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The Frequency of Exotoxin A and Exoenzymes S and U Genes Among Clinical Isolates of Pseudomonas aeruginosa in Shiraz, Iran.

Yousefi-Avarvand A, Khashei R, Sedigh Ebrahim-Saraie H, Emami A, Zomorodian K, Motamedifar M - Int J Mol Cell Med (2015)

Bottom Line: Indeed, genotypes exoS (+)/exoU (+) and exoS (-)/exoU (-) were found with frequencies of 48.7% and 15.3%, respectively.The resistance rate in exoU (+) isolates was 86% compared to 66% in exoS (+) isolates.The high frequencies of virulence genes detected in our clinical isolates with notable antibiotic resistance rates indicate the potential risk of these isolates in nosocomial infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.

ABSTRACT
Pseudomonas aeruginosa as an opportunistic pathogen produces several virulence factors. The most important of these factors are exotoxin A and type III secretion system (T3SS). The aim of this study was to determine the frequency of toxA, exoU and exoS genes among clinical isolates of P. aeruginosa. In this cross-sectional study from September 2011 to February 2012, 156 P. aeruginosa isolates were recovered from different clinical samples. Susceptibility testing against 10 antibiotics was performed on individual isolates by the disc diffusion method according to CLSI guidelines. Extracted DNA was subjected to PCR assay for determining the presence of toxA, exoU and exoS genes. Overall, the frequency of toxA, exoU and exoS genes were 90.4%, 66.7% and 65.4%, respectively. All of the abdominal and eye isolates were exoS (+). The frequency of exoS (+)/exoU (-) and exoS (-)/exoU (+) genotypes was estimated 19.2% and 16.2%, respectively. Indeed, genotypes exoS (+)/exoU (+) and exoS (-)/exoU (-) were found with frequencies of 48.7% and 15.3%, respectively. The highest and lowest antibiotic resistance rate was seen against azteroenam (94.2%) and amikacin (44.9%), respectively. Fluoroqinolone-resistant isolates were isolated with frequency of 45.8%. Multi-drug resistant (MDR) isolates were detected in 62.8% of isolates. The resistance rate in exoU (+) isolates was 86% compared to 66% in exoS (+) isolates. The high frequencies of virulence genes detected in our clinical isolates with notable antibiotic resistance rates indicate the potential risk of these isolates in nosocomial infections.

No MeSH data available.


Related in: MedlinePlus

Amplification of toxA, exoS and exoU genes from clinical isolates of P. aeruginosa by PCR. M: 100 bp ladder; lane 1: toxA gene positive control; lanes 2, 8 and 15: negative control (E. coli ATCC 35218); lanes 3-6 investigated clinical isolates for toxA gene; lane 7: exoU gene positive control; lanes 9-12: investigated clinical isolates for exoU gene; lane 14: exoS gene positive control; lanes 16-18: investigated clinical isolates for exoS gene
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Figure 1: Amplification of toxA, exoS and exoU genes from clinical isolates of P. aeruginosa by PCR. M: 100 bp ladder; lane 1: toxA gene positive control; lanes 2, 8 and 15: negative control (E. coli ATCC 35218); lanes 3-6 investigated clinical isolates for toxA gene; lane 7: exoU gene positive control; lanes 9-12: investigated clinical isolates for exoU gene; lane 14: exoS gene positive control; lanes 16-18: investigated clinical isolates for exoS gene

Mentions: Genomic DNA was extracted from overnight TSB cultures of P. aeruginosa isolates using the small-scale phenol-chloroform extraction method (16). The evaluation of toxA, exoS and exoU genes was accomplished by previously described primers (9,17). PCR amplification was performed in 50 l reaction volume consisting of 5 l 1x PCR buffer, 2 µl of each primer (10 pmol/µl), 1 l MgCl2 (1.5 mM), 0.8 l each of the dNTPs (200 µM), 0.6 l Taq DNA polymerase (1 U), and 2 l DNA (10-1,000 ng) from each isolate. The PCR cycling conditions were: initial denaturation at 95 °C for 10 min, followed by 30 cycles (for exoA gene) and 35 cycles (for exoU and exoS genes) of denaturation at 95 °C for 30 seconds, annealing at 55 °C for 45 seconds, extension at 72 °C for 40 seconds, with final extension at 72 °C for 10 min. All reagents were obtained from Cinnagene Co., Iran. In each run of amplicons, P. aeruginosa ATCC 27853 which is positive for both toxA and exoS, and one clinical isolate which became positive for the presence of exo U gene (428 bp amplicon) and was further confirmed by DNA sequencing, and negative (E. coli ATCC 35218) controls were included in agarose gels. The amplicons were resolved in a 1% horizontal agarose gel, stained with ethidium bromide and photographed under 300 nm UV light (Figure 1).


The Frequency of Exotoxin A and Exoenzymes S and U Genes Among Clinical Isolates of Pseudomonas aeruginosa in Shiraz, Iran.

Yousefi-Avarvand A, Khashei R, Sedigh Ebrahim-Saraie H, Emami A, Zomorodian K, Motamedifar M - Int J Mol Cell Med (2015)

Amplification of toxA, exoS and exoU genes from clinical isolates of P. aeruginosa by PCR. M: 100 bp ladder; lane 1: toxA gene positive control; lanes 2, 8 and 15: negative control (E. coli ATCC 35218); lanes 3-6 investigated clinical isolates for toxA gene; lane 7: exoU gene positive control; lanes 9-12: investigated clinical isolates for exoU gene; lane 14: exoS gene positive control; lanes 16-18: investigated clinical isolates for exoS gene
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4644528&req=5

Figure 1: Amplification of toxA, exoS and exoU genes from clinical isolates of P. aeruginosa by PCR. M: 100 bp ladder; lane 1: toxA gene positive control; lanes 2, 8 and 15: negative control (E. coli ATCC 35218); lanes 3-6 investigated clinical isolates for toxA gene; lane 7: exoU gene positive control; lanes 9-12: investigated clinical isolates for exoU gene; lane 14: exoS gene positive control; lanes 16-18: investigated clinical isolates for exoS gene
Mentions: Genomic DNA was extracted from overnight TSB cultures of P. aeruginosa isolates using the small-scale phenol-chloroform extraction method (16). The evaluation of toxA, exoS and exoU genes was accomplished by previously described primers (9,17). PCR amplification was performed in 50 l reaction volume consisting of 5 l 1x PCR buffer, 2 µl of each primer (10 pmol/µl), 1 l MgCl2 (1.5 mM), 0.8 l each of the dNTPs (200 µM), 0.6 l Taq DNA polymerase (1 U), and 2 l DNA (10-1,000 ng) from each isolate. The PCR cycling conditions were: initial denaturation at 95 °C for 10 min, followed by 30 cycles (for exoA gene) and 35 cycles (for exoU and exoS genes) of denaturation at 95 °C for 30 seconds, annealing at 55 °C for 45 seconds, extension at 72 °C for 40 seconds, with final extension at 72 °C for 10 min. All reagents were obtained from Cinnagene Co., Iran. In each run of amplicons, P. aeruginosa ATCC 27853 which is positive for both toxA and exoS, and one clinical isolate which became positive for the presence of exo U gene (428 bp amplicon) and was further confirmed by DNA sequencing, and negative (E. coli ATCC 35218) controls were included in agarose gels. The amplicons were resolved in a 1% horizontal agarose gel, stained with ethidium bromide and photographed under 300 nm UV light (Figure 1).

Bottom Line: Indeed, genotypes exoS (+)/exoU (+) and exoS (-)/exoU (-) were found with frequencies of 48.7% and 15.3%, respectively.The resistance rate in exoU (+) isolates was 86% compared to 66% in exoS (+) isolates.The high frequencies of virulence genes detected in our clinical isolates with notable antibiotic resistance rates indicate the potential risk of these isolates in nosocomial infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.

ABSTRACT
Pseudomonas aeruginosa as an opportunistic pathogen produces several virulence factors. The most important of these factors are exotoxin A and type III secretion system (T3SS). The aim of this study was to determine the frequency of toxA, exoU and exoS genes among clinical isolates of P. aeruginosa. In this cross-sectional study from September 2011 to February 2012, 156 P. aeruginosa isolates were recovered from different clinical samples. Susceptibility testing against 10 antibiotics was performed on individual isolates by the disc diffusion method according to CLSI guidelines. Extracted DNA was subjected to PCR assay for determining the presence of toxA, exoU and exoS genes. Overall, the frequency of toxA, exoU and exoS genes were 90.4%, 66.7% and 65.4%, respectively. All of the abdominal and eye isolates were exoS (+). The frequency of exoS (+)/exoU (-) and exoS (-)/exoU (+) genotypes was estimated 19.2% and 16.2%, respectively. Indeed, genotypes exoS (+)/exoU (+) and exoS (-)/exoU (-) were found with frequencies of 48.7% and 15.3%, respectively. The highest and lowest antibiotic resistance rate was seen against azteroenam (94.2%) and amikacin (44.9%), respectively. Fluoroqinolone-resistant isolates were isolated with frequency of 45.8%. Multi-drug resistant (MDR) isolates were detected in 62.8% of isolates. The resistance rate in exoU (+) isolates was 86% compared to 66% in exoS (+) isolates. The high frequencies of virulence genes detected in our clinical isolates with notable antibiotic resistance rates indicate the potential risk of these isolates in nosocomial infections.

No MeSH data available.


Related in: MedlinePlus