Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa.

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a "more mobile" and a "less mobile" population: Small particles in cells unable to make polyP are more likely to be "mobile" and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation.

Space and genealogy determine inter-individual differences in siderophore gene expression in bacterial colonies.

Heterogeneity in gene expression is common among clonal cells in bacteria, although the sources and functions of variation often remain unknown. Here, we track cellular heterogeneity in the bacterium Pseudomonas aeruginosa during colony growth by focusing on siderophore gene expression (pyoverdine versus pyochelin) important for iron nutrition. We find that the spatial position of cells within colonies and non-genetic yet heritable differences between cell lineages are significant sources of cellular heterogeneity, while cell pole age and lifespan have no effect. Regarding functions, our results indicate that cells adjust their siderophore investment strategies along a gradient from the colony center to its edge. Moreover, cell lineages with below-average siderophore investment benefit from lineages with above-average siderophore investment, presumably due to siderophore sharing. Our study highlights that single-cell experiments with dual gene expression reporters can identify sources of gene expression variation of interlinked traits and offer explanations for adaptive benefits in bacteria.

Antimicrobial resistance and carbapenemase dissemination in Pseudomonas aeruginosa isolates from Libyan hospitals: a call for surveillance and intervention.

Pseudomonas aeruginosa is a multidrug-resistant bacterium capable of forming biofilms. This study aimed to assess resistance of clinical isolates from Libyan hospitals to antipseudomonal antibiotics, the prevalence of selected extended-spectrum ß-lactamases and carbapenemase genes among these isolates, and the microorganisms' capacity for alginate and biofilm production. Forty-five isolates were collected from four hospitals in Benghazi and Derna, Libya. Antimicrobial susceptibility was determined using agar disc diffusion. The presence of resistance genes (blaCTXM, blaTEM, blaSHV-1, blaGES-1, blaKPC, and blaNDM) was screened using PCR. Biofilm formation was quantified via the crystal violet assay, while alginate production was measured spectrophotometrically. Resistance to antipseudomonal antibiotics ranged from 48.9% to 75.6%. The most prevalent resistance gene was blaNDM (26.7%), followed by blaGES-1 (17.8%). Moreover, all isolates demonstrated varying degrees of biofilm-forming ability and alginate production. No statistically significant correlation was found between biofilm formation and alginate production. The dissemination of resistant genes in P. aeruginosa, particularly carbapenemases, is of great concern. This issue is compounded by the bacteria's biofilm-forming capability. Urgent intervention and continuous surveillance are imperative to prevent further deterioration and the catastrophic spread of resistance among these formidable bacteria.

Adaptive response of Pseudomonas aeruginosa under serial ciprofloxacin exposure.

Understanding the evolution of antibiotic resistance is important for combating drug-resistant bacteria. In this work, we investigated the adaptive response of Pseudomonas aeruginosa to ciprofloxacin. Ciprofloxacin-susceptible P. aeruginosa ATCC 9027, CIP-E1 (P. aeruginosa ATCC 9027 exposed to ciprofloxacin for 14 days) and CIP-E2 (CIP-E1 cultured in antibiotic-free broth for 10 days) were compared. Phenotypic responses including cell morphology, antibiotic susceptibility, and production of pyoverdine, pyocyanin and rhamnolipid were assessed. Proteomic responses were evaluated using comparative iTRAQ labelling LC-MS/MS to identify differentially expressed proteins (DEPs). Expression of associated genes coding for notable DEPs and their related regulatory genes were checked using quantitative reverse transcriptase PCR. CIP-E1 displayed a heterogeneous morphology, featuring both filamentous cells and cells with reduced length and width. By contrast, although filaments were not present, CIP-E2 still exhibited size reduction. Considering the MIC values, ciprofloxacin-exposed strains developed resistance to fluoroquinolone antibiotics but maintained susceptibility to other antibiotic classes, except for carbapenems. Pyoverdine and pyocyanin production showed insignificant decreases, whereas there was a significant decrease in rhamnolipid production. A total of 1039 proteins were identified, of which approximately 25 % were DEPs. In general, there were more downregulated proteins than upregulated proteins. Noted changes included decreased OprD and PilP, and increased MexEF-OprN, MvaT and Vfr, as well as proteins of ribosome machinery and metabolism clusters. Gene expression analysis confirmed the proteomic data and indicated the downregulation of rpoB and rpoS. In summary, the response to CIP involved approximately a quarter of the proteome, primarily associated with ribosome machinery and metabolic processes. Potential targets for bacterial interference encompassed outer membrane proteins and global regulators, such as MvaT.

Biocontrol of multidrug resistant pathogens isolated from fish farms using silver nanoparticles combined with hydrogen peroxide insight to its modulatory effect.

This study was divided into two parts. The first part involved the isolation, and detection of the prevalence and antimicrobial resistance profile of Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio species from Nile tilapia fish and marine aquatic water. One hundred freshly dead Nile tilapia fish were collected from freshwater aquaculture fish farms located in Al-Abbassah district, Sharkia Governorate, and 100 samples of marine aquatic water were collected from fish farms in Port Said. The second part of the study focused on determining the in vitro inhibitory effect of dual-combination of AgNPs-H2O2 on bacterial growth and its down regulatory effect on crucial virulence factors using RT-PCR. The highest levels of A. hydrophila and P. aeruginosa were detected in 43%, and 34% of Nile tilapia fish samples, respectively. Meanwhile, the highest level of Vibrio species was found in 37% of marine water samples. Additionally, most of the isolated A. hydrophila, P. aeruginosa and Vibrio species exhibited a multi-drug resistance profile. The MIC and MBC results indicated a bactericidal effect of AgNPs-H2O2. Furthermore, a transcriptional modulation effect of AgNPs-H2O2 on the virulence-associated genes resulted in a significant down-regulation of aerA, exoU, and trh genes in A. hydrophila, P. aeruginosa, and Vibrio spp., respectively. The findings of this study suggest the effectiveness of AgNPs-H2O2 against drug resistant pathogens related to aquaculture.

A role for the stringent response in ciprofloxacin resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major cause of nosocomial infections and the leading cause of chronic lung infections in cystic fibrosis and chronic obstructive pulmonary disease patients. Antibiotic treatment remains challenging because P. aeruginosa is resistant to high concentrations of antibiotics and has a remarkable ability to acquire mutations conferring resistance to multiple groups of antimicrobial agents. Here we report that when P. aeruginosa is plated on ciprofloxacin (cipro) plates, the majority of cipro-resistant (ciproR) colonies observed at and after 48 h of incubation carry mutations in genes related to the Stringent Response (SR). Mutations in one of the major SR components, spoT, were present in approximately 40% of the ciproR isolates. Compared to the wild-type strain, most of these isolates had decreased growth rate, longer lag phase and altered intracellular ppGpp content. Also, 75% of all sequenced mutations were insertions and deletions, with short deletions being the most frequently occurring mutation type. We present evidence that most of the observed mutations are induced on the selective plates in a subpopulation of cells that are not instantly killed by cipro. Our results suggests that the SR may be an important contributor to antibiotic resistance acquisition in P. aeruginosa.

A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

Antibacterial effect of Moringa oleifera on Staphylococcus aureus and Pseudomonas aeruginosa isolated from raw milk and some dairy products with special reference to biofilm gene expression.

Background: Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are well defined as food poisoning pathogens that are highly resistant and need continuous studies. Aim: The purpose of the work was to examine phenotypic and genotypic characteristics of both P. aeruginosa and S. aureus, and treatment trials with medicinal plants. Methods: Samples were examined for isolation of P. aeruginosa and S. aureus on selective media followed by biochemical confirmation, biofilm formation, genes detection, and expression of P. aeruginosa pslA biofilm gene was performed by quantitative real-time polymerase chain reaction after treatment with 0.312 mg/ml Moringa oleifera aqueous extract as a minimum inhibitory concentration. Results: The highest isolation rate of P. aeruginosa was 20% from both raw milk and Kariesh cheese, followed by 16% and 12% from ice cream and processed cheese, respectively, while the highest isolation rate of S. aureus was 36% from raw milk followed by 28% in ice cream and 16% in both Kariesh cheese and processed cheese. 30% of P. aeruginosa isolates were biofilm producers, while only 21% of S. aureus isolates were able to produce biofilm. The P. aeruginosa isolates harbor virulence-associated genes nan1, exoS, toxA, and pslA at 100%, 80%, 40%, and 40%, respectively. Staphylococcus aureus SEs genes were examined in S. aureus strains, where SEA and SEB genes were detected with 60%, but no isolate harbored SEC, SED, or SEE. The significant fold change of P. aeruginosa pslA expression was 0.40332 after treatment with M. oleifera aqueous extract. Conclusion: Pseudomonas aeruginosa and S. aureus harbor dangerous virulence genes that cause food poisoning, but M. oleifera extract could minimize their action.

Biodiversity of carbapenem-resistant bacteria in clinical samples from the Southwest Amazon region (Rondônia/Brazil).

Brazil is recognized for its biodiversity and the genetic variability of its organisms. This genetic variability becomes even more valuable when it is properly documented and accessible. Understanding bacterial diversity through molecular characterization is necessary as it can improve patient treatment, reduce the length of hospital stays and the selection of resistant bacteria, and generate data for health and epidemiological surveillance. In this sense, in this study, we aimed to understand the biodiversity and molecular epidemiology of carbapenem-resistant bacteria in clinical samples recovered in the state of Rondônia, located in the Southwest Amazon region. Retrospective data from the Central Public Health Laboratories (LACEN/RO) between 2018 and 2021 were analysed using the Laboratory Environment Manager Platform (GAL). Seventy-two species with carbapenem resistance profiles were identified, of which 25 species carried at least one gene encoding carbapenemases of classes A (blaKPC-like), B (blaNDM-like, blaSPM-like or blaVIM-like) and D (blaOXA-23-like, blaOXA-24-like, blaOXA-48-like, blaOXA-58-like or blaOXA-143-like), among which we will highlight Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Serratia marcescens, and Providencia spp. With these results, we hope to contribute to the field by providing epidemiological molecular data for state surveillance on bacterial resistance and assisting in public policy decision-making.

Biodegradation characteristics and genomic analysis of a newly isolated indole-degrading strain Pseudomonas aeruginosa Jade-X / Características de biodegradación y análisis genómico de una cepa degradante de indol recientemente aislada Pseudomonas aeruginosa Jade-X

Indole is a typical heterocyclic compound derived from tryptophan widespread in nature. Pseudomonas aeruginosa is one of the most common opportunistic pathogens everywhere in the world. Indole and P. aeruginosa will encounter inevitably; however, the indole transformation process by P. aeruginosa remains unclear. Herein, an indole-degrading strain of P. aeruginosa Jade-X was isolated from activated sludge. Strain Jade-X could degrade 1 mmol/L indole within 48 h with the inoculum size of 1% (v/v). It showed high efficiency in indole degradation under the conditions of 30–42 °C, pH 5.0–9.0, and NaCl concentration less than 2.5%. The complete genome of strain Jade-X was sequenced which was 6508614 bp in length with one chromosome. Bioinformatic analyses showed that strain Jade-X did not contain the indole oxygenase gene. Three cytochrome P450 genes were identified and up-regulated in the indole degradation process by RT-qPCR analysis, while cytochrome P450 inhibitors did not affect the indole degradation process. It suggested that indole oxidation was catalyzed by an unraveled enzyme. An ant gene cluster was identified, among which the anthranilate 1,2-dioxygenase and catechol 1,2-dioxygenase genes were upregulated. An indole-anthranilate-catechol pathway was proposed in indole degradation by strain P. aeruginosa Jade-X. This study enriched our understanding of the indole biodegradation process in P. aeruginosa.(AU)

Molecular epidemiology of carbapenem-resistant gram-negative bacilli in Ecuador.

INTRODUCTION: Carbapenem-resistant gram-negative bacilli are a worldwide concern because of high morbidity and mortality rates. Additionally, the increasing prevalence of these bacteria is dangerous. To investigate the extent of antimicrobial resistance and prioritize the utility of novel drugs, we evaluated the molecular characteristics and antimicrobial susceptibility profiles of carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii in Ecuador in 2022. METHODS: Ninety-five clinical isolates of carbapenem non-susceptible gram-negative bacilli were collected from six hospitals in Ecuador. Carbapenem resistance was confirmed with meropenem disk diffusion assays following Clinical Laboratory Standard Institute guidelines. Carbapenemase production was tested using a modified carbapenemase inactivation method. Antimicrobial susceptibility was tested with a disk diffusion assay, the Vitek 2 System, and gradient diffusion strips. Broth microdilution assays were used to assess colistin susceptibility. All the isolates were screened for the blaKPC, blaNDM, blaOXA-48, blaVIM and blaIMP genes. In addition, A. baumannii isolates were screened for the blaOXA-23, blaOXA-58 and blaOXA-24/40 genes. RESULTS: Carbapenemase production was observed in 96.84% of the isolates. The blaKPC, blaNDM and blaOXA-48 genes were detected in Enterobacterales, with blaKPC being predominant. The blaVIM gene was detected in P. aeruginosa, and blaOXA-24/40 predominated in A. baumannii. Most of the isolates showed co-resistance to aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. Both ceftazidime/avibactam and meropenem/vaborbactam were active against carbapenem-resistant gram-negative bacilli that produce serin-carbapenemases. CONCLUSION: The epidemiology of carbapenem resistance in Ecuador is dominated by carbapenemase-producing K. pneumoniae harbouring blaKPC. Extensively drug resistant (XDR) P. aeruginosa and A. baumannii were identified, and their identification revealed the urgent need to implement strategies to reduce the dissemination of these strains.

Staphopain mediated virulence and antibiotic resistance alteration in co-infection of Staphylococcus aureus and Pseudomonas aeruginosa: an animal model.

Polymicrobial communities lead to worsen the wound infections, due to mixed biofilms, increased antibiotic resistance, and altered virulence production. Promising approaches, including enzymes, may overcome the complicated condition of polymicrobial infections. Therefore, this study aimed to investigate Staphopain A-mediated virulence and resistance alteration in an animal model of Staphylococcus aureus and Pseudomonas aeruginosa co-infection. S. aureus and P. aeruginosa were co-cultured on the L-929 cell line and wound infection in an animal model. Then, recombinant staphopain A was purified and used to treat mono- and co-infections. Following the treatment, changes in virulence factors and resistance were investigated through phenotypic methods and RT-PCR. Staphopain A resulted in a notable reduction in the viability of S. aureus and P. aeruginosa. The biofilm formed in the wound infection in both animal model and cell culture was disrupted remarkably. Moreover, the biofilm-encoding genes, quorum sensing regulating genes, and virulence factors (hemolysin and pyocyanin) controlled by QS were down-regulated in both microorganisms. Furthermore, the resistance to vancomycin and doripenem decreased following treatment with staphopain A. According to this study, staphopain A might promote wound healing and cure co-infection. It seems to be a promising agent to combine with antibiotics to overcome hard-to-cure infections.

Antimicrobial resistance profiles of Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae strains isolated from broiler chickens.

Globally, the spread of multidrug-resistant Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae from food to humans poses a severe threat to public health. The aim of this study was to assess the co-occurrence of colistin and ß-lactamase resistance genes in E. coli, K. pneumoniae, and P. aeruginosa strains isolated from faeces of abattoir broiler chickens. The E. coli, P. aeruginosa and K. pneumoniae isolates were successfully detected from faecal samples by polymerase chain reaction (PCR) at infection rates of 60.7%, 22.5% and 16.7% respectively. The isolates displayed the highest levels of antibiotic resistance (AR) against ampicillin (82.3%) and amoxicillin-clavulanic acid (74.2%) for E. coli, followed by cefoxitin (70.6%) for K. pneumoniae, whilst P. aeruginosa displayed 26.1% antibiotic resistance (AR) against both ampicillin and colistin sulphate. The colistin mcr-1 gene was harboured by 46.8%, 47.1% and 21.7%, E. coli, K. pneumonia and P. aeruginosa isolates respectively. Ten out of 62 (16.1%), 6/17 (35.3%), 4/23 (17.4%) isolates were phenotypically classified as ESBL E. coli, K. pneumoniae, and P. aeruginosa respectively. The ESBL-E. coli isolates respectively possessed blaCTX-M (60%), blaTEM (20%) and blaCTX-M-9 (10%) genes. The ESBL-K. pneumoniae harboured, blaCTX-M (50%), blaOXA (33%), blaCARB (17%), and blaCTX-M-9 (17%) genes respectively, whilst, P. aeruginosa isolates respectively carried blaTEM (75%), blaCTX-M (50%), blaOXA (25%) and blaCARB (25%) genes. Molecular analysis identified the blaCTX-Mß-lactamase-encoding genes collectively from E. coli, P. aeruginosa, K. pneumoniae isolates. Colistin and ß-lactamase genes were present in only 16.7%, 6.9%, and 2.9% of E. coli, K. pneumoniae, and P. aeruginosa isolates, respectively. A total of 17, 7 and 3 isolates for E. coli, K. pneumoniae and P. aeruginosa respectively carried both colistin and ß-lactamase antibiotics resistant genes. This is a public health threat that points to a challenge in the treatment of infections caused by these zoonotic bacteria. Data generated from this study will contribute to formulation of new strategies for combating spread of E. coli, K. pneumoniae, and P. aeruginosa isolates as well as prevention of their AR development.

Significant role of pyocyanin and exotoxin A in the pathogenesis of Pseudomonas aeruginosa isolated from hospitalized patients.

AIM: Due to the importance of exotoxin A and pyocyanin in the pathogenicity of this bacterium, we decided to evaluate the prevalence of genes encoding these virulence factors in clinical isolates of P.aeruginosa.

Comparative genomic analysis of Pseudomonas aeruginosa strains susceptible and resistant to carbapenems and aztreonam isolated from patients with healthcare-associated infections in a Mexican hospital.

Pseudomonas aeruginosa (PA) is an important opportunistic pathogen that causes different infections on immunocompromised patients. Within PA accessory genome, differences in virulence, antibiotic resistance and biofilm formation have been described between strains, leading to the emergence of multidrug-resistant strains. The genome sequences of 17 strains isolated from patients with healthcare-associated infections in a Mexican hospital were genomically and phylogenetically analyzed and antibiotic resistance genes, virulence genes, and biofilm formation genes were detected. Fifteen of the 17 strains were resistant to at least two of the carbapenems meropenem, imipenem, and the monobactam aztreonam. The antibiotic resistance (mexA, mexB, and oprM) and the biofilm formation (pslA and pslD) genes were detected in all strains. Differences were found between strains in accessory genome size. The strains had different sequence types, and seven strains had sequence types associated with global high risk epidemic PA clones. All strains were represented in two groups among PA global strains. In the 17 strains, horizontally acquired resistance genes to aminoglycosides and beta-lactams were found, mainly, and between 230 and 240 genes that encode virulence factors. The strains under study were variable in terms of their accessory genome, antibiotic resistance, and virulence genes. With these characteristics, we provide information about the genomic diversity of clinically relevant PA strains.

Molecular mechanism of siderophore regulation by the Pseudomonas aeruginosa BfmRS two-component system in response to osmotic stress.

Pseudomonas aeruginosa, a common nosocomial pathogen, relies on siderophores to acquire iron, crucial for its survival in various environments and during host infections. However, understanding the molecular mechanisms of siderophore regulation remains incomplete. In this study, we found that the BfmRS two-component system, previously associated with biofilm formation and quorum sensing, is essential for siderophore regulation under high osmolality stress. Activated BfmR directly bound to the promoter regions of pvd, fpv, and femARI gene clusters, thereby activating their transcription and promoting siderophore production. Subsequent proteomic and phenotypic analyses confirmed that deletion of BfmRS reduces siderophore-related proteins and impairs bacterial survival in iron-deficient conditions. Furthermore, phylogenetic analysis demonstrated the high conservation of the BfmRS system across Pseudomonas species, functional evidences also indicated that BfmR homologues from Pseudomonas putida KT2440 and Pseudomonas sp. MRSN12121 could bind to the promoter regions of key siderophore genes and osmolality-mediated increases in siderophore production were observed. This work illuminates a novel signaling pathway for siderophore regulation and enhances our understanding of siderophore-mediated bacterial interactions and community establishment.

PqsA mutation-mediated enhancement of phage-mediated combat against Pseudomonas aeruginosa.

Phage therapy is a potential approach in the biocontrol of foodborne pathogens. However, the emergence of phage resistance and the narrow host range of most phage isolates continue to limit the antimicrobial efficacy of phages. Here, we investigated the potential of the pqsA gene, encoding the anthranilate-CoA ligase enzyme, as an adjuvant for phage therapy. The knockout of the pqsA gene significantly enhanced the bactericidal effect of phages vB_Pae_QDWS and vB_Pae_S1 against Pseudomonas aeruginosa. Under phage infection pressure, the growth of the PaΔpqsA was significantly inhibited within 8 h compared to the wild-type PAO1. Furthermore, we found that altering phage adsorption is not how PaΔpqsA responds to phage infection. Although pqsA represents a promising target for enhancing phage killing, it may not be applicable to all phages, such as types vB_Pae_W3 and vB_Pae_TR. Our findings provide new material reserves for the future design of novel phage-based therapeutic strategies.

Uncovering the GacS-mediated role in evolutionary progression through trajectory reconstruction in Pseudomonas aeruginosa.

The genetic diversities of subpopulations drive the evolution of pathogens and affect their ability to infect hosts and cause diseases. However, most studies to date have focused on the identification and characterization of adaptive mutations in single colonies, which do not accurately reflect the phenotypes of an entire population. Here, to identify the composition of variant subpopulations within a pathogen population, we developed a streamlined approach that combines high-throughput sequencing of the entire population cells with genotyping of single colonies. Using this method, we reconstructed a detailed quorum-sensing (QS) evolutionary trajectory in Pseudomonas aeruginosa. Our results revealed a new adaptive mutation in the gacS gene, which codes for a histidine kinase sensor of a two-component system (TCS), during QS evolution. This mutation reduced QS activity, allowing the variant to sweep throughout the whole population, while still being vulnerable to invasion by the emerging QS master regulator LasR-null mutants. By tracking the evolutionary trajectory, we found that mutations in gacS facilitated QS-rewiring in the LasR-null mutant. This rapid QS revertant caused by inactive GacS was found to be associated with the promotion of ribosome biogenesis and accompanied by a trade-off of reduced bacterial virulence on host cells. In conclusion, our findings highlight the crucial role of the global regulator GacS in modulating the progression of QS evolution and the virulence of the pathogen population.

Phenotypic and genotypic characterization of resistance and virulence in Pseudomonas aeruginosa isolated from poultry farms in Egypt using whole genome sequencing.

Pseudomonas aeruginosa (P. aeruginosa) is an ESKAPE pathogen that can quickly develop resistance to most antibiotics. This bacterium is a zoonotic pathogen that can be found in humans, animals, foods, and environmental samples, making it a One-Health concern. P. aeruginosa threatens the poultry industry in Egypt, leading to significant economic losses. However, the investigation of this bacterium using NGS technology is nearly non-existent in Egypt. In this study, 38 isolates obtained from broiler farms of the Delta region were phenotypically investigated, and their genomes were characterized using whole genome sequencing (WGS). The study found that 100% of the isolates were resistant to fosfomycin and harbored the fosA gene. They were also resistant to trimethoprim/sulfamethoxazole, although only one isolate harbored the sul1 gene. Non-susceptibility (resistant, susceptible with increased dose) of colistin was observed in all isolates. WGS analysis revealed a high level of diversity between isolates, and MLST analysis allocated the 38 P. aeruginosa isolates into 11 distinct sequence types. The most predominant sequence type was ST267, found in 13 isolates, followed by ST1395 in 8 isolates. The isolates were susceptible to almost all tested antibiotics carrying only few different antimicrobial resistance (AMR) genes. Various AMR genes that confer resistance mainly to ß-lactam, aminoglycoside, sulfonamide, and phenicol compounds were identified. Additionally, several virulence associated genes were found without any significant differences in number and distribution among isolates. The majority of the virulence genes was identified in almost all isolates. The fact that P. aeruginosa, which harbors several AMR and virulence-associated factors, is present in poultry farms is alarming and threatens public health. The misuse of antimicrobial compounds in poultry farms plays a significant role in resistance development. Thus, increasing awareness and implementing strict veterinary regulations to guide the use of veterinary antibiotics is required to reduce health and environmental risks. Further studies from a One-Health perspective using WGS are necessary to trace the potential transmission routes of resistance between animals and humans and clarify resistance mechanisms.

Performance evaluation of rhamnolipid biosurfactant produced by Pseudomonas aeruginosa and its effect on marine oil-spill remediation.

This study aimed to reveal that the effect of biosurfactant on the dispersion and degradation of crude oil. Whole genome analysis showed that Pseudomonas aeruginosa GB-3 contained abundant genes involved in biosurfactant synthesis and metabolic processes and had the potential to degrade oil. The biosurfactant produced by strain GB-3 was screened by various methods. The results showed that the surface tension reduction activity was 28.6 mN·m-1 and emulsification stability was exhibited at different pH, salinity and temperature. The biosurfactant was identified as rhamnolipid by LC-MS and FTIR. The fermentation conditions of strain GB-3 were optimized by response surface methodology, finally the optimal system (carbon source: glucose, nitrogen source: ammonium sulfate, C/N ratio:16:1, pH: 7, temperature: 30-35 °C) was determined. Compared with the initial fermentation, the yield of biosurfactant increased by 4.4 times after optimization. In addition, rhamnolipid biosurfactant as a dispersant could make the dispersion of crude oil reach 38% within seven days, which enhanced the bioavailability of crude oil. As a biostimulant, it could also improve the activity of indigenous microorganism and increase the degradation rate of crude oil by 10-15%. This study suggested that rhamnolipid biosurfactant had application prospect in bioremediation of marine oil-spill.