RpoS Mutation Leads to Prolonged Biofilm Mode of Growth and a Higher Fitness in Pseudomonas Aeruginosa Biofilms

2021 ◽  
Author(s):  
Xiangke Duan ◽  
Yanrong Pan ◽  
Zhao Cai ◽  
Yumei Liu ◽  
Yingdan Zhang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Treatment ◽  
Biofilm Formation ◽  
Point Mutations ◽  
Opportunistic Pathogen ◽  
Antimicrobial Treatment ◽  
Clinical Isolates ◽  
High Dose ◽  
Sequencing Analysis ◽  
Cyclic Treatment

Abstract BackgroundPseudomonas aeruginosa is a notorious opportunistic pathogen causing various biofilm-related infections. Biofilm formation is a unique microbial strategy that allows P. aeruginosa to survive adverse conditions such as antibiotic treatment and human immune responses. ResultsIn this study, we experimentally evolved P. aeruginosa PAO1 biofilms for cyclic treatment in the presence of high dose of imipenem, and enriched hyperbiofilm mutants within six cycles in two independent lineages. The competition assay showed the evolved hyperbiofilm mutants can outcompete the ancestral strain within biofilm by prolonging the biofilm mode of growth but not in planktonic cultures. Whole-genome sequencing analysis revealed the hyperbiofilm phenotype is caused by point mutations in rpoS gene in all independently evolved mutants and the same mutation was found in P. aeruginosa clinical isolates. We further showed that mutation in rpoS enhanced biofilm formation by prolonging the biofilm mode of growth and elevating the intracellular c-di-GMP level. Mutation in rpoS increased pyocyanin production and virulence in both P. aeruginosa laboratory strains and clinical isolates. ConclusionHere, our study revealed that antibiotic treatment of biofilm-related P. aeruginosa infections might induce a hyperbiofilm phenotype via rpoS mutation, which might partially explain antimicrobial treatment failure of many P. aeruginosa biofilm-related infections.

scholarly journals rpoS-mutation variants are selected in Pseudomonas aeruginosa biofilms under imipenem pressure

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiangke Duan ◽  
Yanrong Pan ◽  
Zhao Cai ◽  
Yumei Liu ◽  
Yingdan Zhang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Treatment ◽  
Point Mutations ◽  
Opportunistic Pathogen ◽  
Antimicrobial Treatment ◽  
High Dose ◽  
Sequencing Analysis ◽  
Adverse Conditions ◽  
Diguanylate Cyclases ◽  
Cyclic Treatment

Abstract Background Pseudomonas aeruginosa is a notorious opportunistic pathogen causing various types of biofilm-related infections. Biofilm formation is a unique microbial strategy that allows P. aeruginosa to survive adverse conditions such as antibiotic treatment and human immune clearance. Results In this study, we experimentally evolved P. aeruginosa PAO1 biofilms for cyclic treatment in the presence of high dose of imipenem, and enriched hyperbiofilm mutants within six cycles in two independent lineages. The competition assay showed that the evolved hyperbiofilm mutants can outcompete the ancestral strain within biofilms but not in planktonic cultures. Whole-genome sequencing analysis revealed the hyperbiofilm phenotype is caused by point mutations in rpoS gene in all independently evolved mutants and the same mutation was found in P. aeruginosa clinical isolates. We further showed that mutation in rpoS gene increased the intracellular c-di-GMP level by turning on the expression of the diguanylate cyclases. Mutation in rpoS increased pyocyanin production and virulence in hyperbiofilm variants. Conclusion Here, our study revealed that antibiotic treatment of biofilm-related P. aeruginosa infections might induce a hyperbiofilm phenotype via rpoS mutation, which might partially explain antimicrobial treatment failure of many P. aeruginosa biofilm-related infections.

scholarly journals Effects of Lysozyme, Proteinase K, and Cephalosporins on Biofilm Formation by Clinical Isolates of Pseudomonas aeruginosa

2020 ◽  
Vol 2020 ◽  
pp. 1-9 ◽  
Author(s):  
Mohamed Eladawy ◽  
Mohammed El-Mowafy ◽  
Mohamed Mohamed Adel El-Sokkary ◽  
Rasha Barwa
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Contact Lenses ◽  
Hydrolytic Enzyme ◽  
Opportunistic Pathogen ◽  
Clinical Isolates ◽  
Proteinase K ◽  
Antibiofilm Activity ◽  
Novel Strategy ◽  
Intravascular Devices

Pseudomonas aeruginosa is an opportunistic pathogen that can form biofilms, which confer resistance to immune clearance and antibacterial treatment. Therefore, effective strategies to prevent biofilm formation are warranted. Here, 103 P. aeruginosa clinical isolates were quantitatively screened for biofilm formation ability via the tissue culture plate method. The effects of lysozyme (hydrolytic enzyme) and proteinase K (protease) on biofilm formation were evaluated at different concentrations. Lysozyme (30 μg/mL), but not proteinase K, significantly inhibited biofilm formation (19% inhibition). Treatment of 24-hour-old biofilms of P. aeruginosa isolates with 50 times the minimum inhibitory concentrations (MICs) of ceftazidime and cefepime significantly decreased the biofilm mass by 32.8% and 44%, respectively. Moreover, the exposure of 24-hour-old biofilms of P. aeruginosa isolates to lysozyme (30 μg/mL) and 50 times MICs of ceftazidime or cefepime resulted in a significant reduction in biofilm mass as compared with the exposure to lysozyme or either antibacterial agent alone. The best antibiofilm effect (49.3%) was observed with the combination of lysozyme (30 μg/mL) and 50 times MIC of cefepime. The promising antibiofilm activity observed after treatment with 50 times MIC of ceftazidime or cefepime alone or in combination with lysozyme (30 μg/mL) is indicative of a novel strategy to eradicate pseudomonal biofilms in intravascular devices and contact lenses.

scholarly journals Correlation of quorum sensing and virulence factors in Pseudomonas aeruginosa isolates in Egypt

2015 ◽  
Vol 9 (10) ◽  
pp. 1091-1099 ◽  
Author(s):  
Hamida M. Aboushleib ◽  
Hoda M. Omar ◽  
Rania Abozahra ◽  
Amel Elsheredy ◽  
Kholoud Baraka
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Biofilm Formation ◽  
Point Mutations ◽  
Inhibitory Effect ◽  
Clinical Isolates ◽  
Clove Oil ◽  
Twitching Motility ◽  
Pyocyanin Production

Introduction: Pseudomonas aeruginosa is one of the most virulent nosocomial pathogens worldwide. Quorum sensing (QS) regulates the production of pathogenic virulence factors and biofilm formation in P. aeruginosa. The four genes lasR, lasI, rhlR,and rhlI were found to regulate this QS system. In this study, we aimed to assess the correlation between these four genes and QS-dependent virulence factors and to detect the inhibitory effect of clove oil on QS. Methodology: Fifty P. aeruginosa clinical isolates were collected. Susceptibility to different antibiotics was tested. Virulence factors including biofilm formation, pyocyanin production, and twitching motility were phenotypically detected. QS genes were amplified by polymerase chain reaction (PCR), and one strain subsequently underwent sequencing. The inhibitory effect of clove oil on virulence factors was also tested. Results: A positive correlation was found between biofilm formation and the presence of lasR and rhlI genes. Twitching motility was positively correlated with the presence of lasR, lasI, and rhlI genes. On the other hand, no correlation was found between pyocyanin production and any of the studied genes. Only one isolate amplified all the tested QS gene primers, but it did not express any of the tested virulence factors phenotypically. Sequence analyses of this isolate showed that the four genes had point mutations. Conclusions: Results emphasize the importance of QS in P. aeruginosa virulence; however, QS-deficient clinical isolates occur and are still capable of causing clinical infections in humans. Also, clove oil has an obvious inhibitory effect on QS, which should be clinically exploited.

Antibiotic treatment of Pseudomonas aeruginosa biofilms stimulates expression of the magnesium transporter gene mgtE

Microbiology ◽  
2014 ◽  
Vol 160 (1) ◽  
pp. 165-178 ◽  
Author(s):  
Carly V. Redelman ◽  
Shubham Chakravarty ◽  
Gregory G. Anderson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Treatment ◽  
Type Iii Secretion ◽  
Biofilm Formation ◽  
Opportunistic Pathogen ◽  
Transporter Protein ◽  
Magnesium Transporter ◽  
High Concentrations ◽  
Serious Disease

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with the capacity to cause serious disease, including chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. These infections are treated with high concentrations of antibiotics. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified, including the magnesium transporter protein MgtE. We have recently found that isogenic deletion of mgtE leads to increased cytotoxicity through effects on the type III secretion system. To explore the role of the CF lung environment in MgtE activity, we investigated mgtE transcriptional regulation following antibiotic treatment. Utilizing quantitative real-time-PCR, we have demonstrated an increase in mgtE transcript levels following antibiotic treatment with most of the 12 antibiotics tested. To begin to determine the regulatory network governing mgtE expression, we screened a transposon-mutant library of P. aeruginosa to look for mutants with potentially altered mgtE activity, using cytotoxicity as a readout. In this screen, we observed that AlgR, which regulates production of the biofilm polysaccharide alginate, alters MgtE-mediated cytotoxicity. This cross-talk between MgtE and AlgR suggests that AlgR is involved in linking external inducing signals (e.g. antibiotics) to mgtE transcription and downstream virulence and biofilm activities. Analysing such interactions may lead to a better understanding of how the CF lung environment shapes P. aeruginosa biofilm infections.

Effect of sub-inhibitory concentrations of cefepime on biofilm formation by Pseudomonas aeruginosa

2021 ◽  
pp. 1-8
Author(s):  
Soheir A.A. Hagras ◽  
Alaa El-Dien M.S. Hosny ◽  
Omneya M. Helmy ◽  
Mounir M. Salem-Bekhit ◽  
Faiyaz Shakeel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Clinical Isolates ◽  
Polymeric Chain ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Encoding Gene ◽  
Fimbrial Protein

This study investigated the effect of cefepime at sub-minimum inhibitory concentrations (sub-MICs) on in vitro biofilm formation (BF) by clinical isolates of Pseudomonas aeruginosa. The effect of cefepime at sub-MIC levels (½–1/256 MIC) on in vitro BF by six clinical isolates of P. aeruginosa was phenotypically assessed following 24 and 48 h of challenge using the tissue culture plate (TCP) assay. Quantitative real-time polymeric chain reaction (qRT-PCR) was employed to observe the change in expression of three biofilm-related genes, namely, a protease-encoding gene (lasA), fimbrial protein-encoding gene (cupA1), and alginate-encoding gene (algC), in a weak biofilm-producing strain of P. aeruginosa following 24 and 48 h of challenge with sub-MICs of cefepime. The BF morphology in response to cefepime was imaged using scanning electron microscopy (SEM). The TCP assay showed strain-, time-, and concentration-dependent changes in in vitro BF in P. aeruginosa following challenge with sub-MICs of cefepime, with a profound increase in strains with inherently no or weak biofilm-producing ability. RT-PCR revealed time-dependent upregulation in the expression of the investigated genes following challenge with ½ and ¼ MIC levels, as confirmed by SEM. Cefepime at sub-MICs could upregulate the expression of BF-related genes and enhance BF by P. aeruginosa clinical isolates.

scholarly journals Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 14
Author(s):  
Dina Auliya Amly ◽  
Puspita Hajardhini ◽  
Alma Linggar Jonarta ◽  
Heribertus Dedy Kusuma Yulianto ◽  
Heni Susilowati
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Growth ◽  
Biofilm Formation ◽  
Royal Jelly ◽  
Microtiter Plate ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Antibiotic Tolerance ◽  
Subinhibitory Concentration ◽  
Pyocyanin Production

Background: Pseudomonas aeruginosa, a multidrug-resistant Gram-negative bacterium, produces pyocyanin, a virulence factor associated with antibiotic tolerance. High concentrations of royal jelly have an antibacterial effect, which may potentially overcome antibacterial resistance. However, in some cases, antibiotic tolerance can occur due to prolonged stress of low-dose antibacterial agents. This study aimed to investigate the effect of subinhibitory concentrations of royal jelly on bacterial growth, pyocyanin production, and biofilm formation of P. aeruginosa. Methods: Pseudomonas aeruginosa ATCC 10145 and clinical isolates were cultured in a royal jelly-containing medium to test the antibacterial activity. Pyocyanin production was observed by measuring the absorbance at 690 nm after 36 h culture and determined using extinction coefficient 4310 M-1 cm-1. Static microtiter plate biofilm assay performed to detect the biofilm formation, followed by scanning electron microscopy. Results: Royal jelly effectively inhibited the viability of both strains from a concentration of 25%. The highest production of pyocyanin was observed in the subinhibitory concentration group 6.25%, which gradually decreased along with the decrease of royal jelly concentration. Results of one-way ANOVA tests differed significantly in pyocyanin production of the two strains between the royal jelly groups. Tukey HSD test showed concentrations of 12.5%, 6.25%, and 3.125% significantly increased pyocyanin production of ATCC 10145, and the concentrations of 12.5% and 6.25% significantly increased production of the clinical isolates. Concentrations of 12.5% and 6.125% significantly induced biofilm formation of P. aeruginosa ATCC 10145, in line with the results of the SEM analysis. Conclusions: The royal jelly concentration of 25% or higher inhibits bacterial growth; however, the subinhibitory concentration increases pyocyanin production and biofilm formation in P. aeruginosa. It is advisable to determine the appropriate concentration of royal jelly to obtain beneficial virulence inhibiting activity.

scholarly journals Evaluation of Antibiotic Resistance Pattern, Alginate and Biofilm Production in Clinical Isolates of Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Fateme DAVARZANI ◽  
Navid SAIDI ◽  
Saeed BESHARATI ◽  
Horieh SADERI ◽  
Iraj RASOOLI ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Bacterial Resistance ◽  
Antimicrobial Agents ◽  
Clinical Isolates ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Resistance Pattern ◽  
Alginate Production ◽  
Opportunistic Bacteria

Background: Pseudomonas aeruginosa is one of the most common opportunistic bacteria causing nosocomial infections, which has significant resistance to antimicrobial agents. This bacterium is a biofilm and alginate producer. Biofilm increases the bacterial resistance to antibiotics and the immune system. Therefore, the present study was conducted to investigate the biofilm formation, alginate production and antimicrobial resistance patterns in the clinical isolates of P. aeruginosa. Methods: One hundred isolates of P. aeruginosa were collected during the study period (from Dec 2017 to Jul 2018) from different clinical samples of the patients admitted to Milad and Pars Hospitals at Tehran, Iran. Isolates were identified and confirmed by phenotypic and genotypic methods. Antimicrobial susceptibility was specified by the disk diffusion method. Biofilm formation and alginate production were measured by microtiter plate and carbazole assay, respectively. Results: Sixteen isolates were resistant to all the 12 studied antibiotics. Moreover, 31 isolates were MultidrugResistant (MDR). The highest resistance rate was related to ofloxacin (36 isolates) and the least resistance was related to piperacillin-tazobactam (21 isolates). All the isolates could produce the biofilm and alginate. The number of isolates producing strong, medium and weak biofilms was equal to 34, 52, and 14, respectively. Alginate production was more than 400 μg/ml in 39 isolates, 250-400 μg/ml in 51 isolates and less than 250 μg/ml in 10 isolates. Conclusion: High prevalence of MDR, biofilm formation, and alginate production were observed among the clinical isolates of P. aeruginosa. The results also showed a significant relationship between the amount of alginate production and the level of biofilm formation.

scholarly journals PmtA Regulates Pyocyanin Expression and Biofilm Formation in Pseudomonas aeruginosa

2021 ◽  
Vol 12 ◽  
Author(s):  
Amy V. Thees ◽  
Kathryn M. Pietrosimone ◽  
Clare K. Melchiorre ◽  
Jeremiah N. Marden ◽  
Joerg Graf ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pseudomonas Aeruginosa ◽  
Metal Binding ◽  
Biofilm Formation ◽  
Binding Capacity ◽  
Opportunistic Pathogen ◽  
Small Molecular Weight ◽  
Heavy Metal Binding ◽  
The Impact

The opportunistic pathogen Pseudomonas aeruginosa expresses a small molecular weight, cysteine-rich protein (PmtA), identified as a metallothionein (MT) protein family member. The MT family proteins have been well-characterized in eukaryotes as essential for zinc and copper homeostasis, protection against oxidative stress, and the ability to modify a variety of immune activities. Bacterial MTs share sequence homology, antioxidant chemistry, and heavy metal-binding capacity with eukaryotic MTs, however, the impact of bacterial MTs on virulence and infection have not been well-studied. In the present study, we investigated the role of PmtA in P. aeruginosa PAO1 using a PmtA-deficient strain (ΔpmtA). Here we demonstrated the virulence factor, pyocyanin, relies on the expression of PmtA. We showed that PmtA may be protective against oxidative stress, as an alternative antioxidant, glutathione, can rescue pyocyanin expression. Furthermore, the expression of phzM, which encodes a pyocyanin precursor enzyme, was decreased in the ΔpmtA mutant during early stationary phase. Upregulated pmtA expression was previously detected in confluent biofilms, which are essential for chronic infection, and we observed that the ΔpmtA mutant was disrupted for biofilm formation. As biofilms also modulate antibiotic susceptibility, we examined the ΔpmtA mutant susceptibility to antibiotics and found that the ΔpmtA mutant is more susceptible to cefepime and ciprofloxacin than the wild-type strain. Finally, we observed that the deletion of pmtA results in decreased virulence in a waxworm model. Taken together, our results support the conclusion that PmtA is necessary for the full virulence of P. aeruginosa and may represent a potential target for therapeutic intervention.

scholarly journals Acquisition of multidrug resistance transposon Tn6061 and IS6100-mediated large chromosomal inversions in Pseudomonas aeruginosa clinical isolates

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1448-1458 ◽  
Author(s):  
Sébastien Coyne ◽  
Patrice Courvalin ◽  
Marc Galimand
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Opportunistic Pathogen ◽  
Clinical Isolates ◽  
Chromosomal Inversions ◽  
Gene Acquisition ◽  
Drug Classes ◽  
Horizontal Acquisition

Pseudomonas aeruginosa is a major human opportunistic pathogen, especially for patients in intensive care units or with cystic fibrosis. Multidrug resistance is a common feature of this species. In a previous study we detected the ant(4′)-IIb gene in six multiresistant clinical isolates of P. aeruginosa, and determination of the environment of the gene led to characterization of Tn6061. This 26 586 bp element, a member of the Tn3 family of transposons, carried 10 genes conferring resistance to six drug classes. The ant(4′)-IIb sequence was flanked by directly repeated copies of ISCR6 in all but one of the strains studied, consistent with ISCR6-mediated gene acquisition. Tn6061 was chromosomally located in six strains and plasmid-borne in the remaining isolate, suggesting horizontal acquisition. Duplication-insertion of IS6100, that ended Tn6061, was responsible for large chromosomal inversions. Acquisition of Tn6061 and chromosomal inversions are further examples of intricate mechanisms that contribute to the genome plasticity of P. aeruginosa.

Pseudomonas aeruginosa in Bronchiectasis

2021 ◽  
Vol 42 (04) ◽  
pp. 587-594
Author(s):  
Laia Fernández-Barat ◽  
Victoria Alcaraz-Serrano ◽  
Rosanel Amaro ◽  
Antoni Torres
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Chronic Infection ◽  
Opportunistic Pathogen ◽  
Oxygen Depletion ◽  
Antimicrobial Treatment ◽  
Consensus Definition ◽  
Abiotic Surfaces ◽  
Technological Advances ◽  
Patients At Risk ◽  
Definition Of

Abstract Pseudomonas aeruginosa (PA) in patients with bronchiectasis (BE) is associated with a poor outcome and quality of life, and its presence is considered a marker of disease severity. This opportunistic pathogen is known for its ability to produce biofilms on biotic or abiotic surfaces and to survive environmental stress exerted by antimicrobials, inflammation, and nutrient or oxygen depletion. The presence of PA biofilms has been linked to chronic respiratory infection in cystic fibrosis but not in BE. There is considerable inconsistency in the reported infection/eradication rates of PA and chronic PA. In addition, inadequate antimicrobial treatment may potentiate the progression from intermittent to chronic infection and also the emergence of antibiotic resistance. A better comprehension of the pathophysiology of PA infections and its implications for BE is urgently needed. This can drive improvements in diagnostic accuracy, can move us toward a new consensus definition of chronic infection, can better define the follow-up of patients at risk of PA, and can achieve more successful eradication rates. In addition, the new technological advances regarding molecular diagnostics, -omics, and biomarkers require us to reconsider our traditional concepts.

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