OPTIMIZATION OF GROWTH CONDITIONS AND PURIFICATION OF QUORUM SENSING SIGNAL MOLECULES PRODUCED BY

Expression of virulence factors and biofilm formation in P.aeruginosa is associated with production of quorum sensing signal molecules (QSSMs) belonging to the class of acyl homoserine lactones (AHLs). Besides regulating virulence factors, these molecules also interact with eukaryotic cells and can modulate immune response. In most of the studies, synthetic QSSMs have been employed as therapeutic agents. Although 98-99 % homology exist between synthetic and natural AHLs but the biological response against either may in fact be different in natural host. In the present study, under optimized growth conditions there is increase in the production of natural AHLs. Extracted AHLs were detected using C18 reverse phase analytical thin layer chromatography (RP TLC) by employing Agrobacterium tumefaciens as biosensor strain. Preparative TLC assay was successfully performed to purify the 3oxo-C12-HSL and 3-oxo-C10-HSL. This study provides easy and simple method for purification of natural AHLs under optimized conditions, hence these molecules can be employed for future research involving control of infections associated with P.aeruginosa

T helper cell subsets specific for pseudomonas aeruginosa in healthy individuals and patients with cystic fibrosis

Background: We set out to determine the magnitude of antigen-specific memory T helper cell responses to Pseudomonas aeruginosa in healthy humans and patients with cystic fibrosis. Methods: Peripheral blood human memory CD4+ T cells were co-cultured with dendritic cells that had been infected with different strains of Pseudomonas aeruginosa. The T helper response was determined by measuring proliferation, immunoassay of cytokine output, and immunostaining of intracellular cytokines. Results: Healthy individuals and patients with cystic fibrosis had robust antigen-specific memory CD4+ T cell responses to Pseudomonas aeruginosa that not only contained a Th1 and Th17 component but also Th22 cells. In contrast to previous descriptions of human Th22 cells, these Pseudomonal-specific Th22 cells lacked the skin homing markers CCR4 or CCR10, although were CCR6+. Healthy individuals and patients with cystic fibrosis had similar levels of Th22 cells, but the patient group had significantly fewer Th17 cells in peripheral blood. Conclusions: Th22 cells specific to Pseudomonas aeruginosa are induced in both healthy individuals and patients with cystic fibrosis. Along with Th17 cells, they may play an important role in the pulmonary response to this microbe in patients with cystic fibrosis and other conditions

BIOREMEDIASI LOGAM KROMIUM (VI) PADA LIMBAH MODEL PENYAMAKAN KULIT MENGGUNAKAN BAKTERI PSEUDOMONAS AERUGINOSA

Penelitian ini bertujuan untuk mengetahui potensi bakteri lokal Pseudomonas aeruginosa untuk mereduksi logam Cr (VI) menjadi logam Cr (III) pada limbah model penyamakan kulit. Adapun tahapan penelitian yang dilakukan meliputi sub kultur Pseudomonas aeruginosa, penentuan pengaruh keberadaan logam Cr (VI) terhadap ketahanan hidup Pseudomonas aeruginosa dengan cara penentuan kurva pertumbuhan bakteri pada media dengan dan tanpa suplementasi kalium dikromat (K2Cr2O7) sebagai sumber logam Cr (VI), penentuan konsentrasi hambat minimum (Minimal Inhibitory Concentration/MIC) logam Cr (VI) terhadap Pseudomonas aeruginosa dan pengujian kemampuan Pseudomonas aeruginosa dalam mereduksi logam Cr (VI) menjadi logam Cr (III) pada limbah model penyamakan kulit. Hasil penelitian ini menunjukkan bahwa terlihat adanya pengaruh akibat keberadaan logam Cr (VI) pada media yaitu terjadi penurunan tingkat ketahanan hidup Pseudomonas aeruginosa pada media dengan kandungan logam Cr (VI) sebesar 100 ppm. Konsentrasi hambat minimum (Minimal Inhibitory Concentration/MIC) logam Cr (VI) terhadap Pseudomonas aeruginosa terjadi pada konsentrasi logam Cr (VI) sebesar 70 ppm, hasil uji reduksi logam Cr (VI) oleh Pseudomonas aeruginosa menunjukkan bahwa terjadi penurunan konsentrasi logam Cr (VI) sebesar 100 ppm di media menjadi 5,86 ppm dengan efisiensi sebesar 94,73% selama 48 jam pengujian. Berdasarkan penelitian ini, dapat disimpulkan bahwa Pseudomonas aeruginosa berpotensi sebagai agen bioremediator untuk mereduksi logam kromium heksavalen Cr (VI) menjadi logam kromium trivalen Cr (III) pada limbah penyamakan kulit. ; This research aims at investigating the capability of locally bacterial Pseudomonas aeruginosa to convert chromium hexavalent (Cr (VI)) in to chromium trivalent (Cr (III)) on leather tannery effluent model. This study involved in the sub culture of Pseudomonas aeruginosa, the effect of Cr (VI) on growth of Pseudomonas aeruginosa in the medium contaminated with and without sodium dichromate (K2Cr2O7) as a source of hexavalent chromium, minimum inhibition concentration (MIC) of Cr (VI) on Pseudomonas aeruginosa growth, and the capability of Pseudomonas aeruginosa to switch Cr (VI) into Cr (III) through reduction process. The result show the decreased of Pseudomonas aeruginosa growth rate at the medium with 100 ppm of Cr (VI). The MIC measurements shows the minimum inhibition concetration of Cr (VI) to Pseudomonas aeruginosa found at 70 ppm. In addition, it was monitored the the decreased of Cr (VI) from 100 ppm to 5.86 ppm with the efficiency reach to 94.73% during 48 hours of the treatment. Based on this research, it can be concluded that locally bacterial Pseudomonas aeruginosa is potential to be used as a bioremediation agent chromium hexavalent Cr (VI) reduction to chromium trivalent Cr (III) on leather tannery wastewater

Metallothionein Protein Modeling from Pseudomonas aeruginosa PAO1 as A Metal Biosorber Candidate

Metallothionein is a protein that is well known to play a role in metal metabolism in bacterial cells. Metallothionein is a multifunctional protein that has the potential to be used as a metal adsorbing agent. Pseudomonas aeruginosa is a ubiquitous gram-negative and rapid-growth bacterium. In addition, the complete genome of Pseudomonas aeruginosa has been largely known. Pseudomonas aeruginosa PAO1 is a strain of Pseudomonas aeruginosa that the complete genome of this strain is easily accessible in NCBI. These features make Pseudomonas aeruginosa PAO1 become a common model in bacterial studies. This research aimed to find and model the putative metallothionein of Pseudomonas aeruginosa PAO1. This research was carried out by bioinformatic and protein homology methods. Based on the results, the putative metallothionein of Pseudomonas aeruginosa PAO1 was found in the bacterial genome at base sequence of 2355918 to 2356157. The putative metallothionein-encoding gene of Pseudomonas aeruginosa PAO1 has a size of 240 bp. The translation result of the gene showed that the putative metallothionein of Pseudomonas aeruginosa PAO1 has 79 amino acids. The modeling result showed the 3D structure of the putative metallothionein of Pseudomonas aeruginosa PAO1 is similar to the metallothionein 3D structure of Pseudomonas fluorescens Q2-87. The 3D structure of the putative metallothionein of Pseudomonas aeruginosa PAO1 was dominated by turn and coil, but contained 1 α-helix structure and 2 β-sheet structures. Based on protein analysis, it was found that the putative metallothionein of Pseudomonas aeruginosa PAO1 has 1 metal-binding cluster with 10 amino acids and the most important amino acid residue is Cysteine . Even though, there was 1 Histidine amino acid residue on the metal-binding cluster

Antimicrobial susceptibility pattern and multidrug resistance ındex in Pseudomonas aeruginosa among clinical isolates in Denizli, Turkey

Background: Pseudomonas aeruginosa is an important hospital infection agent causing morbidity and mortality with the ability to gain resistance to many antimicrobials. The objective of this study was to determine the sensitivity profiles of nosocomial P. aeruginosa isolates in Denizli, Turkey. Methods: A total 120 P. aeruginosa strains which were isolated from specimens sent to the microbiology laboratory between January 2015 and December 2015 were investigated. Antimicrobial resistance was determined by agar disc diffusion method using Mueller-Hinton agar according to Clinical and Laboratory Standards Institute recommendations. Results: With respect to sensitivity pattern, the most sensitive antimicrobials were Amikacin, colistin, tobramisin, netilmicin and gentamicin and the resistance rates were detected as 97%, 96%, 92%, 90%, 83%, respectively over 120 P. aeruginosa strains. The sensitivity rates for the other antimicrobials were 56% for Piperacilin and 54% for Tazobactam. P. aeruginosa strains 62 (52%) isolates showed multiple antimicrobial resistance to 13 antimicrobials Conclusion: To prevent the spread of the resistant bacteria, it is critically important to have strict antimicrobial policies while surveillance programmes for multidrug resistant organisms and infection control procedures need to be implemented. In the meantime, it is desirable that the antimicrobial susceptibility pattern of bacterial pathogens like P. aeruginosa in specialized clinical units to be continuously monitored and the results readily made available to clinicians so as to minimize the development of resistance. © 2018, National Institute for Medical Research. All rights reserved

Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs

Jacob G Malone1,21John Innes Centre, Norwich, UK; 2School of Biological Sciences, University of East Anglia, Norwich, UKAbstract: Pseudomonas aeruginosa is an opportunistic pathogen that predominates during the later stages of cystic fibrosis (CF) lung infections. Over many years of chronic lung colonization, P. aeruginosa undergoes extensive adaptation to the lung environment, evolving both toward a persistent, low virulence state and simultaneously diversifying to produce a number of phenotypically distinct morphs. These lung-adapted P. aeruginosa strains include the small colony variants (SCVs), small, autoaggregative isolates that show enhanced biofilm formation, strong attachment to surfaces, and increased production of exopolysaccharides. Their appearance in the sputum of CF patients correlates with increased resistance to antibiotics, poor lung function, and prolonged persistence of infection, increasing their relevance as a subject for clinical investigation. The evolution of SCVs in the CF lung is associated with overproduction of the ubiquitous bacterial signaling molecule cyclic-di-GMP, with increased cyclic-di-GMP levels shown to be responsible for the SCV phenotype in a number of different CF lung isolates. Here, we review the current state of research in clinical P. aeruginosa SCVs. We will discuss the phenotypic characteristics underpinning the SCV morphotype, the clinical implications of lung colonization with SCVs, and the molecular basis and clinical evolution of the SCV phenotype in the CF lung environment.Keywords: small colony variants, cystic fibrosis, cyclic-di-GMP, Pseudomonas aeruginosa, RsmA, antibiotic

Characterization of the newly isolated lytic bacteriophages KTN6 and KT28 and their efficacy against Pseudomonas aeruginosa biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants

Antibiotic Susceptibility Pattern in Clinical Isolates of Pseudomonas aeruginosa

Introduction: Pseudomonas aeruginosa is an opportunistic pathogen and causes of nosocomial infections in hospitals. Due to high antibiotic resistance and the ability to develop new resistance during antibiotic treatment, Pseudomonas aeruginosa infection is difficult to eradicate because the physical treatment becomes difficult and ineffective. This study was conducted to evaluate the antibiotic sensitivity pattern of Pseudomonas aeruginosa strains at two different hospital in Makassar. Methods: This study is a cross-sectional study from March to May 2021. The research samples were taken from the results of culture and antibiotic sensitivity tests conducted at two different hospital, Hasanuddin University Hospital and Dr. Wahidin Sudirohusodo Hospital for the period January 1 to September 30, 2019. A total of 84 samples were cultured and tested for antibiotic sensitivity of Pseudomonas aeruginosa. Results: Antibiotic sensitivity of Pseudomonas aeruginosa was best with aminoglycoside antibiotics, gentamicin (100%) at Hasanuddin University Hospital and amikacin (95.8%) at Dr. Wahidin Sudirohusodo Hospital. At Hasanuddin University Hospital followed by antibiotics amikacin (92.3%) and meropenem (84.6%). At Dr. Wahidin Sudirohusodo Hospital, Pseudomonas aeruginosa also showed good sensitivity to gentamicin (91.5%) and meropenem (77.5%). The sensitivity of Pseudomonas aeruginosa was lowest to piperacillin/tazobactam. Conclusions:  This study shows that the level of effectiveness of the antibiotics meropenem, amikacin and gentamicin is high enough and it can be used as a treatment option in Pseudomonas aeruginosa infection. This study can help as a reference to prevent mortality and morbidity associated with Pseudomonas aeruginosa infection

In Vitro Inhibition Zone Test Of Binahong (Anredera Cordifolia) Towards Staphylococcus Aureus, Enterococcus Faecalis, Escherichia Coli, And Pseudomonas Aeruginosa

This is a true experimental research with post test-only control group design. The study was conducted to test the inhibitory zone of the Binahong leaf extract (Anredera cordifolia) against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Binahong leaf extract is prepared using maceration technique, by soaking it in a sealed jar for 24 hours with 95% methanol. Then subsequently filtered using a funnel with filter paper, and the filtrate is collected inside an erlenmeyer. The filtrate then concentrated using a rotavapor, this concentrated extract dissolved into aquadest with a concentration of 50 ppm, 100 ppm and 1000 ppm. By taking a few colonies with a sterile loop into a stock of Staphylococcus aureus, Enterococcus faecalis, Esherichia coli, Pseudomonas aeruginosa then scratch it into MH blood agar medium, and incubate it for 24 hours with a temperature of 370C. The next day, bacterial suspension was made in test tube, which already contains 0.9% NaCl. The suspension tturbidity is equivalent to 0.5 Mc Farland. Bacterial inhibition zone of binahong leaf extract (Anredera cordifolia) is tested using absorbance disc method or better known as the Kirby-Bauer method. First, pour 10 ml of agar medium (± 400C) into a cup (petridish) and then wait until it's cold. After the medium becomes solid, the suspension of bacteria Staphylococcus aureus, Enterococcus faecalis, Esherichia coli, and Pseudomonas aeruginosa are slowly smeared with sterile cotton sticks on the surface of the media. Soak the paper discs into binahong leaf extract (Anredera cordifolia) with concentrations of 50, 100, and 1000 ppm, for about 5 minutes, and placed it on the surface of the petridish, together with the positive control (amoxicillin) and negative control (aquadest). Then incubate it at 370C for 24 hours. The effectiveness of binahong leaf extract (Anredera cordifolia) inhibition zone, can be determined by measuring the diameter of clear zone around the paper using a sliding-term. Binahong leaf extract (Anredera cordifolia) zone of inhibition is negative, a very slight different is showed by the amoxicillin inhibition zone, for having a clear zone diameter of 28 mm for Staphylococcus aureus and Esherichia coli, and 21 mm for Enterococcus faecalis. This fact is probably caused by several things concerning the mechanism of action of a substance as an anti bacterial of the binahong leaf extract (Anredera cordifolia)