Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus.

Type 1 secretion systems (T1SSs) are broadly distributed among bacteria and translocate effectors with diverse function across the bacterial cell membrane. Legionella pneumophila, the species most commonly associated with Legionellosis, encodes a T1SS at the lssXYZABD locus which is responsible for the secretion of the virulence factor RtxA. Many investigations have failed to detect lssD, the gene encoding the membrane fusion protein of the RtxA T1SS, in non-pneumophila Legionella, which has led to the assumption that this system is a virulence factor exclusively possessed by L. pneumophila. Here we discovered RtxA and its associated T1SS in a novel Legionella taurinensis strain, leading us to question whether this system may be more widespread than previously thought. Through a bioinformatic analysis of publicly available data, we classified and determined the distribution of four T1SSs including the RtxA T1SS and four novel T1SSs among diverse Legionella spp. The ABC transporter of the novel Legionella T1SS Legionella repeat protein secretion system shares structural similarity to those of diverse T1SS families, including the alkaline protease T1SS in Pseudomonas aeruginosa. The Legionella bacteriocin (1-3) secretion systems T1SSs are novel putative bacteriocin transporting T1SSs as their ABC transporters include C-39 peptidase domains in their N-terminal regions, with LB2SS and LB3SS likely constituting a nitrile hydratase leader peptide transport T1SSs. The LB1SS is more closely related to the colicin V T1SS in Escherichia coli. Of 45 Legionella spp. whole genomes examined, 19 (42%) were determined to possess lssB and lssD homologs. Of these 19, only 7 (37%) are known pathogens. There was no difference in the proportions of disease associated and non-disease associated species that possessed the RtxA T1SS (p = 0.4), contrary to the current consensus regarding the RtxA T1SS. These results draw into question the nature of RtxA and its T1SS as a singular virulence factor. Future studies should investigate mechanistic explanations for the association of RtxA with virulence

Shotgun Metagenomics Reveals Taxonomic and Functional Shifts in Hot Water Microbiome Due to Temperature Setting and Stagnation

Hot water premise plumbing has emerged as a critical nexus of energy, water, and public health. The composition of hot water microbiomes is of special interest given daily human exposure to resident flora, especially opportunistic pathogens (OPs), which rely on complex microbial ecological interactions for their proliferation. Here, we applied shotgun metagenomic sequencing to characterize taxonomic and functional shifts in microbiomes as a function of water heater temperature setting, stagnation in distal pipes, and associated shifts in water chemistry. A cross-section of samples from controlled, replicated, pilot-scale hot water plumbing rigs representing different temperature settings (39, 42, and 51°C), stagnation periods (8 h vs. 7 days), and time-points, were analyzed. Temperature setting exhibited an overarching impact on taxonomic and functional gene composition. Further, distinct taxa were selectively enriched by specific temperature settings (e.g., Legionella at 39°C vs. Deinococcus at 51°C), while relative abundances of genes encoding corresponding cellular functions were highly consistent with expectations based on the taxa driving these shifts. Stagnation in distal taps diminished taxonomic and functional differences induced by heating the cold influent water to hot water in recirculating line. In distal taps relative to recirculating hot water, reads annotated as being involved in metabolism and growth decreased, while annotations corresponding to stress response (e.g., virulence disease and defense, and specifically antibiotic resistance) increased. Reads corresponding to OPs were readily identified by metagenomic analysis, with L. pneumophila reads in particular correlating remarkably well with gene copy numbers measured by quantitative polymerase chain reaction. Positive correlations between L. pneumophila reads and those of known protozoan hosts were also identified. Elevated proportions of genes encoding metal resistance and hydrogen metabolism were noted, which was consistent with elevated corrosion-induced metal concentrations and hydrogen generation. This study provided new insights into real-world factors influencing taxonomic and functional compositions of hot water microbiomes. Here metagenomics is demonstrated as an effective tool for screening for potential presence, and even quantities, of pathogens, while also providing diagnostic capabilities for assessing functional responses of microbiomes to various operational conditions. These findings can aid in informing future monitoring and intentional control of hot water microbiomes

To what extent do water reuse treatments reduce antibiotic resistance indicators? A comparison of two full-scale systems

Water reuse is an essential strategy for reducing water demand from conventional sources, alleviating water stress, and promoting sustainability, but understanding the effectiveness of associated treatment processes as barriers to the spread of antibiotic resistance is an important consideration to protecting human health. We comprehensively evaluated the reduction of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in two field-operational water reuse systems with distinct treatment trains, one producing water for indirect potable reuse (ozone/biologically-active carbon/granular activated carbon) and the other for non-potable reuse (denitrification-filtration/chlorination) using metagenomic sequencing and culture. Relative abundances of total ARGs/clinically-relevant ARGs and cultured ARB were reduced by several logs during primary and secondary stages of wastewater treatment, but to a lesser extent during the tertiary water reuse treatments. In particular, ozonation tended to enrich multi-drug ARGs. The effect of chlorination was facility-dependent, increasing the relative abundance of ARGs when following biologically-active carbon filters, but generally providing a benefit in reduced bacterial numbers and ecological and human health resistome risk scores. Relative abundances of total ARGs and resistome risk scores were lowest in aquifer samples, although resistant Escherichia coli and Klebsiella pneumoniae were occasionally detected in the monitoring well 3-days downgradient from injection, but not 6-months downgradient. Resistant E. coli and Pseudomonas aeruginosa were occasionally detected in the nonpotable reuse distribution system, along with increased levels of multidrug, sulfonamide, phenicol, and aminoglycoside ARGs. This study illuminates specific vulnerabilities of water reuse systems to persistence, selection, and growth of ARGs and ARB and emphasizes the role of multiple treatment barriers, including aquifers and distribution systems

An Environmental Science and Engineering Framework for Combating Antimicrobial Resistance

On June 20, 2017, members of the environmental engineering and science (EES) community convened at the Association of Environmental Engineering and Science Professors (AEESP) Biennial Conference for a workshop on antimicrobial resistance. With over 80 registered participants, discussion groups focused on the following topics: risk assessment, monitoring, wastewater treatment, agricultural systems, and synergies. In this study, we summarize the consensus among the workshop participants regarding the role of the EES community in understanding and mitigating the spread of antibiotic resistance via environmental pathways. Environmental scientists and engineers offer a unique and interdisciplinary perspective and expertise needed for engaging with other disciplines such as medicine, agriculture, and public health to effectively address important knowledge gaps with respect to the linkages between human activities, impacts to the environment, and human health risks. Recommendations that propose priorities for research within the EES community, as well as areas where interdisciplinary perspectives are needed, are highlighted. In particular, risk modeling and assessment, monitoring, and mass balance modeling can aid in the identification of “hot spots” for antibiotic resistance evolution and dissemination, and can help identify effective targets for mitigation. Such information will be essential for the development of an informed and effective policy aimed at preserving and protecting the efficacy of antibiotics for future generations

ARGem: a new metagenomics pipeline for antibiotic resistance genes: metadata, analysis, and visualization

Antibiotic resistance is of crucial interest to both human and animal medicine. It has been recognized that increased environmental monitoring of antibiotic resistance is needed. Metagenomic DNA sequencing is becoming an attractive method to profile antibiotic resistance genes (ARGs), including a special focus on pathogens. A number of computational pipelines are available and under development to support environmental ARG monitoring; the pipeline we present here is promising for general adoption for the purpose of harmonized global monitoring. Specifically, ARGem is a user-friendly pipeline that provides full-service analysis, from the initial DNA short reads to the final visualization of results. The capture of extensive metadata is also facilitated to support comparability across projects and broader monitoring goals. The ARGem pipeline offers efficient analysis of a modest number of samples along with affordable computational components, though the throughput could be increased through cloud resources, based on the user’s configuration. The pipeline components were carefully assessed and selected to satisfy tradeoffs, balancing efficiency and flexibility. It was essential to provide a step to perform short read assembly in a reasonable time frame to ensure accurate annotation of identified ARGs. Comprehensive ARG and mobile genetic element databases are included in ARGem for annotation support. ARGem further includes an expandable set of analysis tools that include statistical and network analysis and supports various useful visualization techniques, including Cytoscape visualization of co-occurrence and correlation networks. The performance and flexibility of the ARGem pipeline is demonstrated with analysis of aquatic metagenomes. The pipeline is freely available at https://github.com/xlxlxlx/ARGem

An Environmental Science and Engineering Framework for Combating Antimicrobial Resistance

On June 20, 2017, members of the environmental engineering and science (EES) community convened at the Association of Environmental Engineering and Science Professors (AEESP) Biennial Conference for a workshop on antimicrobial resistance. With over 80 registered participants, discussion groups focused on the following topics: risk assessment, monitoring, wastewater treatment, agricultural systems, and synergies. In this study, we summarize the consensus among the workshop participants regarding the role of the EES community in understanding and mitigating the spread of antibiotic resistance via environmental pathways. Environmental scientists and engineers offer a unique and interdisciplinary perspective and expertise needed for engaging with other disciplines such as medicine, agriculture, and public health to effectively address important knowledge gaps with respect to the linkages between human activities, impacts to the environment, and human health risks. Recommendations that propose priorities for research within the EES community, as well as areas where interdisciplinary perspectives are needed, are highlighted. In particular, risk modeling and assessment, monitoring, and mass balance modeling can aid in the identification of 'hot spots' for antibiotic resistance evolution and dissemination, and can help identify effective targets for mitigation. Such information will be essential for the development of an informed and effective policy aimed at preserving and protecting the efficacy of antibiotics for future generations

Convective Mixing in Distal Pipes Exacerbates Legionella pneumophila Growth in Hot Water Plumbing

Legionella pneumophila is known to proliferate in hot water plumbing systems, but little is known about the specific physicochemical factors that contribute to its regrowth. Here, L. pneumophila trends were examined in controlled, replicated pilot-scale hot water systems with continuous recirculation lines subject to two water heater settings (40 °C and 58 °C) and three distal tap water use frequencies (high, medium, and low) with two pipe configurations (oriented upward to promote convective mixing with the recirculating line and downward to prevent it). Water heater temperature setting determined where L. pneumophila regrowth occurred in each system, with an increase of up to 4.4 log gene copies/mL in the 40 °C system tank and recirculating line relative to influent water compared to only 2.5 log gene copies/mL regrowth in the 58 °C system. Distal pipes without convective mixing cooled to room temperature (23–24 °C) during periods of no water use, but pipes with convective mixing equilibrated to 30.5 °C in the 40 °C system and 38.8 °C in the 58 °C system. Corresponding with known temperature effects on L. pneumophila growth and enhanced delivery of nutrients, distal pipes with convective mixing had on average 0.2 log more gene copies/mL in the 40 °C system and 0.8 log more gene copies/mL in the 58 °C system. Importantly, this work demonstrated the potential for thermal control strategies to be undermined by distal taps in general, and convective mixing in particular

Effect of heat shock on hot water plumbing microbiota and Legionella pneumophila control

Abstract Background Heat shock is a potential control strategy for Legionella pneumophila in hot water plumbing systems. However, it is not consistently effective, with little understanding of its influence on the broader plumbing microbiome. Here, we employed a lab-scale recirculating hot water plumbing rig to compare the pre- and post-“heat shock” (i.e., 40 → 60 → 40 °C) microbiota at distal taps. In addition, we used a second plumbing rig to represent a well-managed system at 60 °C and conducted a “control” sampling at 60 °C, subsequently reducing the temperature to 40 °C to observe the effects on Legionella and the microbiota under a simulated “thermal disruption” scenario. Results According to 16S rRNA gene amplicon sequencing, in the heat shock scenario, there was no significant difference or statistically significant, but small, difference in the microbial community composition at the distal taps pre- versus post-heat shock (both biofilm and water; weighted and unweighted UniFrac distance matrices). While heat shock did lead to decreased total bacteria numbers at distal taps, it did not measurably alter the richness or evenness of the microbiota. Quantitative PCR measurements demonstrated that L. pneumophila relative abundance at distal taps also was not significantly different at 2-month post-heat shock relative to the pre-heat shock condition, while relative abundance of Vermamoeba vermiformis, a known Legionella host, did increase. In the thermal disruption scenario, relative abundance of planktonic L. pneumophila (quantitative PCR data) increased to levels comparable to those observed in the heat shock scenario within 2 months of switching long-term operation at 60 to 40 °C. Overall, water use frequency and water heater temperature set point exhibited a stronger effect than one-time heat shock on the microbial composition and Legionella levels at distal taps. Conclusions While heat shock may be effective for instantaneous Legionella control and reduction in total bacteria numbers, water heater temperature set point and water use frequency are more promising factors for long-term Legionella and microbial community control, illustrating the importance of maintaining consistent elevated temperatures in the system relative to short-term heat shock