Dataset Information

Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing, China.

ABSTRACT: Pseudomonas aeruginosa is a major opportunistic pathogen in hospital-acquired infections and exhibits increasing antibiotic resistance. A rapid and sensitive molecular method for its detection in clinical samples is needed to guide therapeutic treatment and to control P. aeruginosa outbreaks. In this study, we established a polymerase spiral reaction (PSR) method for rapid detection of P. aeruginosa by targeting the toxA gene, which regulates exotoxin A synthesis. Real-time turbidity monitoring and a chromogenic visualization using hydroxynaphthol blue were used to assess the reaction. All 17 non- P. aeruginosa strains tested negative, indicating the high specificity of the PSR primers. The detection limit was 2.3 pg/?l within 60 min at isothermal temperature (65°C), 10-fold more sensitive than conventional PCR. Then, the PSR assay was applied to a clinical surveillance of P. aeruginosa in three top hospitals in Beijing, China. Of the 130 sputum samples collected from ICU patients with suspected multi-resistant infections, 37 P. aeruginosa isolates were identified from the positive samples. All clinical strains belonged to 10 different P. aeruginosa multilocus sequence typing groups and exhibited high resistance to carbapenems, cephalosporins, and aminoglycosides. Interestingly, of the 33 imipenem-resistant isolates, 30 (90.9%) had lost the outer membrane porin oprD gene. Moreover, isolate SY-95, containing multiple antibiotic resistance genes, possessed the ability to hydrolyze all antibiotics used in clinic and was susceptible only to polymyxin B. Our study showed the high level of antibiotic resistance and co-occurrence of resistance genes in the clinical strains, indicating a rapid and continuing evolution of P. aeruginosa. In conclusion, we developed a P. aeruginosa PSR assay, which could be a useful tool for clinical screening, especially in case of poor resources, or for point-of-care testing.

SUBMITTER: Dong D

PROVIDER: S-EPMC4594016 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing, China.

Dong Derong D Zou Dayang D Liu Hui H Yang Zhan Z Huang Simo S Liu Ningwei N He Xiaoming X Liu Wei W Huang Liuyu L

Frontiers in microbiology 20151006

Pseudomonas aeruginosa is a major opportunistic pathogen in hospital-acquired infections and exhibits increasing antibiotic resistance. A rapid and sensitive molecular method for its detection in clinical samples is needed to guide therapeutic treatment and to control P. aeruginosa outbreaks. In this study, we established a polymerase spiral reaction (PSR) method for rapid detection of P. aeruginosa by targeting the toxA gene, which regulates exotoxin A synthesis. Real-time turbidity monitoring ...[more]

PMID: 26500639

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Project description:Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

Project description:IntroductionPreventing carriage of potentially pathogenic micro-organisms from the aerodigestive tract is an infection control strategy used to reduce the occurrence of ventilator-associated pneumonia in intensive care units. However, antibiotic use in selective decontamination protocols is controversial. The purpose of this study was to investigate the effect of oral administration of a probiotic, namely Lactobacillus, on gastric and respiratory tract colonization/infection with Pseudomonas aeruginosa strains. Our hypothesis was that an indigenous flora should exhibit a protective effect against secondary colonization.MethodsWe conducted a prospective, randomized, double-blind, placebo-controlled pilot study between March 2003 and October 2004 in a 17-bed intensive care unit of a teaching hospital in Clermont-Ferrand, France. Consecutive patients with a unit stay of longer than 48 hours were included, 106 in the placebo group and 102 in the probiotic group. Through a nasogastric feeding tube, patients received either 109 colony-forming units unity forming colony of Lactobacillus casei rhamnosus or placebo twice daily, from the third day after admission to discharge. Digestive tract carriage of P. aeruginosa was monitored by cultures of gastric aspirates at admission, once a week thereafter and on discharge. In addition, bacteriological analyses of respiratory tract specimens were conducted to determine patient infectious status.ResultsThe occurrence of P. aeruginosa respiratory colonization and/or infection was significantly delayed in the probiotic group, with a difference in median delay to acquisition of 11 days versus 50 days (P = 0.01), and a nonacquisition expectancy mean of 69 days versus 77 days (P = 0.01). The occurrence of ventilator-associated pneumonia due to P. aeruginosa in the patients receiving the probiotic was less frequent, although not significantly reduced, in patients in the probiotic group (2.9%) compared with those in the placebo group (7.5%). After multivariate Cox proportional hazards modelling, the absence of probiotic treatment increased the risk for P. aeruginosa colonization in respiratory tract (adjusted hazard ratio = 3.2, 95% confidence interval - 1.1 to 9.1).ConclusionIn this pilot study, oral administration of a probiotic delayed respiratory tract colonization/infection by P. aeruginosa.Trial registrationThe trial registration number for this study is NCT00604110.

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Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing, China.

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Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing, China.

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