Blog Entry

Safety In Pharma And Healthcare: Improving Detection Of Legionellosis Causing Bacteria

INTRODUCTION

Many healthcare and pharmaceutical facilities are required to control, monitor and test for the bacteria responsible for causing the collective diseases known as ‘legionellosis’, of which Legionnaires' disease (a form of atypical pneumonia), Pontiac fever (an acute respiratory disease) and Lochgoilhead fever (a variation of Pontiac fever) are the most serious. In particular, Legionnaires' disease is by far the most serious, presenting as a potentially fatal form of pneumonia. While the disease can affect any person, people with impaired lung function (such as those with chronic respiratory and smokers) and the elderly are especially vulnerable.

This IVT article examines the cause of infection and considers emerging technologies for improving testing, which advances the rapid microbiology paradigm.

CAUSATIVE AGENT AND HABITAT

The causative agent is the bacterium Legionella pneumophila, and some other species from the Legionella genera. Globally it is estimated that around 70% of Legionella infections are caused by L. pneumophila serogroup 1. Other serogroups cause 20%, and non-pneumophila species cause 5–10%; these include L. longbeachae, L. micdadei, and L. bozemanii, amongst others. L. pneumophila is a thin, aerobic, pleomorphic, flagellated, non-spore-forming, Gram-negative bacterium (1). In the infected host, the bacterium invades and replicates inside macrophages (2). L. pneumophila has more than 300 toxins that it uses to infect humans, of which SidJ is one of the most virulent (3). Treatment is via antimicrobials such as macrolides (azithromycin or clarithromycin) and fluoroquinolones (levofloxacin or moxifloxacin).

These organisms occur in natural water sources such as rivers, lakes and reservoirs in low numbers. The concern they present to the in-built environment arises because of the design of water systems that enables the concentration of the pathogens to increase. First isolated in 1976 (4), disease surveillance has shown that the common areas of concentrated contamination with the human occupied environment include cooling towers, evaporative condensers, and hot and cold water systems. Numbers are higher in areas where water is of low turbulence or stagnant, and higher numbers are recovered in tap and shower heads (5).

These are environments where the water is maintained at a temperature high enough to encourage bacterial growth (within the range 20-45°C) and where water is stored or recirculated 6). Growth is more likely to take place where there is a nutrient source and where a biofilm develops (within biofilms the bacteria can shield inside amoebae, which makes them difficult to detect) (7). The primary vector for infection is via aerosols, in the form of breathable water droplets that are created by the water system and then dispersed (8).

Risks of infection are lower where daily water usage is high and sufficient to turn over the entire system; or where cold water is directly from a wholesome mains supply (no stored water tanks); and where hot water is fed at 50 °C. The primary risk arises from the use of showers.

PREVENTATIVE MEASURES

Each organization is encouraged to undertake a risk assessment, with design central to the first stage of the risk assessment. Here an attempt should be made to address any poor water system design practices. Thus can include avoiding water temperatures between 20 °C and 45 °C and conditions that favor the growth of legionella bacteria and other microorganisms; avoiding water stagnation which may encourage the growth of biofilm; avoiding the use of materials that harbor bacteria and other microorganisms, or provide nutrients for microbial growth. Suitably hygienic water fittings should be used.  In addition, some researchers have suggested using a probiotic approach which involves intentionally creating conditions that select for a desirable microbial community in order to suppress populations of pathogens (9).

Once design factors have been addressed, the physical and chemical treatment program should be mapped out. This may include temperature control for hot and cold water systems and a periodic chemical treatment program with an appropriate agent (often chorine) together with detail of a suitable concentrations and contact time.

TESTING

With design and control factors addressed, periodic testing needs to be conducted. This will include an assessment of general bacterial numbers (total viable count) to provide an indication of whether microbiological control is being achieved. This should be supported by periodic sampling and testing for the presence of legionella bacteria, although this is technically difficult. With culture-based methods, following membrane filtration (10), suitable culture media includes glycine vancomycin polymyxin B cycloheximide agar, which is recommended in the standard for water quality legionella assessment – ISO 11731: 2017 (11).

With legionella bacteria an alert level is typically set at 100 CFU per liter and an action level at 1000 CFU per liter. More sensitive methods include the increased use of antibody-based methods. While these provide a rapid, non-laborious, and relatively inexpensive method, often these methods are not sensitive enough for establishment as a screening method for surface and drinking water.

METHOD ADVANCEMENTS: RAPID MICROBIOLOGY

In recent years, methods of testing for legionella bacteria in water systems have advanced, with the goal of faster and more rapid testing. This began with the combined use of a multiplex polymerase chain reaction (Multiplex PCR, where two or more target genes are amplified in the same reaction) and pre-treatment with propidium monoazide (PMA). With a PCR test, fragments of DNA are run through a thermocycler, which heats and cools the sample repeatedly to produce multiple copies of these DNA fragments, amplifying them for analysis in just a few hours. The pre-treatment with PMA if followed by concentrating the sample, breaking the bacteria in order to extract its DNA, and then using Multiplex PCR to identify different Legionella species. Multiplex PCR amplifies the DNA of the target species, making it easier to identify them.

Progress with quantitative (qPCR) has led to the monitoring of the amplification of a DNA template in real-time rather than at its end-point, as in conventional PCR (12). This is based on the use of fluorescent reporter molecules which bind to and detect organisms generated during each cycle of the PCR process. As the reaction proceeds, fluorescence increases due to the accumulation of the PCR product with each amplification cycle. The development of ae quantitative qPCR assay method has reduced the testing time to two days (13).

A further advancement, for the medical facility, is a chip that is capable of identifying compounds of the bacterium in the urine of patients ,as part of the "LegioTyper" project funded by the German Federal Ministry of Education and Research. This chip detects pathogens, primarily L. pneumophila, and it can also identify which of the approximately 20 subtypes is present. The chip uses a microarray analysis platform, based on 20 different antibodies. The time-to-result is 34 minutes. The system can also be deployed for environmental hygiene to assess water systems, as well as clinical diagnostics (14).

CONCLUSION

Legionellosis outbreaks present serious risks to people. Often they are caused by evaporative cooling systems. The need for an urgent response highlights the need for rapid screening methods for L. pneumophila and associated organisms in water. This includes developments with qPCR and lab-on-a-chip advances. Continued developments in this field will help to advance public health measures.

REFERENCES

  1. Ensminger AW (2016) Legionella pneumophila, armed to the hilt: justifying the largest arsenal of effectors in the bacterial world. Current Opinion in Microbiology. 29: 74–80
  2. Gomez-Valero L, Rusniok C, Carson D, et al (2019) Legionella genus genome provide multiple, independent combinations for replication in human cells. Proceedings of the National Academy of Sciences. 116 (6): 2265–2273
  3. Bhogaraju, S., Bonn, F., Mukherjee, R. et al (2019) Inhibition of bacterial ubiquitin ligases by SidJ–calmodulin-catalysed glutamylation. Nature, DOI: 10.1038/s41586-019-1440-8
  4. Bopp C.A., et al. (1981) Isolation of Legionella spp. from environmental water samples by low-pH treatment and use of a selective medium. J. Clin. Microbiol. 13 (4): 714–719
  5. Ling, F., Whitaker, R., LeChevallier, M. and Liu, W-T.  (20180 Drinking water microbiome assembly induced by water stagnation. Journal of Microbial Ecology, DOI: 10.1038/s41396-018-0101-5
  6. Correia AM, GonCalves J, Gomes, JP, et al. (2016) Probable Person-to-Person Transmission of Legionnaires’ Diseaseexternal icon. N Engl J Med. 374:497–8
  7. Iversen, O-J., Sommerfelt-Pettersen, J., Sørbø, T.,  et al. (2013) Legionella pneumophila på Sjøforsvarets fartøyer. Tidsskrift for Den norske legeforening, 133 (14): 1445 DOI: 10.4045/tidsskr.12.1459
  8. Public Health England (2013) Legionnaires’ disease: The control of legionella bacteria in water systems, PHE, London: https://www.hse.gov.uk/pubns/priced/l8.pdf
  9. Wang, H., Edwards, M., Falkinham, J., Pruden, A. (2013) Probiotic Approach to Pathogen Control in Premise Plumbing Systems? A Review. Environmental Science & Technology, DOI: 10.1021/es402455r
  10. Smith L. et al., (1993) Comparison of membrane filters for recovery of legionellae from water samples. Appl. Environ. Microbiol. 59 (1): 344–346
  11. ISO 11731:2017 Water quality — Enumeration of Legionella, International Standards Organization, Geneva, Switzerland
  12. Kober, C., Niessner, R., Seidel, M.  (2018) Quantification of viable and non-viable Legionella spp. by heterogeneous asymmetric recombinase polymerase amplification (haRPA) on a flow-based chemiluminescence microarray. Biosensors and Bioelectronics, DOI: 10.1016/j.bios.2017.08.053
  13. Broeders S., Huber I., Grohmann L., et al. (2014). Guidelines for validation of qualitative real-time PCR methods. Trends Food Sci. Technol. 37, 115–126.
  14. Wunderlich, A., Torggler, C., Elsässer,D. et al  (2016)  Rapid quantification method for Legionella pneumophila in surface water. Analytical and Bioanalytical Chemistry, 408 (9): 2203



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