathogen in chronic obstructive pulmonary disease, associated with high morbidity and mortality in patients with cystic fibrosis (CF), is practically impossible to be eradicated from the airways in chronicity. Its extraordinary genomic plasticity is possibly associated with high antimicrobial resistance, virulence factors, and its phenotypic diversity. The occurrence of P. aeruginosa isolates promoting airway infection, showing mucoid, non-mucoid, and small colony variant (SCV) phenotypes, was observed simultaneously, in the present study, in sputum cultures obtained from a male CF young patient with chronic pulmonary infection for over a decade. The isolates belonged to a new ST (2744) were obtained in two moments of exacerbation of the respiratory disease, in which he was hospitalized. Genetic background and phenotypic analysis indicated that the isolates exhibited multi- and pan-antimicrobial resistant profiles, as well as non-susceptible to polymyxin and predominantly hypermutable (HPM) phenotypes. Whole genome sequencing showed variations in genome sizes, coding sequences and their determinants of resistance and virulence. The annotated genomes were compared for antimicrobial resistance, hypermutability, and SCV characteristics. We highlight the lack of reported genetic determinants of SCV emergence and HPM phenotypes, which can be explained in part due to the very short time between collections of isolates. To the best of our knowledge, this is the first report of genome sequencing of P. aeruginosa SCV from a CF patient in Brazil.

Pseudomonas aeruginosa breaches respiratory epithelia through goblet cell invasion in a microtissue model.

Pseudomonas aeruginosa, a leading cause of severe hospital-acquired pneumonia, causes infections with up to 50% mortality rates in mechanically ventilated patients. Despite some knowledge of virulence factors involved, it remains unclear how P. aeruginosa disseminates on mucosal surfaces and invades the tissue barrier. Using infection of human respiratory epithelium organoids, here we observed that P. aeruginosa colonization of apical surfaces is promoted by cyclic di-GMP-dependent asymmetric division. Infection with mutant strains revealed that Type 6 Secretion System activities promote preferential invasion of goblet cells. Type 3 Secretion System activity by intracellular bacteria induced goblet cell death and expulsion, leading to epithelial rupture which increased bacterial translocation and dissemination to the basolateral epithelium. These findings show that under physiological conditions, P. aeruginosa uses coordinated activity of a specific combination of virulence factors and behaviours to invade goblet cells and breach the epithelial barrier from within, revealing mechanistic insight into lung infection dynamics.

Beyond antibiotics: Emerging antivirulence strategies to combat Pseudomonas aeruginosa in cystic fibrosis.

Pseudomonas aeruginosa is an opportunistic pathogen that poses a significant threat to individuals suffering from cystic fibrosis (CF). The pathogen is highly prevalent in CF individuals and is responsible for chronic infection, resulting in severe tissue damage and poor patient outcome. Prolonged antibiotic administration has led to the emergence of multidrug resistance in P. aeruginosa. In this direction, antivirulence strategies achieving targeted inhibition of bacterial virulence pathways, including quorum sensing, efflux pumps, lectins, and iron chelators, have been explored against CF isolates of P. aeruginosa. Hence, this review article presents a bird's eye view on the pulmonary infections involving P. aeruginosa in CF patients by laying emphasis on factors contributing to bacterial colonization, persistence, and disease progression along with the current line of therapeutics against P. aeruginosa in CF. We further collate scientific literature and discusses various antivirulence strategies that have been tested against P. aeruginosa isolates from CF patients.

Carbon source competition within the wound microenvironment can significantly influence infection progression.

It is becoming increasingly apparent that commensal skin bacteria have an important role in wound healing and infection progression. However, the precise mechanisms underpinning many of these probiotic interactions remain to be fully uncovered. In this work, we demonstrate that the common skin commensal Cutibacterium acnes can limit the pathogenicity of the prevalent wound pathogen Pseudomonas aeruginosa in vivo. We show that this impact on pathogenicity is independent of any effect on growth, but occurs through a significant downregulation of the Type Three Secretion System (T3SS), the primary toxin secretion system utilised by P. aeruginosa in eukaryotic infection. We also show a downregulation in glucose acquisition systems, a known regulator of the T3SS, suggesting that glucose availability in a wound can influence infection progression. C. acnes is well known as a glucose fermenting organism, and we demonstrate that topically supplementing a wound with glucose reverses the probiotic effects of C. acnes. This suggests that introducing carbon source competition within the wound microenvironment may be an effective way to prevent or limit wound infection.

Bacteriophage protein Dap1 regulates evasion of antiphage immunity and Pseudomonas aeruginosa virulence impacting phage therapy in mice.

Bacteriophages have evolved diverse strategies to overcome host defence mechanisms and to redirect host metabolism to ensure successful propagation. Here we identify a phage protein named Dap1 from Pseudomonas aeruginosa phage PaoP5 that both modulates bacterial host behaviour and contributes to phage fitness. We show that expression of Dap1 in P. aeruginosa reduces bacterial motility and promotes biofilm formation through interference with DipA, a c-di-GMP phosphodiesterase, which causes an increase in c-di-GMP levels that trigger phenotypic changes. Results also show that deletion of dap1 in PaoP5 significantly reduces genome packaging. In this case, Dap1 directly binds to phage HNH endonuclease, prohibiting host Lon-mediated HNH degradation and promoting phage genome packaging. Moreover, PaoP5Δdap1 fails to rescue P. aeruginosa-infected mice, implying the significance of dap1 in phage therapy. Overall, these results highlight remarkable dual functionality in a phage protein, enabling the modulation of host behaviours and ensuring phage fitness.

A specific innate immune response silences the virulence of Pseudomonas aeruginosa in a latent infection model in the Drosophila melanogaster host.

Microbial pathogenicity often depends on the route of infection. For instance, P. aeruginosa or S. marcescens cause acute systemic infections when low numbers of bacteria are injected into D. melanogaster flies whereas flies succumb much slower to the continuous ingestion of these pathogens, even though both manage to escape from the gut compartment and reach the hemocoel. Here, we have developed a latent P. aeruginosa infection model by feeding flies on the bacteria for a short period. The bacteria stably colonize internal tissues yet hardly cause any damage since latently-infected flies live almost as long as noninfected control flies. The apparently dormant bacteria display particular characteristics in terms of bacterial colony morphology, composition of the outer cell wall, and motility. The virulence of these bacteria can however be reactivated upon wounding the host. We show that melanization but not the cellular or the systemic humoral response is the predominant host defense that establishes latency and may coerce the bacteria to a dormant state. In addition, the lasting activation of the melanization responses in latently-infected flies provides a degree of protection to the host against a secondary fungal infection. Latent infection by an ingested pathogen protects against a variety of homologous or heterologous systemic secondary infectious challenges, a situation previously described for the endosymbiotic Wolbachia bacteria, a guard against viral infections.

Ser/Thr protein kinase Stk1 phosphorylates the key transcriptional regulator AlgR to modulate virulence and resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections worldwide and has emerged as a serious public health threat, due in large part to its multiple virulence factors and remarkable resistance capabilities. Stk1, a eukaryotic-type Ser/Thr protein kinase, has been shown in our previous work to be involved in the regulation of several signalling pathways and biological processes. Here, we demonstrate that deletion of stk1 leads to alterations in several virulence- and resistance-related physiological functions, including reduced pyocyanin and pyoverdine production, attenuated twitching motility, and enhanced biofilm production, extracellular polysaccharide secretion, and antibiotic resistance. Moreover, we identified AlgR, an important transcriptional regulator, as a substrate for Stk1, with its phosphorylation at the Ser143 site catalysed by Stk1. Intriguingly, both the deletion of stk1 and the mutation of Ser143 of AlgR to Ala result in similar changes in the above-mentioned physiological functions. Furthermore, assays of algR expression in these strains suggest that changes in the phosphorylation state of AlgR, rather than its expression level, underlie changes in these physiological functions. These findings uncover Stk1-mediated phosphorylation of AlgR as an important mechanism for regulating virulence and resistance in P. aeruginosa.

Miconazole and phenothiazine hinder the quorum sensing regulated virulence in Pseudomonas aeruginosa.

Antibiotic resistance is a major health problem worldwide. Pseudomonas aeruginosa is a Gram-negative pathogen with an arsenal of virulence factors and elevated antimicrobial resistance. It is a leading cause of nosocomial infections with high morbidity and mortality. The significant time and effort required to develop new antibiotics can be circumvented using alternative therapeutic strategies, including anti-virulence targets. This study aimed to investigate the anti-virulence activity of the FDA-approved drugs miconazole and phenothiazine against P. aeruginosa. The phenotypic effect of sub-inhibitory concentrations of miconazole and phenothiazine on biofilm, pyocyanin, protease, rhamnolipid and hemolysin activities in PAO1 strain was examined. qRT-PCR was used to assess the effect of drugs on quorum-sensing genes that regulate virulence. Further, the anti-virulence potential of miconazole and phenothiazine was evaluated in silico and in vivo. Miconazole showed significant inhibition of Pseudomonas virulence by reducing biofilm-formation approximately 45-48%, hemolytic-activity by 59%, pyocyanin-production by 47-49%, rhamnolipid-activity by approximately 42-47% and protease activity by 36-40%. While, phenothiazine showed lower anti-virulence activity, it inhibited biofilm (31-35%), pyocyanin (37-39%), protease (32-40%), rhamnolipid (35-40%) and hemolytic activity (47-56%). Similarly, there was significantly reduced expression of RhlR, PqsR, LasI and LasR following treatment with miconazole, but less so with phenothiazine. In-silico analysis revealed that miconazole had higher binding affinity than phenothiazine to LasR, RhlR, and PqsR QS-proteins. Furthermore, there was 100% survival in mice injected with PAO1 treated with miconazole. In conclusion, miconazole and phenothiazine are promising anti-virulence agents for P. aeruginosa.

Protocol for survival assay of Caenorhabditis elegans to Pseudomonas aeruginosa PA14 infection.

The nematode Caenorhabditis elegans is a powerful model organism for studying the molecular and cellular mechanisms of innate immunity governed by the intestine. Here, we present a protocol to perform C. elegans survival assays to infection by the bacterial pathogen Pseudomonas aeruginosa PA14. Specifically, we describe steps for preparing C. elegans strains and PA14 bacteria for survival assays. This protocol will assist researchers to study genes involved in intestinal innate immunity and gut defense against pathogen infection. For complete details on the use and execution of this protocol, please refer to Liu et al.1 and Zheng et al.2.

Hospital wastewater as source of human pathogenic bacteria: A phenotypic and genomic analysis of international high-risk clone VIM-2-producing Pseudomonas aeruginosa ST235/O11.

Pseudomonas aeruginosa belong to the special pathogen group capable of causing serious infections, with high mortality rates. The aim of this study was to describe the antibiotic resistance and genomic characteristics of Pseudomonas aeruginosa belonging to international high-risk clone ST235 (GPAE0131 isolate), obtained from hospital wastewater. P. aeruginosa GPAE0131 was isolated from ward tertiary hospital in Brazil and the antibiotic resistance profile was determined by the disc-diffusion method. Genomic characteristics related to antibiotic resistance and virulence factors were evaluated by genomic DNA sequencing on the Illumina MiSeq platform and bioinformatic analysis. GPAE0131 isolate showed resistance to piperacillin-tazobactam, cefepime, ceftazidime, imipenem, meropenem, ciprofloxacin, levofloxacin and tobramycin. Resistome comprehend of resistance genes to ß-lactams (blaVIM-2, blaOXA-4, blaOXA-488, blaPDC-35), aminoglycosides (aph(3')-IIb, aac(6')-IIc, aac(6')-Ib9, aadA1), fosfomycin (fosA), chloramphenicol (catB7) and sulfonamides (sul1). Genome comparisons evidence insertion of blaVIM-2 and blaOXA-4 genes. GPAE0131 isolate was predicted to be pathogenic to humans and several virulence factors were found, including encoding gene for ExoU and exotoxin A. All of these features into a pathogenic international high-risk clone (ST235), classified as critical priority, stands out as public health concern due to the widespread dispersal of human pathogens through wastewater. It is suggested that mitigating measures be implemented, such as the treatment of hospital sewage and the addition of tertiary treatment, to prevent the escape of pathogens at this level into the environment.

Exploring roles of the chitinase ChiC in modulating Pseudomonas aeruginosa virulence phenotypes.

Chitinases are ubiquitous enzymes involved in biomass degradation and chitin turnover in nature. Pseudomonas aeruginosa (PA), an opportunistic human pathogen, expresses ChiC, a secreted glycoside hydrolase 18 family chitinase. Despite speculation about ChiC's role in PA disease pathogenesis, there is scant evidence supporting this hypothesis. Since PA cannot catabolize chitin, we investigated the potential function(s) of ChiC in PA pathophysiology. Our findings show that ChiC exhibits activity against both insoluble (α- and ß-chitin) and soluble chitooligosaccharides. Enzyme kinetics toward (GlcNAc)4 revealed a kcat of 6.50 s-1 and a KM of 1.38 mM, the latter remarkably high for a canonical chitinase. In our label-free proteomics investigation, ChiC was among the most abundant proteins in the Pel biofilm, suggesting a potential contribution to PA biofilm formation. Using an intratracheal challenge model of PA pneumonia, the chiC::ISphoA/hah transposon insertion mutant paradoxically showed slightly increased virulence compared to the wild-type parent strain. Our results indicate that ChiC is a genuine chitinase that contributes to a PA pathoadaptive pathway.IMPORTANCEIn addition to performing chitin degradation, chitinases from the glycoside hydrolase 18 family have been found to play important roles during pathogenic bacterial infection. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing pneumonia in immunocompromised individuals. Despite not being able to grow on chitin, the bacterium produces a chitinase (ChiC) with hitherto unknown function. This study describes an in-depth characterization of ChiC, focusing on its potential contribution to the bacterium's disease-causing ability. We demonstrate that ChiC can degrade both polymeric chitin and chitooligosaccharides, and proteomic analysis of Pseudomonas aeruginosa biofilm revealed an abundance of ChiC, hinting at a potential role in biofilm formation. Surprisingly, a mutant strain incapable of ChiC production showed higher virulence than the wild-type strain. While ChiC appears to be a genuine chitinase, further investigation is required to fully elucidate its contribution to Pseudomonas aeruginosa virulence, an important task given the evident health risk posed by this bacterium.

Bacterial metallothionein, PmtA, a novel stress protein found on the bacterial surface of Pseudomonas aeruginosa and involved in management of oxidative stress and phagocytosis.

Metallothioneins (MTs) are small cysteine-rich proteins that play important roles in homeostasis and protection against heavy metal toxicity and oxidative stress. The opportunistic pathogen, Pseudomonas aeruginosa, expresses a bacterial MT known as PmtA. Utilizing genetically modified P. aeruginosa PAO1 strains (a human clinical wound isolate), we show that inducing pmtA increases levels of pyocyanin and biofilm compared to other PAO1 isogenic strains, supporting previous results that pmtA is important for pyocyanin and biofilm production. We also show that overexpression of pmtA in vitro provides protection for cells exposed to oxidants, which is a characteristic of inflammation, indicating a role for PmtA as an antioxidant in inflammation. We found that a pmtA clean deletion mutant is phagocytized faster than other PAO1 isogenic strains in THP-1 human macrophage cells, indicating that PmtA provides protection from the phagocytic attack. Interestingly, we observed that monoclonal anti-PmtA antibody binds to PmtA, which is accessible on the surface of PAO1 strains using both flow cytometry and enzyme-linked immunosorbent assay techniques. Finally, we investigated intracellular persistence of these PAO1 strains within THP-1 macrophages cells and found that the phagocytic endurance of PAO1 strains is affected by pmtA expression. These data show for the first time that a bacterial MT (pmtA) can play a role in the phagocytic process and can be found on the outer surface of PAO1. Our results suggest that PmtA plays a role both in protection from oxidative stress and in the resistance to the host's innate immune response, identifying PmtA as a potential therapeutic target in P. aeruginosa infection. IMPORTANCE: The pathogen Pseudomonas aeruginosa is a highly problematic multidrug-resistant (MDR) pathogen with complex virulence networks. MDR P. aeruginosa infections have been associated with increased clinical visits, very poor healthcare outcomes, and these infections are ranked as critical on priority lists of both the Centers for Disease Control and Prevention and the World Health Organization. Known P. aeruginosa virulence factors have been extensively studied and are implicated in counteracting host defenses, causing direct damage to the host tissues, and increased microbial competitiveness. Targeting virulence factors has emerged as a new line of defense in the battle against MDR P. aeruginosa strains. Bacterial metallothionein is a newly recognized virulence factor that enables evasion of the host immune response. The studies described here identify mechanisms in which bacterial metallothionein (PmtA) plays a part in P. aeruginosa pathogenicity and identifies PmtA as a potential therapeutic target.

Pseudomonas aeruginosa two-component system LadS/PA0034 regulates macrophage phagocytosis via fimbrial protein cupA1.

Pseudomonas aeruginosa is one of the most common nosocomial pathogens worldwide, known for its virulence, drug resistance, and elaborate sensor-response network. The primary challenge encountered by pathogens during the initial stages of infection is the immune clearance arising from the host. The resident macrophages of barrier organs serve as the frontline defense against these pathogens. Central to our understanding is the mechanism by which bacteria modify their behavior to circumvent macrophage-mediated clearance, ensuring their persistence and colonization. To successfully evade macrophage-mediated phagocytosis, bacteria must possess an adaptive response mechanism. Two-component systems provide bacteria the agility to navigate diverse environmental challenges, translating external stimuli into cellular adaptive responses. Here, we report that the well-documented histidine kinase, LadS, coupled to a cognate two-component response regulator, PA0034, governs the expression of a vital adhesin called chaperone-usher pathway pilus cupA. The LadS/PA0034 system is susceptible to interference from the reactive oxygen species likely to be produced by macrophages and further lead to a poor adhesive phenotype with scantily cupA pilus, impairing the phagocytosis efficiency of macrophages during acute infection. This dynamic underscores the intriguing interplay: as macrophages deploy reactive oxygen species to combat bacterial invasion, the bacteria recalibrate their exterior to elude these defenses. IMPORTANCE: The notoriety of Pseudomonas aeruginosa is underscored by its virulence, drug resistance, and elaborate sensor-response network. Yet, the mechanisms by which P. aeruginosa maneuvers to escape phagocytosis during acute infections remain elusive. This study pinpoints a two-component response regulator, PA0034, coupled with the histidine kinase LadS, and responds to macrophage-derived reactive oxygen species. The macrophage-derived reactive oxygen species can impair the LadS/PA0034 system, resulting in reduced expression of cupA pilus in the exterior of P. aeruginosa. Since the cupA pilus is an important adhesin of P. aeruginosa, its deficiency reduces bacterial adhesion and changes their behavior to adopt a planktonic lifestyle, subsequently inhibiting the phagocytosis of macrophages by interfering with bacterial adhesion. Briefly, reactive oxygen species may act as environmental cues for the LadS/PA0034 system. Upon recognition, P. aeruginosa may transition to a poorly adhesive state, efficiently avoiding engulfment by macrophages.

The interplay of Pseudomonas aeruginosa and Staphylococcus aureus in dual-species biofilms impacts development, antibiotic resistance and virulence of biofilms in in vitro wound infection models.

Due to high tolerance to antibiotics and pronounced virulence, bacterial biofilms are considered a key factor and major clinical challenge in persistent wound infections. They are typically composed of multiple species, whose interactions determine the biofilm's structural development, functional properties and thus the progression of wound infections. However, most attempts to study bacterial biofilms in vitro solely rely on mono-species populations, since cultivating multi-species biofilms, especially for prolonged periods of time, poses significant challenges. To address this, the present study examined the influence of bacterial composition on structural biofilm development, morphology and spatial organization, as well as antibiotic tolerance and virulence on human skin cells in the context of persistent wound infections. By creating a wound-mimetic microenvironment, the successful cultivation of dual-species biofilms of two of the most prevalent wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, was realized over a period of 72 h. Combining quantitative analysis with electron microscopy and label-free imaging enabled a comprehensive evaluation of the dynamics of biofilm formation and matrix secretion, revealing a twofold increased maturation of dual-species biofilms. Antibiotic tolerance was comparable for both mono-species cultures, however, dual-species communities showed a 50% increase in tolerance, mediated by a significantly reduced penetration of the applied antibiotic into the biofilm matrix. Further synergistic effects were observed, where dual-species biofilms exacerbated wound healing beyond the effects observed from either Pseudomonas or Staphylococcus. Consequently, predicting biofilm development, antimicrobial tolerance and virulence for multi-species biofilms based solely on the results from mono-species biofilms is unreliable. This study underscores the substantial impact of a multi-species composition on biofilm functional properties and emphasizes the need to tailor future studies reflecting the bacterial composition of the respective in vivo situation, leading to a more comprehensive understanding of microbial communities in the context of basic microbiology and the development of effective treatments.

Removal of Pseudomonas type IV pili by a small RNA virus.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates has differential impacts on quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans. We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM (PA0718) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Unveiling the ocular battlefield: Insights into Pseudomonas aeruginosa virulence factors and their implications for multidrug resistance.

The research investigates the virulence factors of Pseudomonas aeruginosa (P. aeruginosa), a pathogen known for its ability to cause human infections by releasing various exoenzymes and virulence factors. Particularly relevant in ocular infections, where tissue degeneration can occur, even after bacterial growth has ceased due to the potential role of secreted proteins/enzymes. Clinical isolates of P. aeruginosa, both ocular (146) and non-ocular (54), were examined to determine the frequency and mechanism of virulence factors. Phenotypic characterization revealed the production of alginate, biofilm, phospholipase C, and alkaline protease, while genotypic testing using internal uniplex PCR identified the presence of Exo U, S, T, Y, and LasB genes. Results showed a significant prevalence of Exo U and Y genes in ocular isolates, a finding unique to Indian studies. Additionally, the study noted that ocular isolates often contained all four secretomes, suggesting a potential link between these factors and ocular infections. These findings contribute to understanding the pathogenesis of P. aeruginosa infections, particularly in ocular contexts, and highlights the importance of comprehensive virulence factor analysis in clinical settings.