Health Stream Literature Summary - Issue 50 - June 2008

Characterisation of potential virulence markers in Pseudomonas aeruginosa isolated from drinking water.
da Silva, M.E.Z., Filho, I.C., Endo, E.H., Nakamura, C.V., Ueda-Nakamura, T. and Filho, B.P.D. (2008) Antonie van Leeuwenhoek, 93(4); 323-34.

Pseudomonas aeruginosa is a clinically significant opportunistic pathogen which has the ability to grow in low-nutrient water. The public health significance of larger numbers of Pseudomonas spp. that can develop in water is unclear. P. aeruginosa usually possess virulence-associated factors including the ability to produce cytotoxins, hemolysins and proteases and invasive ability. This study was undertaken to determine the characterisitics of P. aeruginosa isolates from tap water, mineral water and artesian well water in relation to their ability to produce different potential virulence factors or markers such as hemolysins, hemagglutinins and cytotoxins, and their ability to adhere to epithelial cells and to abiotic surfaces. Susceptibility to antibiotics, human serum sensitivity and the survival of P. aeruginosa isolates in a chlorinated environment were also investigated.

The virulence profiles of 30 P. aeruginosa strains isolated from drinking water were investigated with respect to their haemolytic activity, hemagglutination ability (agglutination of red blood cells), cytotoxic effect, multiple-antibiotic resistance, serum resistance and also adhesion to glass, silicon, polyethylene terephtalate (PET), Vero cells and human buccal epithelial cells (BEC). As a comparison, type strains ATCC 15442 (P. aeruginosa isolated from animal room water bottle), ATCC 27853 (P. aeruginosa isolated from blood) and P. aeruginosa C I, C II and C III isolated from blood, sputum and wounds, respectively were included in the study.

All environmental isolates and clinical strains and ATCC type strains included in the study were capable of producing hemolysins. Hemagglutination was seen in 6 of 10 isolates from mineral water and 3 of 10 isolates from tap water whereas only one of the ATCC type strains (isolated from an animal room water bottle) showed the ability to hemagglutinate sheep erythrocytes. Twenty seven of the environmental isolates showed cytotoxicity for the cell line tested. Only 3 of the 10 strains of artesian water showed no cytotoxicity. Resistance to three or more antibiotics was frequently seen in isolates from environmental and clinical strains. The highest prevalence of resistance among all the P. aeruginosa isolates was shown for chloramphenicol, gentamicin and trimethoprim-sulfamethoxazole. Few of the environmental isolates were resistant to cefotaxime. All drinking water isolates were susceptible to aztreonam, cefepime, ceftazidime, ciprofloxacin, imipenem, meropenem, piperacillin-tazabactam and polymyxin. Of the 35 P. aeruginosa strains examined, only 5 isolates from mineral water were found to be serum-sensitive. However all the samples isolated from artesian water were resistant to 50% human serum. No particular bacterial isolates was better able to adhere to abiotic surfaces out of environmental isolates and clinical and ATCC type strains. There was no general relation between the origin of the isolate and the ability to interact with epithelial cells. Three pattens of adhesion to Vero cell surfaces (diffuse, localised and aggregative) were seen. Of the isolates tested the predominant pattern of adhesion was the localised type. Isolates showed a variety of hydrophobicity. Using the bacterial adherence to hydrocarbons (BATH) method, one of the 10 isolates from mineral water was classified as hydrophobic with most of the isolates from environmental sources and collection strains classified as highly hydrophilic.

The chlorine sensitivity tests conducted revealed that 0.2 mg of chlorine per litre at a treatment time of 1 min killed 2 of the 3 clinical strains, whereas only 3 of the 10 isolates from tap water and 4 of the 10 isolates each from the mineral water and artesian water, respectively were killed at this same chlorine concentration. At a chlorine concentration of 0.6 mg/l which is three times the recommended level of free chlorine per litre, 3 of 10 , 2 of 10 and 1 of 3 isolates from tap water, mineral water and clinical collection, respectively were not killed. Bacterial killing in planktonic and biofilm states in response to treatment by chlorine was compared. The viability of planktonic cells of P. aeruginosa isolates was rapidly reduced by 0.5-1.15 mg/l chlorine during the first 5 minutes after which the rate of decrease in viability slowed. After 10 minutes of exposure there was a 100-fold reduction in the viability of planktonic cells at a chlorine concentration of 0.2 mg/ml. Biofilm bacteria were found to be less susceptible to killing in comparison. At the lowest concentration of chlorine (0.2 mg/l) tested, no killing of biofilm bacteria could be seen even after 30 minutes of exposure.

Using Spearman correlation coefficients the inter-relationships between markers of virulence were analysed. A positive correlation was seen between serum-resistance and adherence to Vero cells as well as among hemolysis, cytotoxicity, adherence to silicon and adherence to BEC. Cytotoxicity was positively correlated with adherence to glass, to silicon and to BEC, whereas multiple-antibiotic resistance was positively correlated with adherence to silicon and adherence to BEC. A positive correlation was also seen between adherence to silicon and adherence to BEC.

This study shows that a notable amount of P. aeruginosa isolates from drinking water were able to develop virulence factors and the incidence of virulence properties was not statistically different among the three sources. There was also a strong and statistically significant positive correlation found between adherence and cytotoxicity among the isolates which may indicate that expressing both virulence properties may be essential for P. aeruginosa during infections.

Comment P. aeruginosa is not an enteric pathogen, however this organism can grow in warm water environments (eg spa baths) when disinfection residuals are low or absent. Under these circumstances outbreaks of folliculitis (infection of the hair follicles) have been recorded. Tap water may also be a source of Pseudomonas infection when it is used instead of sterile water to wash medical equipment