The team from the Department of Civil and Environmental Engineering, National University of Singapore, have recently published on the functionality of Pseudomonas aeruginosa following exposure to chlorine and UV light disinfection (Chiang et al., 2022) Their report highlights the major limitations in using cell culture methodology to assess waterborne bacteria pathogenic activity, to validate and/or verify the effectiveness of control measures or to fully understand the risk to health from potable water. This is mainly due to the reluctance of cells recently exposed to environmental stresses, such as disinfection, to show themselves on a culture plate, despite optimal growth conditions. The paper also gives clearer insight on changes in cell function following commonly used disinfection processes.
Rapid Detection of Waterborne Pathogens such as Pseudomonas aeruginosa, Legionella pneumophila and others.
Various alternative, more sensitive and more specific rapid detection technologies for waterborne pathogens such as Legionella spp. and Pseudomonas aeruginosa have been available for more than a decade, however, adoption into practice and within guidance has been at best slow.
Is continued use and dogged recognition of traditional cell culture plate counts driven by the desire to have negative or low count results? Is lack of understanding, confusion over interpretation of the results and implementation of the data generated restricting uptake of alternative technologies?
Many test laboratories have access to rapid detection technologies for various waterborne pathogens, including Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and non-tuberculous mycobacteria, yet demand for routine use remains low.
Polymerase Chain Reaction (PCR/qPCR) techniques have been utilised as one alternative to culture techniques, especially when quick results are needed following diagnosis of a Legionellosis case. However, the foremost weakness with commercially available (and UKAS accredited methodology) qPCR is the inability to differentiate live versus dead cells, as they detect DNA from all bacterial cells. Within all water systems, particularly after a shock biocide treatment, recovery of a level of dead cells is inevitable. Results from qPCR therefore would likely overestimate the presence of viable pathogenic bacteria potentially leading to unnecessary restrictions in water use (creating stagnancy), overuse of systemic disinfectants and implementation of remedial engineering actions.
Viability PCR Detects Active & Viable Cells
Adaptation of qPCR techniques can deliver more relevant results. “Viability PCR” removes spurious amplification of DNA from truly dead cells through use of various dye pre-treatments (e.g. PMA- PCR). These dyes select and exclude cells with membrane damage. Such pre-treatments may be more suitable for monitoring and determining the efficiency of temperature control measures, biocide disinfection and risk assessing water quality. Indeed, Chiang’s paper compares traditional plate cell culture, flow cytometry and dye-treated qPCR methods in detecting various Gram-negative and Gram-positive bacteria following exposure to UV light and chlorine treatments. The research team investigated a dye pre-treatment methodology using DyeTox13 Green C-Z Azide (DT13-PCR) which detects cells with enzymatic activity. These cells are considered to be metabolically active and noted as “Active But Non-Culturable Cells” (ABNC). DT13 treatment clearly differentiated ABNC from “inactivated cells” after UV exposure and chlorine disinfection.
One of the bacteria assessed in Chiang’s studies was Pseudomonas aeruginosa PA01 strain. Focus on Pseudomonas aeruginosa in potable and recreational facility waters has increased since the recent publication of British Standards Institution BS 8580-2:2022 Water Quality Part 2: Risk assessments for Pseudomonas aeruginosa and other waterborne pathogens – Code of Practice which makes this publication so timely. This gram negative opportunistic pathogen is responsible for up to 20% of Health Care Associated Infections, with a 50% mortality rate in Cancer and Burns patients. It is most often associated with infections in the blood, lungs (pneumonia), and wounds. More recently Pseudomonas aeruginosa was found to be one of only six pathogens that were collectively responsible for 73% of Antimicrobial Resistance.
Chiang’s group grew Pseudomonas aeruginosa under laboratory conditions to achieve a concentration of 10E7 Colony Forming Units (CFU)/mL for the disinfection experiments. UV light exposures at up to 198 mJ/cm2 for 30 minutes and 60 minutes were tested, as were sodium hypochlorite (NaOCl) concentrations of 0, 10, 30 and 50 mg/L for 60 minute durations. Test solutions were then plated for traditional culture growth, or analysed by flow cytometry, PMA-PCR or DT13-PCR.
Under culture, Chiang found that 10 mg/L NaOCl was sufficient to impact the majority of Pseudomonas aeruginosa cells, however, exposure at 30 and 50 mg/L did not bring further percentage decline or benefit. There was a 5-6 log reduction in culturability at 10 mg/L NaOCl, and no observable colony growth when concentrations increased to 30 mg/L despite the total cell count under flow cytometry remaining constant. The chlorine concentrations damaged cellular membranes but did not eliminate cell structure. Assessment under PMA- and DT13-PCR for enzymatic activity and membrane integrity illustrated a different picture. There was little or no difference on enzymatic activity and membrane integrity when P. aeruginosa was treated with 10mg/L of chlorine, however, there was significant impact once NaOCl concentrations were greater than 30 mg/L..
This raises the question for those water systems continuously treated with 1 mg/L chlorine, or even up to 5 mg/L the upper allowable potable water limit, does this mean that cell culture results give a false “green light”?
As the applied UV dose increased from 0 to 99 mJ/ cm2 (30 min UV exposure) the percentage of P. aeruginosa cells with intact membranes decreased slightly to approximately 98%. As the UV dose increased further to 198 mJ/cm2 (60 min UV exposure) the percentage of membrane-intact cells remained similar with no further decline regardless of the higher dose and longer exposure. Despite no significant reduction in membrane integrity, the culture plate P. aeruginosa counts showed marked declines of approximately 4 logs. Results indicate that UV treatment below 200 mJ/cm2 disrupted cell reproduction but not membrane integrity. PMA- and DT13-PCR confirmed insignificant membrane damage following UV exposure.
Is Plate Culture Just Fake News, when trying to detect Legionella pneumophila or Pseudomonas aeruginosa?
Following treatment, bacteria that are unable to grow via culture methods may still possess a level of bioactivity and the potential for regrowth after environmental stresses change. Indeed resuscitation of membrane intact bacterial cells from a non-culturable state in the presence of insufficient chlorine levels for example could lead to critically contaminated water accessed by user populations. As chlorine concentrations increased the DT13 was able to identify ABNC cells that retained enzymatic activity, enabling DNA amplification whereas PMA-PCR would count them as membrane compromised. Complementary use of both PMA-PCR and DT13-PCR can provide insights into the function of these living cells after chlorine exposure.
UV exposure has been used as a substitute and as a synergistic method alongside biocide disinfection in drinking water treatment systems. Replication of DNA in bacteria exposed to UV radiation is inhibited, and therefore cell reproduction is inhibited as reflected by lack of culturable colonies. However, UV irradiation does not directly injure cell membranes at lower doses and cells can retain both their membrane integrity and metabolic activity but fail to form colony for plate counting. UV treatment may induce ABNC and VBNC bacteria.
Among the 4 bacteria species tested in Chiang’s study, Pseudomonas aeruginosa had the highest cell population that retained enzymatic activity and intact membranes despite being non-culturable after UV exposure. DT13-PCR indicated metabolic activity. As UV irradiation increased there was a slight increase in the cell population shifting from ABNC to the dormant VBNC state. Dormancy is not the equivalent to cell death. Many bacterial species enter a distinct ABNC state as a strategy to survive under environmental conditions such as UV disinfection, osmotic pressures, chemical disinfection, starvation and extreme temperatures.
Continuing to rely on culture to determine the safety of drinking water which have UV and/or biocidal disinfection will maintain an underestimation of health risks because of cell resuscitation. Who are we kidding when reading culture results? Whilst Viability PCR techniques are not readily or commercially available, there will not be a drive to adopt such technologies until the Duty Holder, written guidance and attitude to measuring real potable water control efficacy changes.
Chiang ELC, Lee S, Medriano CA, Li L & Bae S. (2022) Assessment of physiological responses of bacteria to chlorine and UV disinfection using a plate count method, flow cytometry and viability PCR. Journal of Applied Microbiology, 132(3):1788-1801. doi: 10.1111/jam.15325. PMID: