CRANFIELD UNIVERSITY __________________________________________________________ BIRGITTA BEDFORD LEGIONELLA CONTROL IN SYSTEMS USING COPPER AND SILVER ION GENERATION SYSTEMS INSTITUTE OF BIOSCIENCE AND TECHNOLOGY PhD. THESIS ___________________________________________________________
1.2 Microbiology and Taxonomy Plate 1.1 Legionella bacteria (www.bbc.co.uk) Legionella appear as small rods, faintly staining Gram-negative. They are unencapsulated and non-spore forming with an average size of 0.5-1.0 µm wide and 1.0-3.0 µm long (Barbaree, 1991). Most species exhibit motility through one or more polar or lateral flagella. Legionella cell walls are unique from other Gram-negative bacteria in that they contain significant amounts of branched-chain cellular fatty acids. Legionella require low levels of oxygen for growth (Brenner et al., 1985). Following the initial identification of L. pneumophila after the 1976 outbreak, numerous species have been discovered within the Legionella genus. Currently, the genus consists of at least 50 species, nine of which can be further divided into serogroups that are genetically homologous but can be differentiated by specific reactivity to antibodies. Currently, 70 serogroups are recognised in the genus, including 16 among L. pneumophila (World Health Organisation, 2007). 3
1.3 Transmission Legionella exist naturally in water and moist soil. Transmission to humans normally occurs via inhalation or aspiration of droplets of water containing Legionella (aerosols). Person-to person transmission does not occur (Dennis and Lee, 1988, Fitzgeorge et al., 1993, Fields, 1996, Stout and Yu 1997, Belyi, 1999). Wound infection may be caused by direct entry of Legionella into damaged skin, and has been observed after immersion of a wound in contaminated water (Brabender et al., 1983, Lowry et al., 1991). Transmission hazards have been associated with water distribution systems, cooling towers, humidifiers, whirlpool spas, car wash systems, vegetable water sprayers, and windscreen wiper water. As reviewed by Rathore and Alvarez, (2009), transmission hazards have also been associated with respiratory therapy equipment, nasogastro tubes, dental unit water lines and potting compost (Breiman et al., 1990, Kool et al., 1998, Seenivasan et al., 2005, Kumar et al., 2010, Pravinkumar et al., 2010, www.hcinfo.com, www.nhs.uk). Disease is mainly caused by the transmitted Legionella residing within phagosomes of alveolar macrophages, they multiply intracellularly until the cell ruptures. Liberated Legionella then infect other macrophages (Microbiology Third Edition, 1996). Immuno-compromised people are at increased risk of contracting disease from Legionella because their cell-mediated immunity against Legionella is impaired (Greenberg et al., 2006). 4
whom were treated at home, were suspected cases of Legionnaires’ disease (O’Mahony et al., 1990). 1.5.3 Travel associated Legionnaires’ disease In 2008, the European Surveillance Scheme for Travel Associated Legionnaires’ disease (EWGLINET) received reports of 866 of travel-associated Legionnaires’ disease, which was slightly below the number of cases reported in 2007. 42 of these cases were fatal. Prior to 2007 there had been a steady increase in the number of cases reported to the scheme since its inception in 1987. This was due in part to improved national surveillance and to an increasing number of countries joining the scheme. There is, however, significant under-ascertainment of Legionnaires’ disease within Europe. Especially among the newer Member States of the European Union which include countries where surveillance for Legionnaires’ disease is less well developed. Therefore, there is potential for case numbers to increase (Ricketts et al., 2010). The total reported travel-associated cases of Legionnaires’ disease in England and Wales from 1980 to 2009 was 3343. 2880 of these cases were associated with travel outside the UK and 463 cases were associated with travel within the UK (www.hpa.org.uk). 1.5.4 Legionnaires’ disease in long-term care residents Pneumonia is the leading of death and an important cause of transfer to acute care facilities in long-term care residents (Muder, 1998). There are a number of epidemiological factors that suggest that long-term care residents might be at particular risk for legionellosis should it be present in the environment. Long-term care residents often have swallowing difficulties, and many receive nasogastric tube feedings, predisposing factors to aspiration which is the primary mode of transmission (Seenivasan et al., 2005). 11
1.7 Factors that influence Legionella growth Various factors that influence and promote Legionella growth have been identified since its initial identification back in 1976. 1.7.1 Physio-chemical factors Legionella can survive extreme ranges of environmental conditions. It can survive temperatures of 0ºC to 63ºC and has been found in waters at temperatures between 5ºC and 63ºC but significantly more Legionella has been isolated from warm waters at temperatures of 30ºC and above. Legionella can also survive a wide pH range of and a dissolved oxygen concentration in water of 0.2 to 15 mg/l. L. pneumophila grows best at a pH of 5.5 to 6.2 with an oxygen concentration of 6.0 to 6.2 mg/l. However, Legionella species were found in an extremely acidic, predominantly eukaryotic algal biofilm community in Yellowstone National Park, US, at a pH as low as 2.7. Small concentrations of salt (NaCl 0.1 to 0.5%) may also enhance the survival of Legionella and it has been found in cooling systems using seawater (Fliermans et al., 1981, Nguyen et al.,. 1991, Lee and West, 1991, Newsome, 2001, Sheenan et al., 2005). Metal plumbing components and associated corrosion products are also important factors as these can provide iron and other metals that support the survival and growth of Legionella in plumbing systems (States et al., 1985). 1.7.2 Microbial factors The growth and survival of Legionella is affected by the presence of other organisms. Studies have demonstrated that Legionella were incapable of growing in sterile water because it required nutrients supplied by other micro-organisms, including bacteria (Wadowsky and Yee, 1983), protozoa (Rowbottom, 1980), and cyanobacteria (Tison et al., 1980). Protozoa play an important role in the increase and spread of Legionella in anthropogenic water systems and therefore in the occurrence of Legionnaires’ disease (DeClerck et al., 2009). Legionella can infect and grow, similar as with macrophages, 13 8.5
Cirillo et al., (1994) showed that growth in protozoa also enhanced the ability of L. pneumophila to enter human cells, and Brieland et al., (1997) found that parasitic L. pneumophila were more pathogenic than free living L. pneumophila. Micro-organisms, including Legionella, become attached to surfaces submerged in water and form layers of microbial cells, called biofilms. Biofilms play an important role in the spread of Legionella as they also provide nutrients for their growth and a protective environment enabling them to survive water treatment processes (Kuchta et al., 1993, Lin et al., 1998, Barbeau et al., 1998, Atlas, 1999, Kuiper et al., 2004, Newsome, 2001). A study carried out in 1994 showed that Legionella were more easily detected from swab samples of biofilm than from flowing water, which suggested that the majority of Legionella were essentially biofilm associated (Rogers et al., 1994). Legionella cells persisting in biofilms (sessile Legionella) are also much more resistant to disinfectants than the planktonic, free-living, cells (Cargill et al., 1992, Stewart and Costerton, 2001, Tachikawa et al., 2005). 15
1.9 Detecting Legionella in water systems The association of Legionella with protozoa and its occurrence within biofilms, in they hide, complicates its detection. The current standard and validated technique for detecting Legionella in water is based on culturing for Legionella (ISO 11731:1998). This technique is however lengthy and complex, and detects only planktonic, free-living, and extracellular Legionella. molecular tests for the detection of Legionella Deoxyribonucleic Acid (DNA) have been developed (Mahbubani et al., 1990). Although these tests detect planktonic, sessile, extracellular and intracellular Legionella, nonviable Legionella cells are also detected. Since it is likely that water samples contains nonviable Legionella cells, which were killed by disinfection measures, false-positive readings of Legionella samples are possible which could lead to unnecessary and expensive emergency decontamination procedures (Shih and Lin, 2006). 21
Keevil et al., (1993) that copper supported sparse microbial growth and Legionella comprised a very low proportion of the population. The low colonization of copper surfaces was presumed to be due to the inhibitory effect of copper ions, either by selectively inhibiting Legionella or by inhibiting the organisms that support their growth. The most prolific biofilms were those that developed on the surface of elastomeric materials. The biofilm covered the entire elastomer surfaces after only 24 hours and contained more than 8.9x106cfu/cm2 on latex and 2.7x106cfu/cm2 on ethylene propylene rubber. The highest populations of L. pneumophila were found on latex as well (Keevil et al., 1993). Rogers et al., (1994) found that the lowest concentration of flora was on the stainless steel surface and that the latex and ethylene-propylene rubber were most heavily colonised. Of the plastic materials used, polyethylene appeared to be most heavily colonised (Rogers et al., 1994). full size hot and cold water services rigs were constructed by the Building Services Research and Information Association (BSRIA) in 1996 to evaluate the efficacy of temperature and copper and silver ionization techniques to control Legionella, see Figure 1.3. Each rig replicated the water system of a small home for the elderly. Two of the rigs were fitted with ionization water treatment from silver/copper alloy electrodes and were operating at reduced hot water temperatures; one was filled with hard water and one with soft water. The third rig relied solely upon temperature as disinfection and was filled only with hard water. The cold water system in each rig consisted of a glass reinforced plastic cistern, which served a cold water copper pipework circuit of 30m in length, which included two cold taps and one shower. The hot water system consisted of a vertical hot water storage calorifier of copper construction, which served a hot water copper pipework circuit of 40m length, which included two hot taps and one shower. Copper and glass reinforced plastic coupons were inserted at various locations in each rig. These coupons were regularly removed for biofilm and Legionella analysis. 24
Graph 4.7 below. This graph shows the average Legionella count of all 10 outlets sampled monthly from the 06th March 2008 to the 19th July 2011. Legionella CFU/l 1400 1200 1000 800 600 400 200 0 00 . 8 8 8 9 9 0 0 0 1 011 06 04 00 2 00 26 01 24/11/20101 2 0 0 26/11 28 2 /0 8 0 0 08 /0 /05 /0 1 /0 /0 /12/20 /0 /03/20 3/ 4 6 9 4 3 2 8 9 0 6 Graph 4.7 Study hospital 3 – Average Legionella results from the 06th March 2008 to the 19th July 2011. Graph 4.8 shows the average copper levels found in samples taken monthly from 10 outlets from the 06th March 2008 to the 19th July 2011. The treated water started to discolour in 2009, which may have been due to high phosphate levels in the mains water supply potentially forming precipitates with the higher levels of copper, and the copper side of the ionization system was turned off from the 08th June 2009 onwards. The average copper levels in samples on the 08th June, the 09th July and the 04th August 2009 remained, however, above 0.2mg/l, at 0.26mg/l, 0.241mg/l and 0.35mg/l respectively, which was most likely residual copper from the ionization treatment as well as copper leaching from the copper pipes in the water system, but the levels reduced from the 27th August 2009 onwards. The average copper level from the 27th August to the 19th July 2011 was 0.093mg/l (± 0.025mg/l), which was solely the copper that had leached from the copper pipes. 108
8000 7000 6000 CUF/l 5000 Legionella 4000 3000 2000 1000 0 . 8 /2008 8 9 9 /2009 9 0 0 010 0 0 1 1 /2011 8 0 00 0 0 01 1 1 01 1 00 /20 /2 /2 /2 /2 /2 /2 2 /05/20 /03/20 7 /05/20 5/ 7 0 /02 9 1 1 03 /0 /1 /0 /0 /0 /1 07 /0 1 03/09 27 1 06/07 0 2 14/05 6 10/09/2010 3 28/06 0 0 1 1 0 0 4.10 Study hospital 4 - Average Legionella results of all 6 outlets sampled monthly from the 07th May 2008 to the 28th June 2011. Graphs 4.11 and 4.12 show the averages of the copper and silver levels found in samples taken monthly from 6 outlets from the 07th May 2008 to the 28th June 2011. The average copper level was 0.419mg/l (± 0.008mg/l). The average silver level was 0.043mg/l (± 0.003mg/l). 4.11 Study hospital 4 – Average copper levels from the 07th May 2008 to the 28th June 2011 (the red line is the target level for copper - 0.2mg/l). 114
4.25 Study hospital 9 - Average Legionella counts in samples and re-samples taken monthly from the 02nd September 2009 to the 15th July 2011. After the construction work was started, higher Legionella colony forming unit counts were found in re-samples taken on the 24th June 2010, in samples and re-samples taken on the 29th October 2010, and in re-samples taken on the 15th July 2011, see Tables 4.23, 4.24, and 4.25 below. 148
Graph 4.29 Study hospital 10 – Average copper levels from the 13th January 2010 to the 13th July 2011 (the red line is the target level for copper - 0.2mg/l). 4.30 Study hospital 10 – Average silver levels from the 13th January 2010 to the 13th July 2011 (the red line is the target level for silver - 0.02mg/l). 157
Figure 4.1 Legionella results of sections 1 to 16 of the copper rig – removed simultaneously on day 564. Legionella were found in sections 1 to 15 of the copper rig. No Legionella were found in section 16, which had been in the copper rig for 86 days. The results indicated that Legionella multiplied in biofilm in pipe sections that were in the copper rig for more than 86 days and that multiplication peaked in pipe sections that had been in the rig for 353 days after which multiplication dropped to only 300CFU found in the pipe section that had been in the copper rig for 547 days. Very surprisingly, no Legionella were found in the polyethylene rig sections 1 to 15 but a colony forming unit count of 1800CFU/500ml was found in section 16. Legionella non-pneumophila and L. pneumophila serogroup 2 to 14 were found in the copper rig sections. Only L. pneumophila serogroup 2 to 14 were found in the polyethylene section samples. Legionella results of samples taken from rigs A, after rigs B and C were inoculated with water and sections from rigs A (day 0), and before treatment was started (day 120), are shown in Graph 4.33 below. Page 166 of 229
4.13.2 Rigs B The temperatures in rigs B were maintained below 45ºC, simulating a typical hot water circulating system in which mixing valves are used to blend water to a temperature below 45ºC to avoid scalding. This was effectively the ‘control’ rig in this set of experiments. The temperatures of the polyethylene rig tank recorded daily for 83 days ranged from 36ºC to 49ºC. The most common temperature recorded was 45ºC. The average temperature in the polyethylene rig tank recorded over the 83 days was 43ºC (± 0.4ºC). Graph 4.39 shows the Legionella results of samples taken ones a week from the polyethylene rig draw off point. 160000 rig B 140000 120000 polyethylene 100000 80000 CFU/l - 60000 Legionella 40000 20000 0 0 13 20 27 34 41 48 55 62 69 76 83 Days in rig Graph 4.39 Legionella results of polyethylene rig B. At day 0 a Legionella colony forming unit count of 49500CFU/l was found in the sample taken from the draw off point of the polyethylene rig. The Legionella contamination was variable but increased to a Legionella colony forming unit count of 60800CFU/l found in the sample taken on day 83 (~ 23% increase).The average Legionella count of samples taken from day 13 to day 83 was 64309CFU/l (± 10421CFU/l). Page 175 of 229
biofilms had sheared off and settled elsewhere in the rig, therefore, providing less favourable conditions for Legionella to grow in these sections. Because water was circulated through the rigs continuously at a relatively high rate, and because 3 litres of water were drawn off and 3 litres of fresh water were added to the rigs daily, dispersion of biofilms most likely occurred frequently, which is supported by the variability in the TVC and Legionella results. Less Legionella were found in the samples taken from the copper rig A than in the polyethylene rig A after rigs A were used to inoculate rigs B and C. This could be explained by the removal of sections and water from rigs A for rigs B and C and by doing so a substantial amount of the established biofilms that had formed beforehand in the copper rig A was removed from the rig. The conditions within the copper rig A may have, therefore, become less favourable for Legionella to grow. Although a superior biocidal action of copper compared to polyethylene was not demonstrated in the initial experiment with rigs A, less Legionella were found in the copper rigs B and C than in the polyethylene rigs B and C. The Legionella counts found in polyethylene rigs B and C were ~473% higher than those found in copper rigs B and C. Interestingly, the Legionella found in all rigs during this study, L. pneumophila serogroup 2-14, were different from the species introduced to the rigs, L. pneumophila serogroup 1 and Legionella non-pneumophila. This suggested that the local environment in both rigs promoted the growth of L. pneumophila serogroup 2-14, and that the inoculum introduced could have contained a mixture, including serotypes not readily detectable in the inoculum at the initial stages. Page 203 of 229