Abstract
Water safety and management programs (WSMP) utilize field measurements to evaluate control limits and monitor water quality parameters including Legionella presence. This monitoring is important to verify that the plan is being implemented properly. However, once it has been determined when and how to sample for Legionella, it is important to choose appropriate collection and processing methods. We sought to compare processing immediate and flushed samples, filtration of different volumes collected, and sample hold times. Hot water samples were collected immediately and after a 2-min flush. These samples were plated directly and after filtration of either 100 mL, 200 mL, or 1 L. Additionally, unflushed samples were collected and processed immediately and after 1, 24, and 48 h of hold time. We found that flushed samples had significant reductions in Legionella counts compared to immediate samples. Processing 100 mL of that immediate sample both directly and after filter concentration yielded the highest concentration and percent sample positivity, respectively. We also show that there was no difference in culture values from time 0 compared to hold times of 1 h and 24 h. At 48 h, there were slightly fewer Legionella recovered than at time 0. However, Legionella counts were so variable based on sampling location and date that this hold time effect was minimal. The interpretation of Legionella culture results depends on the sample collection and processing methods used, as these can have a huge impact on the success of sampling and the validation of control measures.
Highlights
Legionella is an opportunistic waterborne pathogen that thrives in warm water environments and typically infects immunocompromised individuals [1]
The interpretation of Legionella culture results depends on the sample collection and processing methods used, as these can have a huge impact on the success of sampling and the validation of control measures
Legionella culture was conducted using a modified ISO method [21] with culture media plates prepared in-house from dehydrated media noted below, and 0.1 mL aliquots of the immediate, unflushed and post-flush samples were directly plated on buffered charcoal yeast extract agar (BCYE) (Remel, Lenexa, KS, USA) and BCYE with dyes, glycine, vancomycin, and polymyxin B (DGVP) [22]
Summary
Legionella is an opportunistic waterborne pathogen that thrives in warm water environments and typically infects immunocompromised individuals [1]. In addition to there being organizationally varied requirements for testing, there are a variety of sample collection volumes, flushing strategies prior to sampling, and sample holding time requirements prior to laboratory processing recommended by different technical guidelines for Legionella recovery (Table 1) [6,9,10,11,12,13]. We evaluated the effect of sample volume processed and use of flushing prior to sample collection on the presence of Legionella and the percentage of positive samples based on these standard recommendations. Ship at ambient temperature within 24 h from collection; if time must exceed 48 h, consult with lab CDC: Centers for Disease Control and Prevention; ISO: International Organization for Standardization; AIHA: American Industrial Hygiene Association; ASTM: American Society for Testing and Materials; ASHRAE: formerly the American Society of Heating, Refrigerating and Air-Conditioning Engineers. 19458 “Water Quality—Sampling for Microbiological Analysis” for these recommendations [20]
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