Clinical cystic fibrosis (CF) Pseudomonas aeruginosa (n = 6) and Burkholderia cenocepacia (n = 4) were inoculated in marine brines from the Dead Sea and the Atlantic Ocean and their survival was monitored over a 1 month duration. In Dead Sea samples, all P. aeruginosa and B. cenocepacia isolates were non-detectable by culture following 24 h incubation, including the non-selective enrichment samples. In the Atlantic Ocean brine, over a 1 month period, mean P. aeruginosa counts decreased by only 0.25 log10 units and mean B. cenocepacia counts decreased by approximately 4 log10 units (10,000 cfu/ml). This study demonstrated that Dead Sea brine exerted a lethal effect within 24 h on planktonic P. aeruginosa and B. cenocepacia. Thus, the Dead Sea effectively purges these organisms from its environment on a daily basis.
Pseudomonas aeruginosa (P. aeruginosa) and Burkholderia cenocepacia (B. cenocepacia) are two important bacterial pathogens in patients with cystic fibrosis (CF), resulting in chronic lung infections with significant morbidity and mortality (LiPuma 2010). Both of these bacteria have been isolated from a wide variety of environmental sources, including waters (LiPuma 2010).
It has been reported previously that the high salinity of the Dead Sea of approximately 347 g/l of mainly divalent cations (Mg) is an inhospitable environment for cellular activity (Rhodes et al. 2012). An older study from a more hypersaline lake, Lake Assal near Djibouti, with a salinity of 400 g/l, showed a diverse population of euryhalines and strict halophiles (Brisou et al. 1974). Notwithstanding this, there have been several previous reports suggesting that the Dead Sea is inhabited primarily by unculturable bacteria (Brisou et al. 1974). Other recent reports indicate that freshwater springs within the Dead Sea are a rich source of bacteria, including Epsilonproteobacteria and Gammaproteobacteria (Ionescu et al. 2012), the latter class, to which the family Pseudomonadaceae and the genera Pseudomonas and Burkholderia belong.
More recently, there have been several reports on improved outcomes for CF patients spending time at the Dead Sea (Falk et al. 2006; Goldbart et al. 2007). Therefore, a combination of these factors could allow for a scenario of CF patients swimming and expectorating P. aeruginosa and B. cenocepacia in their sputum, at common bathing sites along its shoreline.
Therefore, the aim of this study was to compare the planktonic survival of P. aeruginosa and B. cenocepacia in Dead Sea brine with brine from the Atlantic Ocean.
MATERIALS AND METHODS
Bacterial isolates and water microcosm preparation
Clinical isolates (n = 10), consisting of P. aeruginosa (n = 6) and B. cenocepacia (n = 4) were obtained from the Northern Ireland Health and Social Care Microbiology Repository (MicroARK; www.microark.com). All these isolates had been isolated previously from adult CF patients and had been archived at −80 °C. All isolates were recovered on Columbia Blood agar (Oxoid CM0031, Oxoid Ltd, Basingstoke, UK), supplemented with 5% (v/v) defibrinated horse blood for 24 h at 37 °C, under aerobic conditions and passaged a further three times, prior to use.
Water (2 l) was extracted from a regular sampling point at the Dead Sea, with GPS co-ordinates 31°13.941′N, 35°23.858′E. Water (1 l) from the Atlantic Ocean was extracted at GPS co-ordinates 55°10′6.2724″N, 6°47′37.5828″W. Both water samples were transported to the laboratory at ambient temperature. On receipt of water samples, each was filter sterilised individually by passage through a 0.2 μm syringe filter and stored in the dark at ambient temperature. A microcosm of each water type was prepared by preparing 50 ml filter sterilised water into a sterile glass container.
Each isolate was grown aerobically overnight on nutrient agar (Oxoid CM1, Basingstoke, UK) (48 h incubation for the B. cenocepacia isolates) and an homogenous inoculum was prepared in ¼ Ringer's diluent (Oxoid BR0052), equating to approximately 1010 colony forming units (cfu)/ml, after which 0.1 ml of this inoculum was introduced into each prepared water microcosm of both water types. All 20 permutations of organism/water type were stored in the dark for 1 month (30 days), at ambient room temperature (constant 20 °C). Sampling was performed daily for the first week, by the enumeration of organisms employing serial dilutions on nutrient agar for all organisms, except for B. cenocepacia which were enumerated on Columbia Blood Agar, as described above. When direct counts became non-detectable, 0.1 ml water microcosm was non-selectively enriched, following inoculation into nutrient broth (Oxoid CM1; 25 ml). This enrichment was incubated for 48 h at 37 °C and examined/plated at 24 and 48 h for growth.
RESULTS AND DISCUSSION
Survival of organisms in the Dead Sea microcosm is shown in Figure 1(a). All clinical P. aeruginosa and B. cenocepacia isolates were non-detectable following 24 h incubation, including the non-selective enrichment samples, indicating that no culturable organisms could be recovered after 24 h incubation in this water.
Survival of organisms in the Atlantic Ocean microcosm is shown in Figure 1(b). Over the 1 month period, mean P. aeruginosa counts decreased by 0.25 log10 units, which was not statistically significant (p = 0.4966). In contrast, mean counts of B. cenocepacia decreased by approximately 4 log10 units (10,000 cfu/ml), which was statistically significant (p = 0.0002).
This study demonstrated that Dead Sea brine exerted a lethal effect within 24 h on planktonic P. aeruginosa and B. cenocepacia. Thus, the Dead Sea effectively purges these organisms from its environment on a daily basis. In contrast, brine from the Atlantic Ocean, sampled just off the north coast in Northern Ireland, did not have a significant effect on the persistence of clinical P. aeruginosa, with numbers being maintained for 1 month, without any significant decrease in culturable bacterial counts. The relative survival of large numbers of both organisms in Atlantic Ocean water presents several important questions in terms of risk assessment for infection prevention purposes in the CF patient population. What is not known is the infective dose of these pathogens required to establish initial persistent colonisation of the upper airways in CF patients, leading to chronic colonisation and infection of the lower airways. Previous quantitative studies have demonstrated that P. aeruginosa is present in open ocean environments at a cell density of 0 to 8.3 cells/l (Khan et al. 2007), although its apparent marine distribution is with river outlets and shorelines, often associated with fresh water and sewage (Khan et al. 2007).
In conclusion, this study showed that Dead Sea water has a lethal effect on planktonic P. aeruginosa and B. cenocepacia CF clinical isolates, where all cells from both organisms were non-culturable within 24 h. This is in contrast to water from the Atlantic Ocean, where P. aeruginosa culturable cell density was relatively unaltered over the period of 1 month, whereas B. cenocepacia counts were reduced by approximately 4 log cycles.
The authors wish to acknowledge with gratitude Mr George Clulow for collection of the Atlantic Ocean water sample and Mr Alan Murphy, Department of Bacteriology, Belfast City Hospital, for preparation of the specialist media.