DivergentIn vitroMIC Characteristics and underlying isogenic mutations in host-specializedPseudomonas aeruginosa

AbstractClinical isolates ofPseudomonas aeruginosa (Pa)from patients with cystic fibrosis (CF) are known to differ from those associated with infections of non-CF hosts in colony morphology, drug susceptibility patterns, and genomic hypermutability. AlthoughPaisolates from CF have long been recognized for their overall higher resistance rate calculated generally by reduced “percent susceptible”, this study takes the approach to compare and contrast Etest MIC distributions between two distinct cohorts of clinical strains (n=224 from 56 CF patients and n=130 from 68 non-CF patients respectively) isolated in 2013. Logarithmic transformed MIC (logMIC) values of 11 antimicrobial agents were compared between the two groups. CF isolates tended to produce heterogeneous and widely dispersed MICs compared to non-CF isolates. By applying a test for equality of variances, we were able to confirm that the MICs generated from CF isolates against 9 out of the 11 agents were significantly more dispersed than those from non-CF (p<0.02-<0.001). Quantile-quantiles plots indicated little agreement between the two cohorts of isolates. Based on whole genome sequencing of 19 representative CFPaisolates, divergent gain- or loss-of-function mutations in efflux and porin genes and their regulators between isogenic or intra-clonal associates were evident. Not one, not a few, but the net effect all adaptive mutational changes in the genomes of CFPa, both shared and unshared between isogenic strains, are responsible for the divergent heteroresistance patterns. Moreover, the isogenic variations are suggestive of a bacterial syntrophic lifestyle when “lockedȍ inside a host focal airway environment over prolonged periods.Significance statementBacterial heteroresistance is associated with niche specialized organisms interacting with host species for prolonged period of time, medically characterized by “chronic focal infections”. A prime example is found inPseudomonas aeruginosaisogenic/non-homogeneous isolates from patient airways with cystic fibrosis. The development of pseudomonal polarizing MICsin vitroto many actively used antimicrobial agents among isogenic isolates and “Eagle-type” heteroresistance patterns are common and characteristic. Widespread isogenic gene lesions were evident for defects in drug transporters, DNA mismatch repair, and many other structural or cellular functions—a result of pseudomonal symbiotic response to host selection. Co-isolation of extremely susceptible and resistant isogenicPastrains suggests intra-airway evolution of a multicellular syntrophic bacterial lifestyle, which has laboratory interpretation and clinical treatment implications.

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Activities of Tobramycin and Six Other Antibiotics against Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.12.2877 ◽

1999 ◽

Vol 43 (12) ◽

pp. 2877-2880 ◽

Cited By ~ 73

Author(s):

Ribhi M. Shawar ◽

David L. MacLeod ◽

Richard L. Garber ◽

Jane L. Burns ◽

Jenny R. Stapp ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

Active Drug ◽

Resistance Mechanisms ◽

Phase Iii ◽

In Vitro Activity ◽

Phase Iii Clinical Trials ◽

Multiple Antibiotics

ABSTRACT The in vitro activity of tobramycin was compared with those of six other antimicrobial agents against 1,240 Pseudomonas aeruginosa isolates collected from 508 patients with cystic fibrosis during pretreatment visits as part of the phase III clinical trials of tobramycin solution for inhalation. The tobramycin MIC at which 50% of isolates are inhibited (MIC50) and MIC90 were 1 and 8 μg/ml, respectively. Tobramycin was the most active drug tested and also showed good activity against isolates resistant to multiple antibiotics. The isolates were less frequently resistant to tobramycin (5.4%) than to ceftazidime (11.1%), aztreonam (11.9%), amikacin (13.1%), ticarcillin (16.7%), gentamicin (19.3%), or ciprofloxacin (20.7%). For all antibiotics tested, nonmucoid isolates were more resistant than mucoid isolates. Of 56 isolates for which the tobramycin MIC was ≥16 μg/ml and that were investigated for resistance mechanisms, only 7 (12.5%) were shown to possess known aminoglycoside-modifying enzymes; the remaining were presumably resistant by an incompletely understood mechanism often referred to as “impermeability.”

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Evaluation of in vitro activity of ceftolozane-tazobactam compared to other antimicrobial agents against Pseudomonas aeruginosa isolates from cystic fibrosis patients

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2019.01.012 ◽

2019 ◽

Vol 94 (3) ◽

pp. 297-303 ◽

Cited By ~ 4

Author(s):

Giovanni Gherardi ◽

Giulia Linardos ◽

Arianna Pompilio ◽

Ersilia Fiscarelli ◽

Giovanni Di Bonaventura

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

Vitro Activity ◽

In Vitro Activity

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Pseudomonas aeruginosa Syntrophy in Chronically Colonized Airways of Cystic Fibrosis Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01371-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5971-5981 ◽

Cited By ~ 25

Author(s):

Xuan Qin ◽

Danielle M. Zerr ◽

Michael A. McNutt ◽

Jessica E. Berry ◽

Jane L. Burns ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Genotypic Diversity ◽

Content Type ◽

Dna Mismatch ◽

Novel Model ◽

Isogenic Strains ◽

Selection Of

ABSTRACTPseudomonas aeruginosaisolates from cystic fibrosis (CF) patients undergo remarkable phenotypic divergence over time, including loss of pigmentation, hemolysis, motility, and quorum sensing and emergence of antibiotic hypersusceptibility and/or auxotrophism. With prolonged antibiotic treatment and steady decline in lung function in chronically infected patients, the divergent characteristics associated with CF isolates have traditionally been regarded as “adapted/unusual virulence,” despite the degenerative nature of these adaptations. We examined the phenotypic and genotypic diversity in clonally related isogenic strains ofP. aeruginosafrom individual CF patients. Our observations support a novel model of intra-airway pseudomonal syntrophy and accompanying loss of virulence. A 2007 calendar year collection of CFP. aeruginosaisolates (n= 525) from 103 CF patients yieldedin vitroMICs of sulfamethoxazole-trimethoprim (SMX-TMP, which typically has no activity againstP. aeruginosa) ranging from 0.02 to >32 μg/ml (median, 1.5). Coisolation of clonally related SMX-TMP-susceptible and -resistantP. aeruginosastrains from the same host was common (57%), as were isogenic coisolates with mutations in efflux gene determinants (mexR,mexAB-oprM, andmexZ) and genes governing DNA mismatch repair (mutLandmutS). In this cohort, completein vitrogrowth complementation between auxotrophic and prototrophicP. aeruginosaisogenic strains was evident and concurrent with the coding sequence mosaicism in resistance determinants. These observations suggest that syntrophic clonal strains evolvein situin an organized colonial structure. We propose thatP. aeruginosaadopts a multicellular lifestyle in CF patients due to host selection of an energetically favorable, less-virulent microbe restricted within and symbiotic with the airway over the host's lifetime.

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In vitro biofilm formation and antibiotic susceptibility of Pseudomonas aeruginosa isolated from airways of patients with cystic fibrosis

Journal of Medical Science ◽

10.20883/jms.2016.179 ◽

2016 ◽

Vol 85 (4) ◽

pp. 245-253

Author(s):

Jolanta Długaszewska ◽

Marta Antczak ◽

Izabella Kaczmarek ◽

Renata Jankowiak ◽

Malgorzata Buszkiewicz ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Susceptibility ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Susceptibility Testing ◽

Testing Method ◽

Airway Infections ◽

Microdilution Broth

Background: Pseudomonas aeruginosa is the predominant cause of airway infections in patients with cystic fibrosis (CF) as a result of its ability to form biofilm. Resistance to antimicrobial agents is the most important feature of biofilm infection. The aim of this study was to evaluate biofilm formation and to compare antibiotic susceptibility of P. aeruginosa living in two modes of growth: planktonic and biofilm, isolated from respiratory tract of CF patients. Methods: Biofilm formation and biofilm susceptibility to antibiotics were determined using modified microtitere plate method. For susceptibility testing of planktonic culture to antibiotics serial microdilution broth method were used.Results: More than 95% of isolates were capable to form biofilm. Isolates grown as biofilms were more resistant to tested antibiotics compared to those grown planktonically. Ciprofloxacin showed the highest activity against P. aeruginosa biofilm. In contrast, no bacteriostatic activity was obtain for the highest concentration of piperacillin tested against most of P. aeruginosa strains growing in a biofilm (BIC > 4096 mg/L).Conclusions: Our study indicates the need to develop a standardized susceptibility testing method for biofilm mode of growth of pathogens. It appears that it is appropriate to introduce a biofilm susceptibility testing to routinely performed tests, as the effect of antibiotics on biofilm eradication may be variable and unpredictable.

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In Vitro Activity of Doripenem (S-4661) against Multidrug-Resistant Gram-Negative Bacilli Isolated from Patients with Cystic Fibrosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2510-2511.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2510-2511 ◽

Cited By ~ 38

Author(s):

Yunhua Chen ◽

Elizabeth Garber ◽

Qiuqu Zhao ◽

Yigong Ge ◽

Matthew A. Wikler ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Burkholderia Cepacia ◽

Antimicrobial Agents ◽

Multidrug Resistant ◽

Vitro Activity ◽

In Vitro Activity ◽

Gram Negative ◽

Resistant Strains

ABSTRACT Doripenem 50% inhibitory concentrations (MIC50) and 90% inhibitory concentrations (MIC90) for multidrug-resistant strains of mucoid Pseudomonas aeruginosa (n = 200 strains), nonmucoid P. aeruginosa (n = 200), and Burkholderia cepacia complex (n = 200) isolated from patients with cystic fibrosis were 8 and 32, 8 and 64, and 8 and 32 μg/ml, respectively. Doripenem had somewhat better activity than established antimicrobial agents.

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In vitro biofilm formation and antibiotic susceptibility of Pseudomonas aeruginosa isolated from airways of patients with cystic fibrosis

Journal of Medical Science ◽

10.20883/179 ◽

2016 ◽

Vol 85 (4) ◽

pp. 245 ◽

Cited By ~ 2

Author(s):

Jolanta Długaszewska ◽

Marta Antczak ◽

Izabella Kaczmarek ◽

Renata Jankowiak ◽

Malgorzata Buszkiewicz ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Susceptibility ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Susceptibility Testing ◽

Testing Method ◽

Airway Infections ◽

Microdilution Broth

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Pseudomonas aeruginosa PA14 Enhances the Efficacy of Norfloxacin against Staphylococcus aureus Newman Biofilms

Journal of Bacteriology ◽

10.1128/jb.00159-20 ◽

2020 ◽

Vol 202 (18) ◽

Cited By ~ 1

Author(s):

Giulia Orazi ◽

Fabrice Jean-Pierre ◽

George A. O’Toole

Keyword(s):

Cystic Fibrosis ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

Respiratory Infections ◽

Multiple Infection ◽

Bacterial Biofilms ◽

Interspecies Interactions ◽

Chronic Infections ◽

Content Type

ABSTRACT The thick mucus within the airways of individuals with cystic fibrosis (CF) promotes frequent respiratory infections that are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent pathogens that cause CF pulmonary infections, and both are among the most common etiologic agents of chronic wound infections. Furthermore, the ability of P. aeruginosa and S. aureus to form biofilms promotes the establishment of chronic infections that are often difficult to eradicate using antimicrobial agents. In this study, we found that multiple LasR-regulated exoproducts of P. aeruginosa, including 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), siderophores, phenazines, and rhamnolipids, likely contribute to the ability of P. aeruginosa PA14 to shift S. aureus Newman norfloxacin susceptibility profiles. Here, we observe that exposure to P. aeruginosa exoproducts leads to an increase in intracellular norfloxacin accumulation by S. aureus. We previously showed that P. aeruginosa supernatant dissipates the S. aureus membrane potential, and furthermore, depletion of the S. aureus proton motive force recapitulates the effect of the P. aeruginosa PA14 supernatant on shifting norfloxacin sensitivity profiles of biofilm-grown S. aureus Newman. From these results, we hypothesize that exposure to P. aeruginosa PA14 exoproducts leads to increased uptake of the drug and/or an impaired ability of S. aureus Newman to efflux norfloxacin. Surprisingly, the effect observed here of P. aeruginosa PA14 exoproducts on S. aureus Newman susceptibility to norfloxacin seemed to be specific to these strains and this antibiotic. Our results illustrate that microbially derived products can alter the ability of antimicrobial agents to kill bacterial biofilms. IMPORTANCE Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from multiple infection sites, including the lungs of individuals with cystic fibrosis (CF) and nonhealing diabetic foot ulcers. Coinfection with P. aeruginosa and S. aureus has been shown to produce worse outcomes compared to infection with either organism alone. Furthermore, the ability of these pathogens to form biofilms enables them to cause persistent infection and withstand antimicrobial therapy. In this study, we found that P. aeruginosa-secreted products dramatically increase the ability of the antibiotic norfloxacin to kill S. aureus biofilms. Understanding how interspecies interactions alter the antibiotic susceptibility of bacterial biofilms may inform treatment decisions and inspire the development of new therapeutic strategies.

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Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz378 ◽

2019 ◽

Vol 75 (1) ◽

pp. 117-125 ◽

Cited By ~ 8

Author(s):

Odel Soren ◽

Ardeshir Rineh ◽

Diogo G Silva ◽

Yuming Cai ◽

Robert P Howlin ◽

...

Keyword(s):

Nitric Oxide ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Clinical Isolates ◽

Nitric Oxide Donor ◽

Confocal Laser ◽

Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

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In Vitro Newly Isolated Environmental Phage Activity against Biofilms Preformed by Pseudomonas aeruginosa from Patients with Cystic Fibrosis

Microorganisms ◽

10.3390/microorganisms9030478 ◽

2021 ◽

Vol 9 (3) ◽

pp. 478

Author(s):

Ersilia Vita Fiscarelli ◽

Martina Rossitto ◽

Paola Rosati ◽

Nour Essa ◽

Valentina Crocetta ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Multidrug Resistant ◽

In Vitro Test ◽

Chronic Infections ◽

Hospital Environments ◽

Vitro Test ◽

General Reliability ◽

Phage Activity

As disease worsens in patients with cystic fibrosis (CF), Pseudomonas aeruginosa (PA) colonizes the lungs, causing pulmonary failure and mortality. Progressively, PA forms typical biofilms, and antibiotic treatments determine multidrug-resistant (MDR) PA strains. To advance new therapies against MDR PA, research has reappraised bacteriophages (phages), viruses naturally infecting bacteria. Because few in vitro studies have tested phages on CF PA biofilms, general reliability remains unclear. This study aimed to test in vitro newly isolated environmental phage activity against PA isolates from patients with CF at Bambino Gesù Children’s Hospital (OBG), Rome, Italy. After testing in vitro phage activities, we combined phages with amikacin, meropenem, and tobramycin against CF PA pre-formed biofilms. We also investigated new emerging morphotypes and bacterial regrowth. We obtained 22 newly isolated phages from various environments, including OBG. In about 94% of 32 CF PA isolates tested, these phages showed in vitro PA lysis. Despite poor efficacy against chronic CF PA, five selected-lytic-phages (Φ4_ZP1, Φ9_ZP2, Φ14_OBG, Φ17_OBG, and Φ19_OBG) showed wide host activity. The Φ4_ZP1-meropenem and Φ14_OBG-tobramycin combinations significantly reduced CF PA biofilms (p < 0.001). To advance potential combined phage-antibiotic therapy, we envisage further in vitro test combinations with newly isolated phages, including those from hospital environments, against CF PA biofilms from early and chronic infections.

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Murepavadin antimicrobial activity against and resistance development in cystic fibrosis Pseudomonas aeruginosa isolates

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa529 ◽

2020 ◽

Author(s):

María Díez-Aguilar ◽

Marta Hernández-García ◽

María-Isabel Morosini ◽

Ad Fluit ◽

Michael M Tunney ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Activity ◽

Spontaneous Mutation ◽

Lung Surfactant ◽

Vitro Activity ◽

In Vitro Activity ◽

Broth Microdilution ◽

Agar Dilution

Abstract Background Murepavadin, a novel peptidomimetic antibiotic, is being developed as an inhalation therapy for treatment of Pseudomonas aeruginosa respiratory infection in people with cystic fibrosis (CF). It blocks the activity of the LptD protein in P. aeruginosa causing outer membrane alterations. Objectives To determine the in vitro activity of murepavadin against CF P. aeruginosa isolates and to investigate potential mechanisms of resistance. Methods MIC values were determined by both broth microdilution and agar dilution and results compared. The effect of artificial sputum and lung surfactant on in vitro activity was also measured. Spontaneous mutation frequency was estimated. Bactericidal activity was investigated using time–kill assays. Resistant mutants were studied by WGS. Results The murepavadin MIC50 was 0.125 versus 4 mg/L and the MIC90 was 2 versus 32 mg/L by broth microdilution and agar dilution, respectively. Essential agreement was >90% when determining in vitro activity with artificial sputum or lung surfactant. It was bactericidal at a concentration of 32 mg/L against 95.4% of the strains within 1–5 h. Murepavadin MICs were 2–9 two-fold dilutions higher for the mutant derivatives (0.5 to >16 mg/L) than for the parental strains. Second-step mutants were obtained for the PAO mutS reference strain with an 8×MIC increase. WGS showed mutations in genes involved in LPS biosynthesis (lpxL1, lpxL2, bamA2, lptD, lpxT and msbA). Conclusions Murepavadin characteristics, such as its specific activity against P. aeruginosa, its unique mechanism of action and its strong antimicrobial activity, encourage the further clinical evaluation of this drug.

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