Reducing the public health impact of infections caused by waterborne pathogens

Many opportunistic waterborne pathogens, including Legionella species, non-tuberculous mycobacteria, and Pseudomonas aeruginosa, can thrive in hot water systems despite municipal and traditional on-site disinfection. These organisms can cause healthcare-acquired infections in immunocompromised and elderly patients. This project aimed to assess and reduce the impact of waterborne pathogens (WBPs) in these populations. In this study I developed a LAMP based assay that is specific for L. pneumophila that does not cross-react with other Legionella species or bacteria commonly found in either water or urine samples. This assay can detect L. pneumophila at a concentration of 400 cfu/mL and higher in contaminated water. Evaluation of on-site monochloramine treatment over a two year period demonstrated a significant reduction in Legionella and total bacterial counts. The growth of other WBPs did not increase and the negative consequences seen in municipal monochloramine addition were not observed. Using Illumina sequencing I showed that the resulting shift in water microbial ecology over the course of monochloramine treatment was immediate and not gradual over time. This sequencing analysis revealed an increase in the relative abundance of certain non-Legionella WBPs throughout the course of chloramination. While molecularly the relative abundance increased, the total culturable bacterial counts decreased, likely resulting in no change overall. I conducted a different sequencing study to look at the comparison of monochloramine treated and control water sampled at the same time points. This analysis showed significant differences in the richness, evenness, and composition of microbes present, related to treatment. A field evaluation of a new point-of-use faucet filters showed them to be effective in preventing exposure to Legionella for 17 weeks. While these filters did not exclude all heterotrophs, there was a significant reduction in the amount of total bacteria and the three species present in filtered samples have not been found to cause human disease. These studies have public health significance because they aid in the rapid detection of L. pneumophila, the cause of most cases of Legionnaires’ disease. They have also evaluated the effects of on-site monochloramine disinfection and point-of-use filtration to prevent exposure to Legionella and other opportunistic waterborne pathogens

Evaluation of a new culture medium for isolation of nontuberculous mycobacteria from environmental water samples

Nontuberculous mycobacteria (NTM) are waterborne pathogens commonly found in building water systems where they are a primary concern to vulnerable patient populations and can cause severe disease. The recovery of NTM from environmental samples can be a laborious undertaking and current pre-treatment methods and selective media lack sensitivity. We explored the use of the highly selective Rapidly Growing Mycobacteria (RGM) medium for culturing NTM from environmental water samples compared to existing methods. In total, 223 environmental water samples, including potable and non-potable water, were cultured for NTM using three culture media. In addition to direct culture on RGM medium, each sample was cultured on Middlebrook 7H10 medium and Mitchison 7H11 medium after pre-treatment with 0.2M KCl-HCl. Additionally, 33 distinct species of NTM were inoculated onto RGM medium and 7H10 medium in parallel to directly compare their growth. The use of RGM medium alone without pre-treatment provided a sensitivity (91%) comparable to that offered by culture on both 7H10 and 7H11 with acid pretreatment (combined sensitivity; 86%) with significantly less overgrowth and interference from other organisms on RGM medium. The average concentration of NTM observed on RGM medium alone was comparable to or greater than the NTM concentration on either medium alone or combined. Thirty-three species were examined in parallel and all tested strains of 27 of these species successfully grew on RGM medium, including 19 of 21 from the CDC’s healthcare-associated infections species list. RGM medium was successful at recovering environmental NTM without a pre-treatment, greatly reducing labor and materials required to process samples. Simplification of culture processing for environmental NTM will allow for a better assessment of their presence in building water systems and the potential for reduced exposure of susceptible populations

Diagnostic testing for Legionnaires’ disease

Abstract Legionnaires’ disease is commonly diagnosed clinically using a urinary antigen test. The urinary antigen test is highly accurate for L. pneumophila serogroup 1, however other diagnostic tests should also be utilized in conjunction with the urinary antigen as many other Legionella species and serogroups are pathogenic. Culturing of patient specimens remains the gold standard for diagnosis of Legionnaires’ disease. Selective media, BYCE with the addition of antibiotics, allows for a high sensitivity and specificity. Culturing can identify all species and serogroups of Legionella. A major benefit of culturing is that it provides the recovery of a patient isolate, which can be used to find an environmental match. Other diagnostic tests, including DFA and molecular tests such as PCR and LAMP, are useful tests to supplement culturing. Molecular tests provide much more rapid results in comparison to culture, however these tests should not be a primary diagnostic tool given their lower sensitivity and specificity in comparison to culturing. It is recommended that all laboratories develop the ability to culture patient specimens in-house with the selective media

Water Quality as a Predictor of Legionella Positivity of Building Water Systems

Testing drinking water systems for the presence of Legionella colonization is a proactive approach to assess and reduce the risk of Legionnaires’ disease. Previous studies suggest that there may be a link between Legionella positivity in the hot water return line or certain water quality parameters (temperature, free chlorine residual, etc.) with distal site Legionella positivity. It has been suggested that these measurements could be used as a surrogate for testing for Legionella in building water systems. We evaluated the relationship between hot water return line Legionella positivity and other water quality parameters and Legionella colonization in premise plumbing systems by testing 269 samples from domestic cold and hot water samples in 28 buildings. The hot water return line Legionella positivity and distal site positivity only demonstrated a 77.8% concordance rate. Hot water return line Legionella positivity compared to distal site positivity had a sensitivity of 55% and a specificity of 96%. There was poor correlation and a low positive predictive value between the hot water return line and distal outlet positivity. There was no correlation between Legionella distal site positivity and total bacteria (heterotrophic plate count), pH, free chlorine, calcium, magnesium, zinc, manganese, copper, temperature, total organic carbon, or incoming cold-water chlorine concentration. These findings suggest that hot water return line Legionella positivity and other water quality parameters are not predictive of distal site positivity and should not be used alone to determine the building’s Legionella colonization rate and effectiveness of water management programs

Evaluation of Recommended Water Sample Collection Methods and the Impact of Holding Time on Legionella Recovery and Variability from Healthcare Building Water Systems

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

Comparison of the number of OTUs (97% similarity) for each month.

<p>Bars represent standard deviation. Each sample pool was normalized to 7,000 sequences. Samples from B3 and B0 represent those taken three months and immediately prior to monochloramine treatment, respectively. Samples from M1, M2, M3, M4, M5, and M6 were taken monthly during the first six months of treatment.</p

Taxonomic assignments of sequences from HWT (hot water tank samples) (Panel A), F3 (floors 3–5) (Panel B), F6A (floors 6 and 7 automatic faucets) (Panel C), F6S (floors 6 and 7 standard faucets) (Panel D), F8 (floors 8–12) and F8rep (replicate barcoded PCRs of samples from floors 8–12) (Panel E).

<p>Samples from B3 and B0 represent those taken three months and immediately prior to monochloramine treatment, respectively. Samples from M1, M2, M3, M4, M5, and M6 were taken monthly during the first six months of treatment. Black lines in Panel E separate pairs of replicates.</p

Relative abundance of genera containing nitrifying (<i>Nitrospira</i> and Nitrosomonadacea) and denitrifying bacteria (<i>Thiobacillus</i>, <i>Micrococcus</i>, and <i>Paracoccus</i>).

<p>No other genera associated with nitrification (<i>Nitrosococcus</i>, <i>Nitrobacter</i>, <i>Nitrospina</i>, or <i>Nitrococcus</i>,) or denitrification (<i>Rhizobiales</i> and <i>Rhodanobacter</i>) were found in any of our samples. The x-axis represents sampling months with months B3 and B0 being before monochloramine treatment and months M1–M6 representing the first six months of treatment. The y-axis represents the relative abundance.</p

Sample pool description, abbreviati<b>on, and number of pooled sites.</b>

<p>Hot water was collected after a one-minute flush from the following locations throughout the hospital.</p

PCoA analysis of samples pools.

<p>Samples that cluster more closely together share a greater similarity in microbial community structure. Colors represent months sampled whereas shapes represent sample pool. Samples from B3 and B0 represent those taken three months and immediately prior to monochloramine treatment, respectively. Samples from M1, M2, M3, M4, M5, and M6 were taken monthly during the first six months of treatment.</p