DNA hybridization analysis of thePseudomonas aeruginosaelastase gene [lasB) from different clinical isolates

1995 ◽  
Vol 41 (10) ◽  
pp. 910-917 ◽  
Author(s):  
Abdul N. Hamood ◽  
John Griswold
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Structural Gene ◽  
Dna Hybridization ◽  
Restriction Site ◽  
Clinical Isolates ◽  
Upstream Region ◽  
Gene Probe ◽  
Chromosomal Dna ◽  
Hybridization Pattern ◽  
Hybridization Band

Pseudomonas aeruginosa produces several extracellular virulence factors including elastase (which is encoded by lasB). Recently, we examined several clinical isolates of P. aeruginosa for the production of toxin A, elastase, exoenzyme S, and phospholipase C. Although the majority of the isolates produced a high level of elastase, a few isolates produced either very low or no detectable elastase. In this study, we tried to determine the presence of restriction site heterogeneity within lasB from these isolates and the possible correlation between such heterogeneity and the observed variation in elastase production. Chromosomal DNA from the isolates was digested with different restriction enzymes and examined by Southern blot hybridization experiments using two lasB probes. One lasB probe covers 636 bp of lasB structural gene while the other covers 240 bp of the lasB upstream region. Chromosomal DNA from P. aeruginosa PAO1 and PA103 was used as controls. Results indicate that chromosomal DNA from all isolates hybridized to both lasB probes. Depending on the restriction enzyme used for DNA digestion, lasB from 3 to 12% of the isolates showed different patterns of hybridization with the lasB structural gene probe. However, no difference in the hybridization pattern was seen with the lasB upstream probe. With the exception of one isolate, hybridization of genomic DNA from different isolates (with both probes) produced a single hybridization band. In that isolate, an additional hybridization band was detected. Immunoblotting experiments confirmed that elastase protein is not produced by 6 out of 67 isolates. However, lasB from four of these elastase-deficient strains showed no difference in the hybridization pattern with either lasB probe. These results suggest that (i) lasB is present as a single copy in all but one isolate; (ii) with the exception of one, the lasB upstream region from different P. aeruginosa isolates contains no restriction site polymorphism; (iii) the observed heterogeneity within lasB structural genes is limited; and (iv) variations in the hybridization patterns of lasB from different isolates do not correlate with the differences between these isolates in elastase production.Key words: Pseudomonas aeruginosa, clinical isolates, DNA hybridization, elastase, lasB.

scholarly journals Comparison of the exoS Gene and Protein Expression in Soil and Clinical Isolates of Pseudomonas aeruginosa

2001 ◽  
Vol 69 (4) ◽  
pp. 2198-2210 ◽  
Author(s):  
Michael W. Ferguson ◽  
Jill A. Maxwell ◽  
Timothy S. Vincent ◽  
Jack da Silva ◽  
Joan C. Olson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Structural Gene ◽  
Gene Sequence ◽  
Specific Activity ◽  
Clinical Isolates ◽  
Secretory Process ◽  
Sequence Comparisons ◽  
Exoenzyme S ◽  
Gene And Protein Expression ◽  
Adp Ribosyltransferase

ABSTRACT Exoenzyme S (ExoS) is translocated into eukaryotic cells by the type III secretory process and has been hypothesized to function in conjunction with other virulence factors in the pathogenesis ofPseudomonas aeruginosa. To gain further understanding of how ExoS might contribute to P. aeruginosa survival and virulence, ExoS expression and the structural gene sequence were determined in P. aeruginosa soil isolates and compared with ExoS of clinical isolates. Significantly higher levels of ExoS ADP-ribosyltransferase (ADPRT) activity were detected in culture supernatants of soil isolates compared to those of clinical isolates. The higher levels of ADPRT activity of soil isolates reflected both the increased production of ExoS and the production of ExoS having a higher specific activity. ExoS structural gene sequence comparisons found the gene to be highly conserved among soil and clinical isolates, with the greatest number of nonsynonymous substitutions occurring within the region of ExoS encoding GAP function. The lack of amino acid changes in the ADPRT region in association with a higher specific activity implies that other factors produced by P. aeruginosa or residues outside the ADPRT region are affecting ExoS ADPRT activity. The data are consistent with ExoS being integral to P. aeruginosa survival in the soil and suggest that, in the transition of P. aeruginosa from the soil to certain clinical settings, the loss of ExoS expression is favored.

scholarly journals AAC(6′)-Iaf, a Novel Aminoglycoside 6′-N-Acetyltransferase from Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates

2009 ◽  
Vol 53 (6) ◽  
pp. 2327-2334 ◽  
Author(s):  
Tomoe Kitao ◽  
Tohru Miyoshi-Akiyama ◽  
Teruo Kirikae
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Decubitus Ulcer ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Upstream Region ◽  
Aminoglycoside Resistance ◽  
Class 1 Integron ◽  
Class 1 ◽  
Layer Chromatography

ABSTRACT We report here the characterization of a novel aminoglycoside resistance gene, aac(6′)-Iaf, present in two multidrug-resistant (MDR) Pseudomonas aeruginosa clinical isolates. These isolates, IMCJ798 and IMCJ799, were independently obtained from two patients, one with a urinary tract infection and the other with a decubitus ulcer, in a hospital located in the western part of Japan. Although the antibiotic resistance profiles of IMCJ798 and IMCJ799 were similar to that of MDR P. aeruginosa IMCJ2.S1, which caused outbreaks in the eastern part of Japan, the pulsed-field gel electrophoresis patterns for these isolates were different from that for IMCJ2.S1. Both IMCJ798 and IMCJ799 were found to contain a novel chromosomal class 1 integron, In123, which included aac(6′)-Iaf as the first cassette gene. The encoded protein, AAC(6′)-Iaf, was found to consist of 183 amino acids, with 91 and 87% identity to AAC(6′)-Iq and AAC(6′)-Im, respectively. IMCJ798, IMCJ799, and Escherichia coli transformants carrying a plasmid containing the aac(6′)-Iaf gene and its upstream region were highly resistant to amikacin, dibekacin, and kanamycin but not to gentamicin. The production of AAC(6′)-Iaf in these strains was confirmed by Western blot analysis. Thin-layer chromatography indicated that AAC(6′)-Iaf is a functional acetyltransferase that specifically modifies the amino groups at the 6′ positions of aminoglycosides. Collectively, these findings indicate that AAC(6′)-Iaf contributes to aminoglycoside resistance.

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

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
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.

Pharmacodynamics of once- versus twice-daily dosing of nebulized amikacin in an in vitro Hollow-Fiber Infection Model against 3 clinical isolates of Pseudomonas aeruginosa

2021 ◽  
Vol 100 (2) ◽  
pp. 115329
Author(s):  
Aaron James Heffernan ◽  
Fekade Bruck Sime ◽  
Saiyuri Naicker ◽  
Katherine Andrews ◽  
David Ellwood ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Hollow Fiber ◽  
Clinical Isolates ◽  
Infection Model

Widespread of ESBL- and carbapenemase GES-type genes on carbapenem-resistant Pseudomonas aeruginosa clinical isolates: a multicenter study in Mexican hospitals

2015 ◽  
Vol 81 (2) ◽  
pp. 135-137 ◽  
Author(s):  
Ulises Garza-Ramos ◽  
Humberto Barrios ◽  
Fernando Reyna-Flores ◽  
Elsa Tamayo-Legorreta ◽  
Juan C. Catalan-Najera ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multicenter Study ◽  
Clinical Isolates ◽  
Carbapenem Resistant

scholarly journals Detection of Extended-Spectrum β-Lactamases in Clinical Isolates of Pseudomonas aeruginosa

2006 ◽  
Vol 50 (9) ◽  
pp. 2990-2995 ◽  
Author(s):  
Xiaofei Jiang ◽  
Zhe Zhang ◽  
Min Li ◽  
Danqiu Zhou ◽  
Feiyi Ruan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Screening Methods ◽  
Extended Spectrum ◽  
Pcr Product ◽  
Amoxicillin Clavulanate ◽  
Synergy Test ◽  
Esbl Producers ◽  
Disk Test

ABSTRACT With the occurrence of extended-spectrum β-lactamases (ESBLs) in Pseudomonas aeruginosa being increasingly reported worldwide, there is a need for a reliable test to detect ESBLs in clinical isolates of P. aeruginosa. In our study, a total of 75 clinical isolates of P. aeruginosa were studied. Nitrocefin tests were performed to detect the β-lactamase enzyme; isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to further characterize the contained ESBLs. Various ESBL-screening methods were designed to compare the reliabilities of detecting ESBLs in clinical isolates of P. aeruginosa whose β-lactamases were well characterized. Thirty-four of 36 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBLs. bla VEB-3 was the most prevalent ESBL gene in P. aeruginosa in our study. Among the total of 34 isolates that were considered ESBL producers, 20 strains were positive using conventional combined disk tests and 10 strains were positive using a conventional double-disk synergy test (DDST) with amoxicillin-clavulanate, expanded-spectrum cephalosporins, aztreonam, and cefepime. Modifications of the combined disk test and DDST, which consisted of shorter distances between disks (20 mm instead of 30 mm) and the use of three different plates that contained cloxacillin (200 μg/ml) alone, Phe-Arg β-naphthylamide dihydrochloride (MC-207,110; 20 μg/ml) alone, and both cloxacillin (200 μg/ml) and MC-207,110 (20 μg/ml) increased the sensitivity of the tests to 78.8%, 91.18%, 85.29%, and 97.06%.

scholarly journals Development of Multi-Drug Resistant Mutants of Clinical Isolates of Pseudomonas aeruginosa after Exposure to Fluoroquinolone

1996 ◽  
Vol 70 (2) ◽  
pp. 123-131 ◽  
Author(s):  
Miyuki HASEGAWA ◽  
Intetsu KOBAYASHI ◽  
Takeshi SAIKA ◽  
Mitsunobu SHIMAZU ◽  
Minoru NISHIDA
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Isolates ◽  
Drug Resistant ◽  
Resistant Mutants

Validation of MALDI-TOF for the early detection of the ST175 high-risk clone of Pseudomonas aeruginosa in clinical isolates belonging to a Spanish nationwide multicenter study

2020 ◽  
Author(s):  
Xavier Mulet ◽  
Marta Fernández-Esgueva ◽  
Cristina Norte ◽  
Laura Zamorano ◽  
Ester del Barrio-Tofiño ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Risk ◽  
Early Detection ◽  
Multicenter Study ◽  
Clinical Isolates ◽  
Maldi Tof

New integron gene arrays from multiresistant clinical isolates of members of the Enterobacteriaceae and Pseudomonas aeruginosa from hospitals in Malaysia

2013 ◽  
Vol 62 (3) ◽  
pp. 412-420 ◽  
Author(s):  
Sue-Bee Kor ◽  
Quok-Cheong Choo ◽  
Choy-Hoong Chew
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Isolates ◽  
Gene Arrays
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