Tolerance of Pseudomonas aeruginosa in in-vitro biofilms to high-level peracetic acid disinfection

The purpose of this study was to assess the antimicrobial efficacy of a peracetic acid-based disinfectant for decontamination of heat-polymerized, chemically activated and microwave-polymerized acrylic resins. Resin plates were contaminated in vivo upon intraoral use by 10 volunteers for 7 nights and slabs were contaminated in vitro by contact with Bacillus subtilis and Bacillus stearothermophilus. The contaminated acrylic resin specimens were immersed in a 0.2% peracetic acid-based disinfectant (Sterilife®; Lifemed) for 5 min or 10 min and placed in a BHI culture medium. After incubation at 37°C for 48 h, bacterial growth was assessed by analyzing turbidity of the medium. For all types of acrylic resin, no turbidity of the medium was observed for any of the resin specimens immersed in the peracetic acid-based disinfectant for either 5 or 10 min. On the other hand, the media with specimens that were not immersed in the disinfectant (control) showed turbidity in 100% of the cases, indicating the presence of microorganisms in both tested conditions. In conclusion, immersion for at least 5 min in a 0.2% peracetic acid-based disinfectant promoted high-level disinfection of heat-polymerized, chemically activated and microwave-polymerized acrylic resins contaminated with either human saliva or Bacillus subtilis or Bacillus stearothermophilus.

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Efficacy of a ciprofloxacin/amikacin combination against planktonic and biofilm cultures of susceptible and low-level resistant Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz355 ◽

2019 ◽

Vol 74 (11) ◽

pp. 3252-3259 ◽

Cited By ~ 3

Author(s):

Anaïs Soares ◽

Kévin Alexandre ◽

Fabien Lamoureux ◽

Ludovic Lemée ◽

François Caron ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Biofilm ◽

The Other ◽

Clinical Strain ◽

Bacterial Reduction ◽

Low Level ◽

Mechanical Dispersion ◽

Resistant Mutants ◽

High Level

Abstract Background Eradicating bacterial biofilm without mechanical dispersion remains a challenge. Combination therapy has been suggested as a suitable strategy to eradicate biofilm. Objectives To evaluate the efficacy of a ciprofloxacin/amikacin combination in a model of in vitro Pseudomonas aeruginosa biofilm. Methods The antibacterial activity of ciprofloxacin and amikacin (alone, in combination and successively) was evaluated by planktonic and biofilm time–kill assays against five P. aeruginosa strains: PAO1, a WT clinical strain and three clinical strains overexpressing the efflux pumps MexAB-OprM (AB), MexXY-OprM (XY) and MexCD-OprJ (CD), respectively. Amikacin MIC was 16 mg/L for XY and ciprofloxacin MIC was 0.5 mg/L for CD. The other strains were fully susceptible to ciprofloxacin and amikacin. The numbers of total and resistant cells were determined. Results In planktonic cultures, regrowth of high-level resistant mutants was observed when CD was exposed to ciprofloxacin alone and XY to amikacin alone. Eradication was obtained with ciprofloxacin or amikacin in the other strains, or with the combination in XY and CD strains. In biofilm, bactericidal reduction after 8 h followed by a mean 4 log10 cfu/mL plateau in all strains and for all regimens was noticed. No regrowth of resistant mutants was observed whatever the antibiotic regimen. The bacterial reduction obtained with a second antibiotic used simultaneously or consecutively was not significant. Conclusions The ciprofloxacin/amikacin combination prevented the emergence of resistant mutants in low-level resistant strains in planktonic cultures. Biofilm persister cells were not eradicated, either with monotherapy or with the combination.

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In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa206 ◽

2020 ◽

Vol 75 (9) ◽

pp. 2508-2515 ◽

Cited By ~ 1

Author(s):

María A Gomis-Font ◽

Gabriel Cabot ◽

Irina Sánchez-Diener ◽

Pablo A Fraile-Ribot ◽

Carlos Juan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Resistance Mechanisms ◽

Infection Model ◽

Mechanisms Of Resistance ◽

Resistance Development ◽

C Elegans ◽

Imipenem Resistance ◽

High Level

Abstract Objectives We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in Pseudomonas aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. Methods PAO1 or PAOMS strains were incubated for 24 h in Mueller–Hinton Broth with 0.125–64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. Results Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulatoras of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. Conclusions Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

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Inhibitory Effect of Biocides on the Viable Masses and Matrices of Staphylococcus aureus and Pseudomonas aeruginosa Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.02095-09 ◽

2010 ◽

Vol 76 (10) ◽

pp. 3135-3142 ◽

Cited By ~ 96

Author(s):

K. Toté ◽

T. Horemans ◽

D. Vanden Berghe ◽

L. Maes ◽

P. Cos

Keyword(s):

Hydrogen Peroxide ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Sodium Hypochlorite ◽

Peracetic Acid ◽

Inhibitory Effect ◽

European Guidelines ◽

The Matrix ◽

Antibiofilm Agents

ABSTRACT Bacteria and matrix are essential for the development of biofilms, and assays should therefore target both components. The current European guidelines for biocidal efficacy testing are not adequate for sessile microorganisms; hence, alternative discriminatory test protocols should be used. The activities of a broad range of biocides on Staphylococcus aureus and Pseudomonas aeruginosa biofilms were evaluated using such in vitro assays. Nearly all selected biocides showed a significant decrease in S. aureus biofilm viability, with sodium hypochlorite and peracetic acid as the most active biocides. Only hydrogen peroxide and sodium hypochlorite showed some inhibitory effect on the matrix. Treatment of P. aeruginosa biofilms was roughly comparable to that of S. aureus biofilms. Peracetic acid was the most active on viable mass within 1 min of contact. Isopropanol ensured a greater than 99.999% reduction of P. aeruginosa viability after at least 30 min of contact. Comparable to results with S. aureus, sodium hypochlorite and hydrogen peroxide markedly reduced the P. aeruginosa matrix. This study clearly demonstrated that despite their aspecific mechanisms of action, most biocides were active only against biofilm bacteria, leaving the matrix undisturbed. Only hydrogen peroxide and sodium hypochlorite were active on both the biofilm matrix and the viable mass, making them the better antibiofilm agents. In addition, this study emphasizes the need for updated and standardized guidelines for biofilm susceptibility testing of biocides.

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Efficacy of single and multiple oral doses of fosfomycin against Pseudomonas aeruginosa urinary tract infections in a dynamic in vitro bladder infection model

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa127 ◽

2020 ◽

Vol 75 (7) ◽

pp. 1879-1888 ◽

Cited By ~ 2

Author(s):

Iain J Abbott ◽

Elke van Gorp ◽

Rixt A Wijma ◽

Jordy Dekker ◽

Peter D Croughs ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Single Dose ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Infection Model ◽

Post Exposure ◽

High Level ◽

Tract Infections ◽

Emergence Of Resistance

Abstract Objectives We used a dynamic bladder infection in vitro model with synthetic human urine (SHU) to examine fosfomycin exposures to effectively kill, or prevent emergence of resistance, among Pseudomonas aeruginosa isolates. Methods Dynamic urinary fosfomycin concentrations after 3 g oral fosfomycin were simulated, comparing single and multiple (daily for 7 days) doses. Pharmacodynamic response of 16 P. aeruginosa (MIC range 1 to >1024 mg/L) were examined. Baseline disc diffusion susceptibility, broth microdilution MIC and detection of heteroresistance were assessed. Pathogen kill and emergence of resistance over 72 h following a single dose, and over 216 h following daily dosing for 7 days, were investigated. The fAUC0–24/MIC associated with stasis and 1, 2 and 3 log10 kill were determined. Results Pre-exposure high-level resistant (HLR) subpopulations were detected in 11/16 isolates after drug-free incubation in the bladder infection model. Five of 16 isolates had >2 log10 kill after single dose, reducing to 2/16 after seven doses. Post-exposure HLR amplification occurred in 8/16 isolates following a single dose and in 11/16 isolates after seven doses. Baseline MIC ≥8 mg/L with an HLR subpopulation predicted post-exposure emergence of resistance following the multiple doses. A PK/PD target of fAUC0–24/MIC >5000 was associated with 3 log10 kill at 72 h and 7 day-stasis. Conclusions Simulated treatment of P. aeruginosa urinary tract infections with oral fosfomycin was ineffective, despite exposure to high urinary concentrations and repeated daily doses for 7 days. Emergence of resistance was observed in the majority of isolates and worsened following prolonged therapy. Detection of a baseline resistant subpopulation predicted treatment failure.

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Structure of BrlR reveals a potential pyocyanin binding site

10.1101/188136 ◽

2017 ◽

Author(s):

Harikiran Raju ◽

Rukmini Sundararajan ◽

Rohan Sharma

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Site ◽

Transcriptional Regulator ◽

Drug Tolerance ◽

Binding Partner ◽

Direct Binding ◽

High Level

AbstractThe transcriptional regulator BrlR from Pseudomonas aeruginosa is a member of the MerR family of multidrug transport activators. Studies have shown BrlR plays an important role in high level drug tolerance of P. aeruginosa in biofilm. Its drug tolerance ability can be enhanced by 3′,5′-cyclic diguanylic acid (c-di-GMP). Here, we show the apo structure of BrlR and the direct binding between GyrI-like domain of BrlR and P. aeruginosa toxin pyocyanin. Furthermore, pyocyanin can enhance the binding between BrlR and DNA in vitro. These findings suggest BrlR can serve as the binding partner for both c-di-GMP and pyocyanin.

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Evolution of Antibiotic Tolerance Shapes Resistance Development in Chronic Pseudomonas aeruginosa Infections

10.1101/2020.10.23.352104 ◽

2020 ◽

Author(s):

Isabella Santi ◽

Pablo Manfredi ◽

Enea Maffei ◽

Adrian Egli ◽

Urs Jenal

Keyword(s):

Pseudomonas Aeruginosa ◽

Drug Tolerance ◽

Antibiotic Tolerance ◽

Bacterial Survival ◽

Chronic Infections ◽

Tolerance Mechanisms ◽

Resistance Development ◽

Evolutionary Trajectories ◽

High Level

AbstractThe widespread use of antibiotics promotes the evolution and dissemination of resistance and tolerance mechanisms. To assess the relevance of tolerance and its implications for resistance development, we used in vitro evolution and analyzed inpatient microevolution of Pseudomonas aeruginosa, an important human pathogen causing acute and chronic infections. We show that the development of tolerance precedes and promotes the acquisition of resistance in vitro and we present evidence that similar processes shape antibiotic exposure in human patients. Our data suggest that during chronic infections, P. aeruginosa first acquires moderate drug tolerance before following distinct evolutionary trajectories that lead to high-level multi-drug tolerance or to antibiotic resistance. Our studies propose that the development of antibiotic tolerance predisposes bacteria for the acquisition of resistance at early stages of infection and that both mechanisms independently promote bacterial survival during antibiotic treatment at later stages of chronic infections.

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Constitutive High Expression of Chromosomal β-Lactamase in Pseudomonas aeruginosa Caused by a New Insertion Sequence (IS1669) Located in ampD

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.11.3406-3411.2002 ◽

2002 ◽

Vol 46 (11) ◽

pp. 3406-3411 ◽

Cited By ~ 77

Author(s):

Niels Bagge ◽

Oana Ciofu ◽

Morten Hentzer ◽

Joan I. A. Campbell ◽

Michael Givskov ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Insertion Sequence ◽

Enterobacter Cloacae ◽

High Level Expression ◽

Dramatic Decrease ◽

Strain Pao1 ◽

Genetic Changes ◽

High Level

ABSTRACT The expression of chromosomal AmpC β-lactamase in Pseudomonas aeruginosa is negatively regulated by the activity of an amidase, AmpD. In the present study we examined resistant clinical P. aeruginosa strains and several resistant variants isolated from in vivo and in vitro biofilms for mutations in ampD to find evidence for the genetic changes leading to high-level expression of chromosomal β-lactamase. A new insertion sequence, IS1669, was found located in the ampD genes of two clinical P. aeruginosa isolates and several biofilm-isolated variants. The presence of IS1669 in ampD resulted in the expression of high levels of AmpC β-lactamase. Complementation of these isolates with ampD from the reference P. aeruginosa strain PAO1 caused a dramatic decrease in the expression of AmpC β-lactamase and a parallel decrease of the MIC of ceftazidime to a level comparable to that of PAO1. One highly resistant, constitutive β-lactamase-producing variant contained no mutations in ampD, but a point mutation was observed in ampR, resulting in an Asp-135→Asn change. An identical mutation of AmpR in Enterobacter cloacae has been reported to cause a 450-fold higher AmpC expression. However, in many of the isolates expressing high levels of chromosomal β-lactamase, no changes were found in either ampD, ampR, or in the promoter region of ampD, ampR, or ampC. Our results suggest that multiple pathways may exist leading to increased antimicrobial resistance due to chromosomal β-lactamase.

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Activity of a New Cephalosporin, CXA-101 (FR264205), against β-Lactam-Resistant Pseudomonas aeruginosa Mutants Selected In Vitro and after Antipseudomonal Treatment of Intensive Care Unit Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01104-09 ◽

2010 ◽

Vol 54 (3) ◽

pp. 1213-1217 ◽

Cited By ~ 66

Author(s):

Bartolome Moya ◽

Laura Zamorano ◽

Carlos Juan ◽

José L. Pérez ◽

Yigong Ge ◽

...

Keyword(s):

Intensive Care Unit ◽

Pseudomonas Aeruginosa ◽

Intensive Care ◽

Efflux Pump ◽

Multidrug Resistant ◽

Icu Patients ◽

Resistant Strains ◽

High Level ◽

Level Resistance

ABSTRACT CXA-101, previously designated FR264205, is a new antipseudomonal cephalosporin. We evaluated the activity of CXA-101 against a highly challenging collection of β-lactam-resistant Pseudomonas aeruginosa mutants selected in vitro and after antipseudomonal treatment of intensive care unit (ICU) patients. The in vitro mutants investigated included strains with multiple combinations of mutations leading to several degrees of AmpC overexpression (ampD, ampDh2, ampDh3, and dacB [PBP4]) and porin loss (oprD). CXA-101 remained active against even the AmpD-PBP4 double mutant (MIC = 2 μg/ml), which shows extremely high levels of AmpC expression. Indeed, this mutant showed high-level resistance to all tested β-lactams, except carbapenems, including piperacillin-tazobactam (PTZ), aztreonam (ATM), ceftazidime (CAZ), and cefepime (FEP), a cephalosporin considered to be relatively stable against hydrolysis by AmpC. Moreover, CXA-101 was the only β-lactam tested (including the carbapenems imipenem [IMP] and meropenem [MER]) that remained fully active against the OprD-AmpD and OprD-PBP4 double mutants (MIC = 0.5 μg/ml). Additionally, we tested a collection of 50 sequential isolates that were susceptible or resistant to penicillicins, cephalosporins, carbapenems, or fluoroquinolones that emerged during treatment of ICU patients. All of the mutants resistant to CAZ, FEP, PTZ, IMP, MER, or ciprofloxacin showed relatively low CXA-101 MICs (range, 0.12 to 4 μg/ml; mean, 1 to 2 μg/ml). CXA-101 MICs of pan-β-lactam-resistant strains ranged from 1 to 4 μg/ml (mean, 2.5 μg/ml). As described for the in vitro mutants, CXA-101 retained activity against the natural AmpD-PBP4 double mutants, even when these exhibited additional overexpression of the MexAB-OprM efflux pump. Therefore, clinical trials are needed to evaluate the usefulness of CXA-101 for the treatment of P. aeruginosa nosocomial infections, particularly those caused by multidrug-resistant isolates that emerge during antipseudomonal treatments.

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A Survival Strategy for Pseudomonas aeruginosa That Uses Exopolysaccharides To Sequester and Store Iron To Stimulate Psl-Dependent Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.01307-16 ◽

2016 ◽

Vol 82 (21) ◽

pp. 6403-6413 ◽

Cited By ~ 20

Author(s):

Shan Yu ◽

Qing Wei ◽

Tianhu Zhao ◽

Yuan Guo ◽

Luyan Z. Ma

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Iron Concentration ◽

Survival Strategy ◽

Iron Storage ◽

Content Type ◽

High Level ◽

Biofilm Development ◽

Biofilm Matrix

ABSTRACTExopolysaccharide Psl is a critical biofilm matrix component inPseudomonas aeruginosa, which forms a fiber-like matrix to enmesh bacterial communities. Iron is important forP. aeruginosabiofilm development, yet it is not clearly understood how iron contributes to biofilm development. Here, we showed that iron promoted biofilm formation via elevating Psl production inP. aeruginosa. The high level of iron stimulated the synthesis of Psl by reducing rhamnolipid biosynthesis and inhibiting the expression of AmrZ, a repressor ofpslgenes. Iron-stimulated Psl biosynthesis and biofilm formation held true in mucoidP. aeruginosastrains. Subsequent experiments indicated that iron bound with Pslin vitroand in biofilms, which suggested that Psl fibers functioned as an iron storage channel inP. aeruginosabiofilms. Moreover, among three matrix exopolysaccharides ofP. aeruginosa, Psl is the only exopolysaccharide that can bind with both ferrous and ferric ion, yet with higher affinity for ferrous iron. Our data suggest a survival strategy ofP. aeruginosathat uses exopolysaccharide to sequester and store iron to stimulate Psl-dependent biofilm formation.IMPORTANCEPseudomonas aeruginosais an environmental microorganism which is also an opportunistic pathogen that can cause severe infections in immunocompromised individuals. It is the predominant airway pathogen causing morbidity and mortality in individuals affected by the genetic disease cystic fibrosis (CF). Increased airway iron and biofilm formation have been proposed to be the potential factors involved in the persistence ofP. aeruginosain CF patients. Here, we showed that a high level of iron enhanced the production of the key biofilm matrix exopolysaccharide Psl to stimulate Psl-dependent biofilm formation. Our results not only make the link between biofilm formation and iron concentration in CF, but also could guide the administration or use of iron chelators to interfere with biofilm formation inP. aeruginosain CF patients. Furthermore, our data also imply a survival strategy ofP. aeruginosaunder high-iron environmental conditions.

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