Water Age Effects on the Occurrence and Concentration of Legionella Species in the Distribution System, Premise Plumbing, and the Cooling Towers

In this study, droplet digital PCRTM (ddPCRTM) was used to characterize total Legionella spp. and five specific Legionella species from source (groundwater) to exposure sites (taps and cooling towers). A total of 42–10 L volume water samples were analyzed during this study: 12 from a reservoir (untreated groundwater and treated water storage tanks), 24 from two buildings (influents and taps), and six from cooling towers, all part of the same water system. The approximate water age (time in the system) for all sample locations are as follows: ~4.5, 3.4, 9.2, 20.8, and 23.2 h (h) for the groundwater to the reservoir influent, reservoir influent to the reservoir effluent, reservoir effluent to building Fa (building names are abbreviated to protect the privacy of site location), building ERC and the cooling towers, respectively. Results demonstrated that gene copies of Legionella spp. (23S rRNA) were significantly higher in the cooling towers and ERC building (p < 0.05) relative to the reservoir and building Fa (closest to reservoir). Legionella spp. (23S rRNA) were found in 100% (42/42) of water samples at concentrations ranging from 2.2 to 4.5 Log10 GC/100 mL. More specifically, L. pneumophila was found in 57% (24/42) of the water samples, followed by L. bozemanii 52% (22/42), L. longbeachae 36% (15/42), L. micdadei 23% (10/42) and L. anisa 21% (9/42) with geometric mean concentrations of 1.7, 1.7, 1.4, 1.6 and 1.7 Log10 GC/100 mL, respectively. Based on this study, it is hypothesized that water age in the distribution system and the premise-plumbing system as well as building management plays a major role in the increase of Legionella spp., (23S rRNA) and the diversity of pathogenic species found as seen in the influent, and at the taps in the ERC building—where the building water quality was most comparable to the industrial cooling towers. Other pathogenic Legionella species besides L.pneumophila are also likely amplifying in the system; thus, it is important to consider other disease relevant species in the whole water supply system—to subsequently control the growth of pathogenic Legionella in the built water environment.

274. Legionella bozemanii (Fluoribacter bozemanae) Brain Abscess in a Renal Transplant Recipient

Background Legionnaires’ disease is a potentially fatal multi-system disease caused by Legionella species. However, extra-pulmonary Legionella disease is rare and is typically associated with Legionella species other than L. pneumophila in immunocompromised patients. Methods We present a 55-year-old immunocompromised male with history of living-related renal transplant secondary to IgA nephropathy (day 0) which was complicated by T-cell mediated rejection requiring anti-thymocyte globulin and elotuzumab (day 130). Results Patient was hospitalized on day 184 with community-acquired pneumonia and treated with piperacillin-tazobactam and azithromycin. Three weeks later (day 214), he presented with new-onset seizures and was found to have a frontal brain abscess on MRI. His clinical course and brain imaging worsened despite undergoing multiple operative drainage procedures, placement of an extra ventricular drain, and receiving broad-spectrum antimicrobials. L. bozemanii was first identified from cerebrospinal fluid (CSF) on buffered charcoal yeast extract (BCYE) agar from day 240 and was also later confirmed by 16S rRNA sequencing. Susceptibilities were unavailable due to poor organism growth. Of note, his allergy history was significant for rash with ciprofloxacin and levofloxacin. Based on the low severity of the allergic reaction and need for central nervous system penetration, moxifloxacin 400 mg intravenously every 24 hours was initiated on day 244 in addition to broad-spectrum antibiotics. Subsequent CSF cultures were positive for L. bozemanii until the CSF culture on day 250. Due to poor clinical response, azithromycin and intrathecal polymyxin B were added for salvage therapy on day 255. His neurological status continued to worsen and he eventually succumbed to his illness on day 262. 08/31/20 MRI Brain New solitary ring-enhancing lesion with significant surrounding vasogenic edema within the anterior right frontal lobe. 09/23/20 MRI Brain Post-Surgical right frontal lobe with edema, persistent cerebritis, and mass effect on the lateral ventricles. Fluoribacter Bozemanae Formerly known as Legionella bozemanii, an intracellular GNR grown on BCYE. Conclusion We present a diagnostically challenging case of L. bozemanii brain abscess in an immunocompromised patient. To our knowledge, this is the first case of culture proven L. bozemanii brain abscess in the literature. Considering the fastidious growth of the organism, fatal nature of the infection, and narrow therapeutic profile, Legionella infection should be considered in a multi-system disease in immunocompromised patients. Disclosures Wesley Kufel, PharmD, Melinta (Grant/Research Support)Merck (Grant/Research Support)Theratechnologies, Inc. (Advisor or Review Panel member)

Cooccurrence of Five Pathogenic Legionella spp. and Two Free-Living Amoebae Species in a Complete Drinking Water System and Cooling Towers

Pathogenic Legionella species grow optimally inside free-living amoebae to concentrations that increase risks to those who are exposed. The aim of this study was to screen a complete drinking water system and cooling towers for the occurrence of Acanthamoeba spp. and Naegleria fowleri and their cooccurrence with Legionella pneumophila, Legionella anisa, Legionella micdadei, Legionella bozemanii, and Legionella longbeachae. A total of 42 large-volume water samples, including 12 from the reservoir (water source), 24 from two buildings (influents to the buildings and exposure sites (taps)), and six cooling towers were collected and analyzed using droplet digital PCR (ddPCR). N. fowleri cooccurred with L. micdadei in 76 (32/42) of the water samples. In the building water system, the concentrations of N. fowleri and L. micdadei ranged from 1.5 to 1.6 Log10 gene copies (GC)/100 mL, but the concentrations of species increased in the cooling towers. The data obtained in this study illustrate the ecology of pathogenic Legionella species in taps and cooling towers. Investigating Legionella’s ecology in drinking and industrial waters will hopefully lead to better control of these pathogenic species in drinking water supply systems and cooling towers.

Intracellular Behaviour of Legionella Non-pneumophila Strains within Three Amoeba Strains, Including Willaertia magna C2c Maky

Legionellosis, an often-lethal pneumonia, is generally associated with contamination by Legionella pneumophila. This bacterium can persist in the environment and resist chemical treatment when it is internalized by amoebae. In addition, there is increasing medical proof that other Legionella species can be causative agents of Legionellosis. The objective of this study was to evaluate whether Legionella non-pneumophila (Lnp) strains were able to use the machinery of amoeba to multiply, or whether amoebae were able to limit their proliferation. Seven strains belonging to the species L. longbeachae, L. anisa, L. bozemanae, L. taurinensis, and L. dumoffii were cocultured with three amoebae, Acanthamoeba castellanii, Willaertia magna T5(S)44, and Willaertia magna C2c Maky, at two temperatures, 22 and 37 °C. We found that at 22 °C, all amoebae were able to phagocytose the seven Lnp strains and to avoid intracellular development, except for L. longbeachae, which was able to multiply inside W. magna T5(S)44. At 37 °C, four Lnp strains were able to hijack the machinery of one or two amoebae and to use it to proliferate, but none were able to multiply inside W. magna C2c Maky.

Advances in Environmental Detection and Clinical Diagnostic Tests for Legionella Species

Legionella is a fastidious organism that is difficult to culture in the lab but is widely distributed in environmental, domestic, and hospital settings. The clinical manifestations due to Legionella infections range from mild fever to fatal pneumonia and multiorgan pathologies. Legionella outbreaks though prevalent globally are not reported in developing countries due to difficulties in isolating this organism and the lack of simple diagnostic protocols. Here, we review the literature from across countries to present various methods used to detect Legionella from environmental and clinical samples. We compare the sensitivity and the specificity of the conventional culture-based assays with the recent methods and discuss approaches to develop better detection and diagnostic tests. With better cost-effective detection techniques and regular monitoring of the susceptible sites, which may harbor Legionella colonies, most of the Legionella infections can be prevented. As a result, considerable burden, caused by Legionella infections, on the healthcare system, in especially economically weaker countries, can be mitigated.

Re-opening hairdressing salons, barber shops and gyms following COVID-19 lockdown: reducing risks from Legionella species through successful domestic steam disinfection of showerheads

Given the importance of disinfecting showerheads from Legionella species and the lack of instructions as to how to successfully achieve this, the aim of this study was to examine the ability of domestic steam disinfection to successfully disinfect showerheads from Legionella species. Steam disinfection of Legionella pneumophila [n=3; L. pneumophila serogroup 2–15 (wildtype environmental water isolate); L. pneumophila serogroup 1 NCTC11192 (reference strain); L. pneumophila serogroup 1 (wildtype environmental water isolate)], L. erythra (wildtype environmental water isolate) and L. bozemanii CRM11368M (reference strain) were examined in this study. Steam disinfection employing a baby bottle steam disinfector device eradicated all Legionella organisms tested. Steam disinfection, when performed properly under the manufacturer’s instructions, offers a relatively inexpensive, simple, versatile and widely available technology for the elimination of Legionella species from contaminated showerheads. We therefore advocate the employment of such devices to regularly disinfect showerheads and shower tubing in hairdressing salons, barber shops and gyms, as a critical control in the elimination of these organisms from these sources, thereby enhancing customer/client/staff safety.

Legionellosis Caused by Non-Legionella pneumophila Species, with a Focus on Legionella longbeachae

Although known as causes of community-acquired pneumonia and Pontiac fever, the global burden of infection caused by Legionella species other than Legionella pneumophila is under-recognised. Non-L. pneumophila legionellae have a worldwide distribution, although common testing strategies for legionellosis favour detection of L. pneumophila over other Legionella species, leading to an inherent diagnostic bias and under-detection of cases. When systematically tested for in Australia and New Zealand, L. longbeachae was shown to be a leading cause of community-acquired pneumonia. Exposure to potting soils and compost is a particular risk for infection from L. longbeachae, and L. longbeachae may be better adapted to soil and composting plant material than other Legionella species. It is possible that the high rate of L. longbeachae reported in Australia and New Zealand is related to the composition of commercial potting soils which, unlike European products, contain pine bark and sawdust. Genetic studies have demonstrated that the Legionella genomes are highly plastic, with areas of the chromosome showing high levels of recombination as well as horizontal gene transfer both within and between species via plasmids. This, combined with various secretion systems and extensive effector repertoires that enable the bacterium to hijack host cell functions and resources, is instrumental in shaping its pathogenesis, survival and growth. Prevention of legionellosis is hampered by surveillance systems that are compromised by ascertainment bias, which limits commitment to an effective public health response. Current prevention strategies in Australia and New Zealand are directed at individual gardeners who use potting soils and compost. This consists of advice to avoid aerosols generated by the use of potting soils and use masks and gloves, but there is little evidence that this is effective. There is a need to better understand the epidemiology of L. longbeachae and other Legionella species in order to develop effective treatment and preventative strategies globally.