The engineering of a home-made portable double-layer filtration and concentration device with the common syringe for rapid analysis of water samples is reported. The core elements of the device were two installed filtration membranes with different pore sizes for respective functions. The upper filtration membrane was used for preliminary intercepting large interfering impurities (interception membrane), while the lower filtration membrane was used for collecting multiple target pathogens (enrichment membrane) for determination. This combination can make the contaminated environmental water, exemplified by surface water, filtrated quickly through the device and just retained the target bacteria of Escherichia coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes on the lower enrichment membrane. Integrating with surface-enhanced Raman spectra (SERS) platform to decode the SERS-Tags (SERS-TagCVa, SERS-TagR6G, and SERS-TagMB) already labeled on each of the enriched bacteria based the antibody-mediated immuno-recognition effect, fast separation, concentration, and detection of multiple pathogenic bacteria from the bulk of contaminated environmental water were realized. Results show that within 30 min, all target bacteria in the lake water can be simultaneously and accurately measured in the range from 101 to 106 CFU mL-1 with detection limit of 10.0 CFU mL-1 without any pre-culture procedures. This work highlights the simplicity, rapidness, cheapness, selectivity, and the robustness of the constructed method for simultaneous detecting multiple pathogens in aqueous samples. This protocol opens a new avenue for facilitating the development of versatile analytical tools for drinking water and food safety monitoring in underdeveloped or developing countries.

Sea urchins are primary herbivores on coral reefs, regulating algal biomass and facilitating coral settlement and growth.1,2,3,4,5,6,7,8,9,10,11,12 Recurring mass mortality events (MMEs) of Diadema species Gray, 1825 have been recorded globally,13,14,15,16,17,18,19,20,21,22,23 the most notorious and ecologically significant of which occurred in the Caribbean in 1983,14,17,19,20 contributing to the shift from coral to algal-dominated ecosystems.17,24,25 Recently, first evidence of Diadema setosum mass mortality was reported from the eastern Mediterranean Sea.23 Here, we report extensive mass mortalities of several diadematoid species inhabiting the Red Sea and Western Indian Ocean (WIO)26,27,28 including first evidence of mortalities in the genus Echinothrix Peters, 1853. Mortalities initiated in the Gulf of Aqaba on December 2022 and span the Red Sea, the Gulf of Oman, and the Western Indian Ocean (Réunion Island), with population declines reaching 100% at some sites. Infected individuals are characterized by spine loss and tissue necrosis, resulting in exposed skeletons (i.e., tests) and mortality. Molecular diagnostics of the 18S rRNA gene confirm the presence of a waterborne scuticociliate protozoan most closely related to Philaster apodigitiformis in infected specimens-identical to the pathogen found in the 2022 Caribbean mass mortality of Diadema antillarum.13,15,18 Collapse of these key benthic grazers in the Red Sea and Western Indian Ocean may lead to algal dominance over corals, threatening the stability of coral reefs on a regional scale.29,30,31,32 We issue a warning regarding the further expansion of mortalities and call for immediate monitoring and conservation efforts for these key ecological species.

OBJECTIVE: To assess the residual risk of waterborne contamination by Pseudomonas aeruginosa from a water network colonized by a single genotype [sequence type (ST) 299] despite the presence of antimicrobial filters in a medical intensive care unit (ICU).
METHODS: During the first 19-month period since the ICU opened, contamination of the water network was assessed monthly by collecting water upstream of the filters. Downstream water was also sampled to assess the efficiency of the filters. P. aeruginosa isolates from patients were collected and compared with the waterborne ST299 P. aeruginosa by multiplex-rep polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. Cross-transmission events by other genotypes of P. aeruginosa were also assessed.
RESULTS: Overall, 1.3% of 449 samples of filtered water were positive for P. aeruginosa in inoculum, varying between 1 and 104 colony-forming units/100 mL according to the tap. All P. aeruginosa hydric isolates belonged to ST299 and displayed fewer than two single nucleotide polymorphisms (SNPs). Among 278 clinical isolates from 122 patients, 10 isolates in five patients showed identical profiles to the hydric ST299 clone on both multiplex-rep PCR and PFGE, and differed by an average of fewer than five SNPs, confirming the water network reservoir as the source of contamination by P. aeruginosa for 4.09% of patients. Cross-transmission events by other genotypes of P. aeruginosa were responsible for the contamination of 1.75% of patients.
CONCLUSIONS: Antimicrobial filters are not sufficient to protect patients from waterborne pathogens when the water network is highly contaminated. A microbiological survey of filtered water may be needed in units hosting patients at risk of P. aeruginosa infections, even when all water points-of-use are fitted with filters.

Accurate, rapid, and multiplexed nucleic acid detection is critical for environmental and biomedical monitoring. In recent years, CRISPR-Cas12a has shown great potential in improving the performance of DNA biosensing. However, the nonspecific trans-cleavage activity of Cas12a complicates the multiplexing capability of Cas12a biosensing. We report a 3D-printed composable microfluidic plate (cPlate) device that utilizes miniaturized wells and microfluidic loading for a multiplexed CRISPR-Cas12a assay. The device easily combines loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12a readout in a simple and high-throughput workflow with low reagent consumption. To ensure the maximum performance of the device, the concentration of Cas12a and detection probe was optimized, which yielded a four-fold sensitivity improvement. Our device demonstrates sensitive detection to the fg mL- 1 level for four waterborne pathogens including shigella, campylobacter, cholera, and legionella within 1 h, making it suitable for low-resource settings.

The present study investigated the water quality index, microbial composition and antimicrobial resistance genes in urban water habitats. Combined chemicals testing, metagenomic analyses and qualitative PCR (qPCR) were conducted on 20 locations, including rivers from hospital surrounds (n = 7), community surrounds (n = 7), and natural wetlands (n = 6). Results showed that the indexes of total nitrogen, phosphorus, and ammonia nitrogen of hospital waters were 2-3 folds high than that of water from wetlands. Bioinformatics analysis revealed a total of 1,594 bacterial species from 479 genera from the three groups of water samples. The hospital-related samples had the greatest number of unique genera, followed by those from wetlands and communities. The hospital-related samples contained a large number of bacteria associated with the gut microbiome, including Alistipes, Prevotella, Klebsiella, Escherichia, Bacteroides, and Faecalibacterium, which were all significantly enriched compared to samples from the wetlands. Nevertheless, the wetland waters enriched bacteria from Nanopelagicus, Mycolicibacterium and Gemmatimonas, which are typically associated with aquatic environments. The presence of antimicrobial resistance genes (ARGs) that were associated with different species origins in each water sample was observed. The majority of ARGs from hospital-related samples were carried by bacteria from Acinetobacter, Aeromonas and various genera from Enterobacteriaceae, which each was associated with multiple ARGs. In contrast, the ARGs that were exclusively in samples from communities and wetlands were carried by species that encoded only 1 to 2 ARGs each and were not normally associated with human infections. The qPCR showed that water samples of hospital surrounds had higher concentrations of intI1 and antimicrobial resistance genes such as tetA, ermA, ermB, qnrB, sul1, sul2 and other beta-lactam genes. Further genes of functional metabolism reported that the enrichment of genes associated with the degradation/utilization of nitrate and organic phosphodiester were detected in water samples around hospitals and communities compared to those from wetlands. Finally, correlations between the water quality indicators and the number of ARGs were evaluated. The presence of total nitrogen, phosphorus, and ammonia nitrogen were significantly correlated with the presence of ermA and sul1. Furthermore, intI1 exhibited a significant correlation with ermB, sul1, and blaSHV, indicating a prevalence of ARGs in urban water environments might be due to the integron intI1\'s diffusion-promoting effect. However, the high abundance of ARGs was limited to the waters around the hospital, and we did not observe the geographical transfer of ARGs along with the river flow. This may be related to water purifying capacity of natural riverine wetlands. Taken together, continued surveillance is required to assess the risk of bacterial horizontal transmission and its potential impact on public health in the current region.

Environmental waters (EW) substantially lend to the transmission of Helicobacter pylori (Hp). But the increase in Hp infections and antimicrobial resistance is often attributed to socioeconomic status. The connection between socioeconomic status and Hp prevalence in EW is however yet to be investigated. This study aimed to assess the impacts of socioeconomic indices (SI: continent, world bank region (WBR), world bank income (WBI), WHO region, Socio-demographic Index (SDI quintile), Sustainable Development Index (SuDI), and Human Development Index (HDI)) on the prevalence of Hp in EW. Hp-EW data were fitted to a generalized linear mixed-effects model and SI-guided meta-regression models with a 1000-resampling test. The worldwide prevalence of Hp in EW was 21.76% [95% confidence interval [CI]: 10.29-40.29], which declined significantly from 59.52% [43.28-74.37] in 1990-99 to 19.36% [3.99-58.09] in 2010-19 and with increasing trend in 2020-22 (33.33%, 22.66-45.43). Hp prevalence in EW was highest in North America (45.12%, 17.07-76.66), then Europe (22.38%, 5.96-56.74), South America (22.09%, 13.76-33.49), Asia (2.98%, 0.02-85.17), and Africa (2.56%, 0.00-99.99). It was negligibly different among sampling settings, WBI, and WHO regions demonstrating highest prevalence in rural location [42.62%, 3.07-94.56], HIEs [32.82%, 13.19-61.10], and AMR [39.43%, 19.92-63.01], respectively. However, HDI, sample size, and microbiological method robustly predict Hp prevalence in EW justifying 26.08%, 21.15%, and 16.44% of the true difference, respectively. In conclusion, Hp is highly prevalence in EW across regional/socioeconomic strata and thus challenged the uses of socioeconomic status as surrogate for hygienic/sanitary practices in estimating Hp infection prevalence.

Building on a previously developed workflow for rapid and sensitive pathogen detection by qPCR, this work has established a sample treatment strategy that produces consistent quantification efficiencies (QEs) for Campylobacter jejuni against a complex and highly variable sample matrix from a suburban river. The individual treatments most effective at minimizing the inhibitory effects of the sample matrix were pH buffering with HEPES (50 mM, pH 5.7) and addition of the surfactant Tween 20 (2% v/v). Unexpectedly, sample acidification (pH 4-5) resulting from the use of aged Tween 20 that had undergone partial hydrolysis, appeared to play a key role in enhancing QE. This effect could be replicated by direct pH adjustment with dilute hydrochloric acid and may be linked to the solubilization and removal of inhibitory particles at an acidic pH. While the effectiveness of each individual treatment method varied, a combined treatment of either HEPES buffer + Tween 20, or direct pH adjustment + Tween 20, consistently produced QEs of 60%-70% and up to 100%, respectively, over a sampling period of one year. The consistency and scalability of this workflow make it a suitable alternative to culture-based ISO methods for detecting Campylobacter spp.

Cryptosporidiosis is a water- and food-borne zoonotic disease caused by the protozoon parasite of the genus Cryptosporidium. C. hominis and C. parvum are the main two species causing infections in humans and animals. The disease can be transmitted by the fecal-oral route as well as the respiratory route. The infective stage (sporulated oocysts) is resistant to different disinfectants including chlorine. Currently, no effective therapeutic drugs or vaccines are available to treat and control Cryptosporidium infection. To prevent cryptosporidiosis in humans and animals, we need to understand better how the disease is spread and transmitted, and how to interrupt its transmission cycle. This review focuses on understanding cryptosporidiosis, including its infective stage, pathogenesis, life cycle, genomics, epidemiology, previous outbreaks, source of the infection, transmission dynamics, host spectrum, risk factors and high-risk groups, the disease in animals and humans, diagnosis, treatment and control, and the prospect of an effective anti-Cryptosporidium vaccine. It also focuses on the role of the One Health approach in managing cryptosporidiosis at the animal-human-environmental interface. The summarized data in this review will help to tackle future Cryptosporidium infections in humans and animals and reduce the disease occurrence.

Metagenomics offers the highest level of strain discrimination of bacterial pathogens from complex food and water microbiota. With the rapid evolvement of assembly algorithms, defining an optimal assembler based on the performance in the metagenomic identification of foodborne and waterborne pathogens is warranted. We aimed to benchmark short-read assemblers for the metagenomic identification of foodborne and waterborne pathogens using simulated bacterial communities. Bacterial communities on fresh spinach and in surface water were simulated by generating paired-end short reads of Illumina HiSeq, MiSeq, and NovaSeq at different sequencing depths. Multidrug-resistant Salmonella Indiana SI43 and Pseudomonas aeruginosa PAO1 were included in the simulated communities on fresh spinach and in surface water, respectively. ABySS, IDBA-UD, MaSuRCA, MEGAHIT, metaSPAdes, and Ray Meta were benchmarked in terms of assembly quality, identifications of plasmids, virulence genes, Salmonella pathogenicity island, antimicrobial resistance genes, chromosomal point mutations, serotyping, multilocus sequence typing, and whole-genome phylogeny. Overall, MEGHIT, metaSPAdes, and Ray Meta were more effective for metagenomic identification. We did not obtain an optimal assembler when using the extracted reads classified as Salmonella or P. aeruginosa for downstream genomic analyses, but the extracted reads showed consistent phylogenetic topology with the reference genome when they were aligned with Salmonella or P. aeruginosa strains. In most cases, HiSeq, MiSeq, and NovaSeq were comparable at the same sequencing depth, while higher sequencing depths generally led to more accurate results. As assembly algorithms advance and mature, the evaluation of assemblers should be a continuous process.

Helicobacter pylori (Hp) transmission dynamics via drinking water (DW) has a far much higher direct and indirect public health disease burden than previously thought. This study aimed to assess the global prevalence of Hp in DW, distributions across regions and socioeconomic indices (continent, world bank income, Human Development Index (HDI), Sustainable Development Index (SuDI), Socio-Demographic Index (SDI) quintile, and WHO regions). Hp-DW related data mined from five databases until 10/12/2022 according to PRISMA standard were quality-appraised and fitted to a generalized linear mixed-effects model. Sub-group analysis and meta-regression-modelling coupled with a 1000-permutation test (⁎) were conducted. The global prevalence of Hp in DW was 15.7% (95% confidence interval [CI]: 7.98-27.5), which varied significantly by sampling methods (Moore swabbing (61.0% [0.00-100.0]) vs. grab sampling (13.68%[6.99-25.04])) and detection technique (non-culture (21.35%[9.13-42.31]) vs. cultured-based methods (Psubgroup < 0.01)). The period 1990-99 had the highest prevalence (41.24% [0.02-99.97]). Regarding regional designations, Hp prevalence in DW was significantly different being highest in North America (61.82% [41.03-79.02]) by continents, AMR (42.66% [20.81-67.82]) by WHO group, high HDI (24.64% [10.98-46.43]) by HDI group and North America (61.90% [2.79-98.93]) by world bank region (Psubgroup < 0.01). Generally, sample preparation, SuDI grouping, and detection/confirmation techniques, have significant effects on the detection/prevalence of Hp in DW (Psubgroup < 0.01). Hp prevalence in DW was not significantly different among rural and urban DW (Psubgroup = 0.90), world bank income groups (Psubgroup = 0.15), and SDI quintiles (Psubgroup = 0.07). Among the predictors examined, only sample size (p < 0.1, R∗2(coefficient of determinant) = 15.29%), continent (p∗val = 0.04), HDI (p∗val = 0.02), HDI group (p∗val = 0.05), and microbiological methods (p < 0.1; R∗2=28.09 %) predicted Hp prevalence in DW robustly. In conclusion, Hp prevalence is still endemic in DW regardless of the regional designations/improve DW supplies.