Development and evaluation of a new PCR assay for detection of Pseudomonas aeruginosa D genotype.

This report describes a new PCR-based assay for the detection of Pseudomonas aeruginosa genotype D in occupational saturation diving systems in the North Sea. This genotype has persisted in these systems for 11 years (1993-2003) and represents 18% of isolates from infections analysed during this period. The new PCR assay was based on sequences obtained after randomly amplified polymorphic DNA (RAPD)-PCR analysis of a group of isolates related to diving that had been identified previously by pulsed-field gel electrophoresis (PFGE). The primer set for the D genotype targets a gene that codes for a hypothetical class 4 protein in the P. aeruginosa PAO1 genome. A primer set able to detect P. aeruginosa at the species level was also designed, based on the 23S-5S rDNA spacer region. The two assays produced 382-bp and 192-bp amplicons, respectively. The PCR assay was evaluated by analysing 100 P. aeruginosa isolates related to diving, representing 28 PFGE genotypes, and 38 clinical and community P. aeruginosa isolates and strains from other species. The assay identified all of the genotype D isolates tested. Two additional diving-relevant genotypes (TP2 and TP27) were also identified, as well as three isolates of non-diving origin. It was concluded that the new PCR assay is a useful tool for early detection and prevention of infections with the D genotype.

An in-field demonstration of the true relationship between skin infections and their sources in occupational diving systems in the North Sea.

INTRODUCTION: Skin infections in saturation diving are caused by microbes that flourish in saturation environments. Improvements in the prevention of infections must therefore be based on environmental control and elimination. Furthermore, only a few genotypes seem to be responsible for the majority of infections in the Norwegian sector of the North Sea, and these have all been demonstrated in saturation systems for many years. Although reservoirs of infectious genotypes have been identified, their true sources have not been identified. OBJECTIVES: The purpose of this field study was to log the contamination by Pseudomonas aeruginosa of the saturation system throughout a diving operation. MATERIALS AND METHODS: Daily water samples from the vessels drinking water system and from the heated seawater systems to divers suits were taken throughout the diving period of 1 month in the summer of 2001. All P.aeruginosa isolates were genotyped by pulsed field gel electrophoresis. RESULTS: A total of 17 P.aeruginosa genotypes were identified in the course of this field study. None of the most common infectious genotypes previously observed in the Norwegian sector were among these strains. Two genotypes were involved in skin infections during the period of operation: TP2 and TP12. TP2 was shown to be an inhabitant of the diving systems throughout the investigation period, while TP12 was introduced from seawater in the course of the operation and rapidly spread and established itself throughout the diving system. CONCLUSIONS: The study has demonstrated seawater as a true source of an infectious P.aeruginosa genotype in occupational diving systems.

The impact of environmental Pseudomonas aeruginosa genotypes on skin infections in occupational saturation diving systems.

Skin infection caused by Pseudomonas aeruginosa is the most frequent health problem associated with occupational saturation diving on the Norwegian continental shelf. In the course of 14-y surveillance of infection and environmental control in occupational offshore saturation diving systems, a collection of approximately 1000 P. aeruginosa isolates has been amassed. Retrospective genomic analyses using restriction enzyme fragmentation and pulsed-field gel electrophoresis have identified 24 of 76 environmental P. aeruginosa genotypes as being of significance for single infections, outbreaks of infections and recurrent skin infections in occupational diving systems. In addition, these genomic analyses have made it possible to separate outbreaks of infection into outbreaks with 1 single genotype and clusters of infections where different genotypes are involved. We conclude that the established, assumed diver-to-diver contagion vector ought to be replaced by a environmental contagion vector as the most likely vector within these specific occupational environments. Furthermore, consecutive presence of the frequent environmental/infectious genotypes demands specific improvement of infection prevention and control in these systems.

Survival of infectious Pseudomonas aeruginosa genotypes in occupational saturation diving environment and the significance of these genotypes for recurrent skin infections.

BACKGROUND: Occupational saturation divers suffer from various skin disorders, of which skin infections are the most serious and frequent. Pseudomonas aeruginosa is the microbe most often isolated. METHODS: P. aeruginosa isolates from 292 skin infections in operational saturation divers and about 800 isolates from occupational saturation diving systems have been collected during the period 1986 to 1998. Genotyping of the isolates has been performed by using restriction enzyme fragmentation and pulsed field gel electrophoresis. RESULTS: Four hundred and seventy-two P. aeruginosa isolates have been analyzed, of which 181 originate from skin infections in divers. Ninety-seven significantly different P. aeruginosa genotypes have been defined. Some of these genotypes are solely found from skin infections, some solely from the saturation environment and about 25% were found both from infections and from the saturation environment. Eight frequent infectious genotypes have been identified, and these are shown to be present over several years, both in infections and in the saturation environment. CONCLUSIONS: The study suggests that skin infections in occupational saturation divers are commonly caused by environmental strains.

Identification of infectious Pseudomonas aeruginosa strains in an occupational saturation diving environment.

OBJECTIVES: Occupational saturation divers have various skin disorders, of which skin infections are the most serious and frequent. Pseudomonas aeruginosa is the microbe most often isolated from skin infections in divers. The purpose of the present work was (a) to report the occurrence of P aeruginosa in skin infections in operational saturation diving in the North Sea from 1987 to 1995; (b) to report the environmental occurrence of P aeruginosa in saturation diving systems, and finally (c) to identify possible relations between infection related to strains of P aeruginosa and environmental isolates of the microbe. RESULTS: During the period 1987-95, P aeruginosa was isolated from 257 skin infections in operational saturation divers. Most of the isolates related to infection by P aeruginosa show a unique growth inhibition pattern towards the normal skin flora, and the serotype pattern of P aeruginosa from skin infections is limited compared with similar infections in non-divers. In a mini-epidemiological study on board one diving vessel during one operational diving period, five significantly different DNA fragment profiles were found among the 12 isolates related to infection by P aeruginosa obtained from the saturation system. In two cases the infectious genotypes were detected in the fresh water for the saturation chambers weeks before the arrival of the infected diver. CONCLUSIONS: The most commonly used epidemiological marker for P aeruginosa world wide, also used in earlier studies, is serotyping, but with pulsed field gel electrophoresis (PFGE) miniepidemiology it was shown to be insufficient for epidemiological purposes in saturation environments. PFGE analyses were shown to be superior both to antibacterial factor and to serotyping in epidemiological analyses of P aeruginosa infections in saturation diving.