An In-field Demonstration of the True Relationship between Skin Infections and their Sources in Occupational Diving Systems in the North Sea

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1 Ann. occup. Hyg., Vol. 47, No. 3, pp , British Occupational Hygiene Society Published by Oxford University Press DOI: /annhyg/meg034 An In-field Demonstration of the True Relationship between Skin Infections and their Sources in Occupational Diving Systems in the North Sea C. AHLÉN 1 *, L. H. MANDAL 1 and O. J. IVERSEN 2 1 Division of Microbial Exposure and Indoor Air, SINTEF Unimed, N-7034 Trondheim; 2 Department of Laboratory Medicine, Norwegian University of Science and Technology, N-7006 Trondheim, Norway Received 15 July 2002; in final form 10 January 2003 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 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. Keywords: Pseudomonas aeruginosa; saturation diving; skin infections; source identification INTRODUCTION Occupational saturation diving is extensively employed in the installation, inspection and maintenance of offshore sub-sea oil production systems in the North Sea. The occupational living and working environment is unique (Freitag and Woods, 1983). During saturation, teams of divers live for weeks at a time in confined steel chambers, pressurized with helium to the actual working depth of the diving operation. The partial pressure of oxygen is kept between 40 and 60 kpa. The ambient absolute pressure is dependent on the working depth, which in the North Sea *Author to whom correspondence should be addressed. Tel: ; fax: ; catrine.ahlen@sintef.no normally is between 50 and 200 m ( MPa). The ambient temperature is typically between 28 and 30 C and relative humidity may reach 80 90% during periods of intense diving activity. In order to maintain thermal balance while working in the sea, the divers wear protective suits, through which heated ( 36 C) seawater is continuously flushed onto the skin. All in all, saturation diving involves several unusual environmental factors, any or all of which may be of significance for occupational health (Alcock, 1977; Hope et al., 1994; Ahlén et al., 1998a,b). Superficial skin infections are a dominant health problem during occupational diving in the Norwegian offshore sector (Norwegian Petroleum Directorate, 1999). The microbial flora of the living and working environment is very rich, in terms of both numbers 227

2 228 C. Ahlén, L. H. Mandal and O. J. Iversen Fig. 1. Identification of P.aeruginosa isolates from the Upgrade study using PFGE and the endonuclease SpeI. The PFGE patterns are consecutively coded (capital letters). The sources of the isolates are shown in Table 2. and genera. Nevertheless, the dominant microbe in divers skin infections is Pseudomonas aeruginosa, a very common bacterium in waters all around the world, and known as a very common challenge to divers health since the beginning of saturation diving (Thalman, 1974). In the course of a long-term process of infection monitoring and environmental control in Norwegian occupational offshore saturation diving systems, a great deal of specific knowledge about the infectionrelated P.aeruginosa strains has been gained. This knowledge includes both phenotypic and genotypic characteristics and patterns (Ahlén et al., 1998b). The most important epidemiological findings from this surveillance process have been gained from retrospective genetic studies of our collection of more than 1000 field-related P.aeruginosa strains. A total of 250 genotypes have been identified from infections, of which a few have been identified as frequent infectious genotypes (Ahlén et al., 2000). These have been shown to occur frequently both within the saturation environment and in infections over many years. The relationships and significance of these environmental isolates on the spectrum of infections in occupational diving have recently been discussed (Ahlén et al., 2001). Potential reservoirs of these strains, involving both water and gas systems on board diving vessels, have been identified. In spite of the long period during which monitoring has continued, it has so far proved impossible to identify the source of the specific frequent genotypes in saturation systems. This paper describes seawater as a candidate true source of an infection-related genotype of P.aeruginosa. MATERIALS AND METHODS Field study The field study was undertaken during a 1 month diving operation in July 2001 in the western part of the Norwegian offshore sector of the North Sea. The diving vessel was British, relatively new and had never been to the Norwegian sector before. The specific diving operation was pipe laying of a new gas pipeline, and involved three different fields and locations at which the vessel was stationary for periods. The respective periods were 3 14 July, July and, finally, 26 July 3 August. Mobilization for the operation was carried out in harbour in Haugesund, Norway. Diving vessels usually have two separate water production systems on board; one for drinking water

3 Pseudomonas aeruginosa in diving systems 229 Table 1. Pseudomonas aeruginosa genotypes and isolation sites during the field study Genotype Date of first occurrence Dates of subsequent occurrence Sites of occurrence TP 5 29 June 4, 5, 8 July Galley, Ch3 TP 6 29 June 15, 21, 24 July Galley, Ch3 TP 7/TP June Sw systems TP 8 29 June 3, 21, 24 July Ch3 TP9 7 July 8 July Ch1 TP10 7 July Sw systems TP11 14 July Sw intake TP12 15 July 16, 21, 24, 25, 27 July Sw intake, Ch1, Ch2, Ch4, Ch5, Equip. room, Sat ctr, Suit rinse, infection TP13 15 July 16 July Sw intake TP15 18 July Sw intake TP16 18 July Sw systems TP17 21 July Sw systems Ch, saturation chamber; Equip. room, equipment room; Sat ctr, saturation control room; Sw, seawater. and one for hot water (seawater) for the divers. In addition to the seawater production system, this vessel had two separate drinking water production systems on board; one employing evaporation and the other reverse osmosis. Potable water was bunkered from an onshore supply during mobilization on 29 June 2 July. Except for a certain amount of evaporation, bunkered water was the main source of water during the first 2 weeks of operation. Full water production on board was first introduced on the morning of 15 July, upon arrival at the second stationary location. Microbiological sampling and analyses The field study focused entirely on P.aeruginosa. The total microbial flora observed in the samples is therefore not reported here. The quality of microbiological sampling was assured by the presence of licensed medical personnel (nurses) on board throughout the operation. Microbiological analyses of samples were performed at the same clinical microbiological laboratory onshore as has been employed since the start of monitoring in Details of microbiological sampling procedures, analyses and identification have been described elsewhere (Ahlén et al., 1998b), and are only described briefly here. Sampling period. The sampling period included mobilization, operation and demobilization, and lasted from 29 June, the date of arrival of the diving vessel at the quay in Norway, to 3 August, the date of return to the same quay. Daily water samples. Daily samples of fresh water (potable water from both the general system on board the vessel and from the water supply to the saturation system) were taken from the following sites. External saturation systems: taps in galley, saturation control Table 2. Pseudomonas aeruginosa genotype patterns and site of isolation: Upgrade study Date Sampling site PFGE pattern (TP) March 2001 Galley March 2001 EL1 shower March 2001 EL1 shower March 2001 EL1 shower March 2001 EL4 shower March 2001 EL4 shower March 2001 EL3 shower, swab March 2001 EL4 shower, swab March 2001 EL5 hose toilet, swab 4 EL, entry lock. room and equipment maintenance room. Internal saturation systems: taps and showers in saturation chambers 1 5. Daily samples from the hot water system for divers at work were taken from the seawater intake. Sampling protocols were distributed to the vessel prior to project start. Transport of samples to laboratory. Daily delivery of samples to the laboratory was not possible due to infrequent helicopter transport schedules. The daily

4 230 C. Ahlén, L. H. Mandal and O. J. Iversen Table 3. Pseudomonas aeruginosa genotypes present during the first part of the field study Date Sampling site PFGE pattern (TP) 29 June 2001 Galley dish hw, UK baseline 5 29 June 2001 Galley dish hw, UK baseline 6 29 June 2001 Sw, U1 AFT, UK baseline 7 29 June 2001 EL4 tap cw 3 3 July 2001 EL3 shower mix 8 4 July 2001 EL3 shower mix 5 7 July 2001 EL1 shower mix 9 7 July 2001 EL1 tap cw 9 7 July 2001 EL3 tap hw 5 7 July 2001 Sw, FWD bell U July 2001 EL1 shower mix 9 8 July 2001 EL3 shower mix 5 14 July 2001 Sw intake July 2001 Sw intake July 2001 EL2 shower mix 2 15 July 2001 EL3 shower mix 6 15 July 2001 EL4 shower mix 3 15 July 2001 Sw intake 13 cw, cold water; EL, entry lock; hw, hot water; Sw, seawater. water samples were therefore stored at 4 C until they were sent to the laboratory ashore. Samples were collected for five working days at most. Microbiological analyses. A 500 ml aliquot of each sample of water was filtered (Millipore 0.45 µm) and cultured on Pseudomonas agar (Cetrimidine). Specific microbial findings were biochemically typed (API systems) to species level. All P.aeruginosa isolates were further analysed by genotyping. Genotyping was done by means of pulsed-field gel electrophoresis (PFGE). The method has been described in detail in earlier reports and papers (Ahlén et al., 1998b). Briefly, DNA was digested by means of a rare-cutter endonuclease SpeI (Boehringer Mannheim Biochemica) and electrophoresis was performed in a commercial Gene Navigator (Pharmacia Biotech). The DNA fragment patterns were visually compared with each other and with previously identified genotypes in our material from saturation diving. All new genotypes were labelled TP with a consecutive number. Upgrade study : fitness for diving operations in the Norwegian sector The regulations for manned diving operations are set on a national basis, which results in differences in Table 4. Pseudomonas aeruginosa genotypes present in the last part of the field study Date Sampling site PFGE pattern (TP) 24 July 2001 Sat ctr tap mix July 2001 Equip. room, tap mix July 2001 Suit rinse, tap mix July 2001 EL1 shower mix July 2001 EL2 shower mix 2 24 July 2001 EL3 shower mix 8 24 July 2001 EL3 shower mix 6 24 July 2001 EL4 shower mix July 2001 EL5 shower mix July 2001 Sw intake July 2001 Sat ctr tap mix July 2001 Equip. room tap mix July 2001 Suit rinse, tap mix July 2001 EL2 shower mix July 2001 EL4 shower mix July 2001 EL5 shower mix 4 27 July 2001 D.E., infection July 2001 D.E., infection 12 Equip. room, equipment room; EL, entry lock; Sat ctr, saturation control room; Sw, seawater. the regulations applied by individual countries. For this specific project, harmonization of the UK and the Norwegian procedures for hygiene and infection control was undertaken as a separate project: the Upgrade study. This study was performed towards the end of April 2001 while the vessel was in harbour in the UK, and included a thorough survey of contamination status on board the vessel, particularly in the saturation systems. A very high level of contamination of P.aeruginosa was demonstrated in the saturation systems, with massive contamination of the freshwater supply to the saturation chambers. Cleaning and disinfection regimes were implemented by the vessel management in accordance with previously described guidelines (Ahlén et al., 1991). The genotypes found in the saturation system on board the diving vessel during the Upgrade study were four different genotypes, none of which were related to the frequent infectious genotypes known from Norwegian saturation systems. The genotypes isolated in the Upgrade study were labelled TP1, TP2, TP3 and TP4. The sampling sites and PFGE patterns of these genotypes are shown in Table 2 and Fig. 1.

5 Pseudomonas aeruginosa in diving systems 231 Fig. 2. Pseudomonas aeruginosa genotypes, identified by PFGE, present during the first part of the field study. The sources of the isolates are shown in Table 3. RESULTS None of the most common P.aeruginosa infectious genotypes seen in other studies in the Norwegian sector were identified in this study. Two of the genotypes identified from the Upgrade study (TP2 and TP3) were also found in the field study. TP2 was identified in the showers in one of the chambers on 15 July, and skin infections caused by the same genotype were seen on 18 July. TP3 was regularly observed throughout the field study, but has not been involved in infection. A total of 13 P.aeruginosa genotypes not previously observed in the Norwegian sector were isolated in the course of the field study. These were consecutively labelled TP5 TP17 on the basis of their first occurrence in the diving systems (with the exception of TP14, which was present during the mobilization period but was analysed at a later stage). In Table 1, the first occurrence, later occurrences throughout the field study and sites of isolation of each genotype are shown. Table 3 and Fig. 2 list the genotypes identified during the first part of the field study. Genotypes TP5, TP6, TP7 and T14 had already been seen in the mobilization period (29 30 June). TP5 and TP6 were identified from the drinking water system outside the chamber systems and TP7 and TP14 were identified from the hot seawater system. Genotypes TP8 and TP9 were identified early in the diving operations period, and both of these were found in the drinking water supplied to the chamber system. In Table 4 and Fig. 3, the genotypes present during the last part of the field study are shown. Genotypes TP10 TP17 were all initially identified from the seawater intake throughout the diving operations period. Genotypes TP10 and TP11 were introduced into the system during operations at the first location and the remaining genotypes (TP12 TP17) were all introduced at the second diving location. Although several genotypes were introduced into the systems in the course of the period of operation, only one genotype, TP12, was widely disseminated throughout the systems, spreading to both the external vessel systems and the saturation systems (Table 1). TP12 was demonstrated as a pure culture from skin infections on 21 July (Fig. 3). DISCUSSION We have previously reported that skin infections in occupational saturation divers in the Norwegian offshore sector are related to certain frequent infectious genotypes of P.aeruginosa (Ahlén et al., 1998a,b). These genotypes have been shown to be present both in the saturation environment and as

6 232 C. Ahlén, L. H. Mandal and O. J. Iversen Fig. 3. Pseudomonas aeruginosa genotypes, identified by PFGE, present during the last part of the field study. The dominance of TP12 can be seen both in the external systems and in the saturation systems. The sources of the isolates are shown in Table 4. skin infection strains over a period of many years (Ahlén et al., 2000, 2001). While several potential reservoirs of the frequent infectious genotypes have been determined, it has hitherto been impossible to identify the respective sources of these genotypes. Knowledge of sources is of great importance for improving control and elimination, and thereby prevention, of occupational infections. The field study presented in this paper was performed on a diving vessel which had not previously been employed in the Norwegian sector. None of the common frequent infectious genotypes previously observed were seen during this field study. The fact that this operation involved the laying of a new pipeline in fields not earlier exposed to oil exploration might be of relevance for this result. Including the genotypes from the Upgrade study in the UK sector, a total of 17 genotypes were identified from the diving system investigated in the field study. The absence of the earlier well-known genotypes together with the new patterns from the UK sector may suggest that P.aeruginosa genotypes are to a certain extent field-specific. A total of 13 P.aeruginosa genotypes were identified throughout the field study period. As mentioned above, four of these had already been seen during the mobilization period and are therefore not relevant to the field study. These may have been introduced in the UK sector, during the trip to Norway or in the Norwegian harbour during mobilization. Although diving operations started on 3 July, it was not until 15 July that the contamination reached significant levels. From that date, contamination increased in terms both of number of genotypes and number of bacteria. More than 50% of the genotypes were introduced into the systems during the mid diving period between 15 and 25 July. The change in location and the introduction of full on-board water production may have been factors of significance for the level of contamination. Most of the genotypes described in this study were introduced into the vessel system through the seawater systems, particularly via the seawater intake. This study has thus demonstrated that seawater is a potential source of contamination by P.aeruginosa. Nevertheless, only one genotype (TP12) was demonstrated as being widespread in both the general vessel water systems and the satura-

7 Pseudomonas aeruginosa in diving systems 233 tion water systems on board. After its first occurrence on 15 July, a massive spread could be observed in the course of the following days (Fig. 3). This might indicate that certain P.aeruginosa genotypes have greater potential to survive and spread within such systems than do others. The fact that this genotype also caused infection demonstrates that P.aeruginosa obtained from seawater represents an infection threat in diving systems. The demonstration that TP2 could survive for several months, as could the infection caused by that genotype, are examples of the contamination and contagion mechanisms seen in this specific niche. Finally, the results also strengthen the probability that certain genotypes are more infectious than others. In conclusion, this field study has for the first time demonstrated seawater as a true source of a P.aeruginosa infectious genotype in a saturation diving system. It is therefore reasonable to consider seawater as a potential source of the frequent infectious genotypes seen in Norwegian diving systems in the course of the past 18 years. Acknowledgements We thank all participating divers and the health personnel on board the diving vessels for their skilled cooperation and Marianne Aas, Grethe Lysholm Iversen, Evy Mellemsæther and Therese Ahlén for skilled technical assistance. We wish to thank Statoil, Norsk Hydro, Saga Petroleum and the Norwegian Petroleum Directorate for financial support. REFERENCES Ahlén C, Leinan I, Berg M. (1991) Guidelines for cleaning/disinfection in chamber systems used in operational saturation diving, SPE Richardson, TX: Society of Petroleum Engineers. Ahlén C, Iversen OJ, Risberg J, Volden G, Aarseth H. (1998a) Diver s hand a skin disorder common in occupational saturation diving. Occup Environ Med; 55: Ahlén C, Mandal LH, Iversen OJ. (1998b) Identification of infectious strains of Pseudomonas aeruginosa in occupational saturation environment. Occup Environ Med; 55: Ahlén C, Mandal LH, Johannessen, L, Iversen OJ. (2000) Survival of infectious Pseudomonas aeruginosa genotypes in occupational saturation environments and the significance of these genotypes for recurrent skin infections. Am J Ind Med; 37: Ahlén C, Mandal LH, Iversen OJ. (2001) The impact of environmental Pseudomonas aeruginosa genotypes on skin infections in occupational saturation diving systems; Scand J Infect Dis; 33: Alcock SR. (1977) Acute otitis externa in divers in the North Sea. A microbiological survey of seven saturation dives. J Hyg (Camb); 78: Freitag M, Woods A. (1983) Commercial diving. Reference and operations handbook. Bath: Wiley. Hope A, Lund T, Elliott D, Halsey M, Wiig H. (1994) Longterm health effects of diving. An international consensus conference at Godøysund, Norway, 6 10 June Bergen: NUTEC and University of Bergen. Norwegian Petroleum Directorate. (1999) Report from the dive database, DSYS Stavanger: Graphic Centre, NPD. Thalman ED. (1974) A prophylactic program for the prevention of otitis externa in saturation divers, research report Washington, DC: US Navy Experimental Diving Unit, Washington Navy Yard.