Genome sequences of Enterobacter asburiae mcr-9 harboring and Enterobacter roggenkampii mcr-10 harboring isolated from a wastewater treatment plant in Japan.

Severe infections caused by multi-drug-resistant Gram-negative rods pose a clinical threat due to their high mortality risk. The global spread of plasmid-mediated colistin-resistance genes has become a serious problem. In this study, we identified Enterobacter spp. harboring mcr-9 and mcr-10 from a wastewater treatment plant in Japan.

Cell Extracts Derived from Cypress and Cedar Show Antiviral Activity against Enveloped Viruses.

The antiviral efficacy of cell-extracts (CEs) derived from cypress (Chamaecyparis obtusa (Siebold & Zucc.) Endl., C. obtusa) and cedar (Cryptomeria japonica (Thunb. ex. L.) D.Don, C. japonica) was assessed using phi6 and MS2 bacteriophages, which are widely accepted surrogate models for enveloped and non-enveloped viruses, in order to verify their potential use as antiviral agents. Our results indicate that CEs derived from C. obtusa are dominantly composed of terpinen-4-ol (18.0%), α-terpinyl acetate (10.1%), bornyl acetate (9.7%), limonene (7.1%), and γ-terpinene (6.7%), while CEs derived from C. japonica are dominantly composed of terpinen-4-ol (48.0%) and α-pinene (15.9%), which exhibited robust antiviral activity against phi6 bacteriophage. Both CEs successfully inactivated the phi6 bacteriophage below the detection limit (10 PFU/mL) within a short exposure time of 30 s (log reduction value, LRV > 4). Through exposure experiments utilizing CEs with content ratios prepared via 2-fold serial dilutions (ranging from 3.13% to 100%), we demonstrated that the antiviral effect could be sustained up to a concentration of 25% (C. obtusa LRV = 3.8, C. japonica LRV > 4.3 at a 25% CE content ratio for each species). However, CEs with content ratios below 12.5% did not produce a significant reduction in bacteriophage concentration and consequently lost their antiviral effects. Conversely, both CEs did not exhibit antiviral activity against MS2 bacteriophage, a non-enveloped virus. Our findings reveal for the first time the potential of CEs derived from C. obtusa and C. japonica for use as antiviral agents specifically targeting enveloped viruses.

Exploring indoor and outdoor dust as a potential tool for detection and monitoring of COVID-19 transmission.

This study investigated the potential of using SARS-CoV-2 viral concentrations in dust as an additional surveillance tool for early detection and monitoring of COVID-19 transmission. Dust samples were collected from 8 public locations in 16 districts of Bangkok, Thailand, from June to August 2021. SARS-CoV-2 RNA concentrations in dust were quantified, and their correlation with community case incidence was assessed. Our findings revealed a positive correlation between viral concentrations detected in dust and the relative risk of COVID-19. The highest risk was observed with no delay (0-day lag), and this risk gradually decreased as the lag time increased. We observed an overall decline in viral concentrations in public places during lockdown, closely associated with reduced human mobility. The effective reproduction number for COVID-19 transmission remained above one throughout the study period, suggesting that transmission may persist in locations beyond public areas even after the lockdown measures were in place.

Tracing the transmission of mpox through wastewater surveillance in Southeast Asia.

High population density and tourism in Southeast Asia increase the risk of mpox due to frequent interpersonal contacts. Our wastewater surveillance in six Southeast Asian countries revealed positive signals for Monkeypox virus (MPXV) DNA, indicating local transmission. This alerts clinicians and helps allocate resources like testing, vaccines and therapeutics in resource-limited countries.

COVID-19 monitoring with sparse sampling of sewered and non-sewered wastewater in urban and rural communities.

Equitable SARS-CoV-2 surveillance in low-resource communities lacking centralized sewers is critical as wastewater-based epidemiology (WBE) progresses. However, large-scale studies on SARS-CoV-2 detection in wastewater from low-and middle-income countries is limited because of economic and technical reasons. In this study, wastewater samples were collected twice a month from 186 urban and rural subdistricts in nine provinces of Thailand mostly having decentralized and non-sewered sanitation infrastructure and analyzed for SARS-CoV-2 RNA variants using allele-specific RT-qPCR. Wastewater SARS-CoV-2 RNA concentration was used to estimate the real-time incidence and time-varying effective reproduction number (Re). Results showed an increase in SARS-CoV-2 RNA concentrations in wastewater from urban and rural areas 14-20 days earlier than infected individuals were officially reported. It also showed that community/food markets were "hot spots" for infected people. This approach offers an opportunity for early detection of transmission surges, allowing preparedness and potentially mitigating significant outbreaks at both spatial and temporal scales.

Multiple traces of monkeypox detected in non-sewered wastewater with sparse sampling from a densely populated metropolitan area in Asia.

The monkeypox virus is excreted in the feces of infected individuals. Therefore, there is an interest in using viral load detection in wastewater for sentinel early surveillance at a community level and as a complementary approach to syndromic surveillance. We collected wastewater from 63 sewered and non-sewered locations in Bangkok city center between May and August 2022. Monkeypox viral DNA copy numbers were quantified using real-time polymerase chain reaction (PCR) and confirmed positive by Sanger sequencing. Monkeypox viral DNA was first detected in wastewater from the second week of June 2022, with a mean copy number of 16.4 copies/ml (n = 3). From the first week of July, the number of viral DNA copies increased to a mean copy number of 45.92 copies/ml. Positive samples were Sanger sequenced and confirmed the presence of the monkeypox virus. Our study is the first to detect monkeypox viral DNA in wastewater from various locations within Thailand. Results suggest that this could be a complementary source for detecting viral DNA and predicting upcoming outbreaks.

Draft Genome Sequence of Stutzerimonas stutzeri NT-I, Which Reduces Selenium Oxyanions into Elemental Selenium and Volatile Selenium Species.

Stutzerimonas stutzeri strain NT-I effectively reduces selenate and selenite into elemental selenium and volatile selenium species. It is thus a promising biological agent for treatment of selenium-contaminated wastewater. We here report the draft genome sequence of this strain.

Prevalence of antibiotic resistance genes in drinking and environmental water sources of the Kathmandu Valley, Nepal.

Antibiotic-resistant bacteria-associated infections are responsible for more than 1.2 million annual deaths worldwide. In low- and middle-income countries (LMICs), the consumption of antibiotics for human and veterinary uses is not regulated effectively. Overused and misused antibiotics can end up in aquatic environments, which may act as a conduit for antibiotic resistance dissemination. However, data on the prevalence of antibiotic resistance determinants in aquatic environments are still limited for LMICs. In this study, we evaluated the prevalence and concentration of antibiotic resistance genes (ARGs) in different drinking and environmental water sources collected from the Kathmandu Valley, Nepal, using droplet digital polymerase chain reaction to understand the current situation of ARG contamination. River water and shallow dug well water sources were the most contaminated with ARGs. Almost all samples contained sul1 (94%), and intI1 and tet(A) were detected in 83 and 60% of the samples, respectively. Maximum ARG concentration varied between 4.2 log10 copies/100 ml for mecA and 9.3 log10 copies/100 ml for sul1. Significant positive correlations were found between ARGs (r > 0.5, p < 0.01), except for mecA, qnrS, and vanA. As sul1 and intI1 were detected in almost all samples, the presence of these genes in a given sample may need to be considered as background antibiotic resistance in LMICs. Therefore, monitoring of ARGs, such as ß-lactam ARGs, quinolone resistance genes, and vancomycin resistance genes, may provide a better picture of the antibiotic resistance determinants in aquatic environments of LMICs.

Concentration and reduction efficiency of vancomycin-resistant heterotrophic bacteria and vanA and vanB genes in wastewater treatment unit processes.

OBJECTIVES: This study elucidated the distribution and fate of vancomycin (VCM)-resistant heterotrophic bacteria (HTB) and resistance genes, vanA and vanB, during each treatment unit process of a wastewater treatment plant (WWTP). METHODS: Several bacterial counts as well as copy numbers of vanA and vanB genes were determined in each wastewater and sludge sample. In addition, HTB strains isolated from wastewater and sludge were analyzed for VCM susceptibility. Then, the fate and reduction ratios of each bacterial count, copy number of vanA and vanB genes, and the existence ratio of VCM-resistant HTB strains in the wastewater treatment unit process were evaluated. RESULTS: VCM-resistant HTB were detected in all wastewater and sludge samples, and their existence ratio decreased along the treatment process (92.9% in influent wastewater to 39.4% in chlorinated water). Notably, most of the HTB isolated from the influent wastewater were resistant to 8.0 µg/mL of VCM, strongly suggesting that a significant number of antibiotic-resistant bacteria are flowing into the WWTP from urban areas through the sewage system. The vanA and vanB genes were also detected in all wastewater and sludge, with high copy numbers (102-104 copies/mL) even in chlorinated water samples. CONCLUSIONS: Results revealed that residual VCM-resistant HTB, and resistance genes, which could not be completely removed, were ubiquitously released into the aquatic environment. Furthermore, a high existence ratio of VCM-resistant HTB and high copy numbers of resistance genes were also detected in the sludge, indicating that they are constantly circulating in the WWTP via the returned sludge.

Inactivation of antibiotic resistant bacteria and their resistance genes in sewage by applying pulsed electric fields.

We evaluated the suitability of pulsed electric field (PEF) technology as a new disinfection option in the sewage treatment plants (STPs) that can inactivate antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). It was shown that PEF applied disinfection could inactivate not only vancomycin-resistant enterococci (VRE), but also vanA resistance gene. Cultivable VRE could be effectively inactivated by PEF applied disinfection, and were reduced to below the detection limit (log reduction value of VRE > 5 log). Although the vanA also showed a reduction of more than 4 log, it remained in the order of 105 copies/mL, suggesting that ARGs are more difficult to be inactivated than ARB in PEF applied disinfection. Among parameters in each applying condition verified in this study, the initial voltage was found to be the most important for inactivation of ARB and ARGs. Furthermore, frequency was a parameter that affects the increase or decrease of the duration time, and it was suggested that the treatment time could be shortened by increasing the frequency. Our results strongly suggested that PEF applied disinfection may be a new disinfection technology option for STPs that contributes to the control of ARB and ARGs contamination in the aquatic environments.

Pathogens and disease vectors/hosts monitoring in aquatic environments: Potential of using eDNA/eRNA based approach.

Infectious diseases are spreading in to previously unreported geographical regions, and are reappeared in regions 75 or 100 years after their last reported case, as a result of environmental changes caused by anthropogenic activities. A pathogen, vector/host monitoring methodology is therefore indispensable in identifying potential transmission sites, providing early warnings and evaluating the human health risks of these infectious diseases in a given area. Recently, environmental DNA (eDNA) and environmental RNA approach (eRNA) have become widespread in monitoring organisms in the environment due to advantages like lower cost, time, and labour requirements. However, eDNA/eRNA based monitoring of pathogens and vectors/hosts using aquatic samples is limited to very few studies. In this review, we summarized the currently available eDNA/eRNA based human and non-human pathogens and vectors/hosts detection studies in aquatic samples. Species-specific shedding, transport, and decay of eDNA/eRNA in aquatic environments which is essential in estimating the abundance of pathogen, vectors/host in focus is also summarized. We also suggest the usage of eDNA/eRNA approach in urban aquatic samples like runoff in identifying the disease vectors/hosts inhabiting in locations which are not accessible easily.

Complete genome sequencing and comparative plasmid analysis of KPC-2-producing Klebsiella pneumoniae isolated from hospital sewage water in Japan.

OBJECTIVES: The Klebsiella pneumoniae carbapenemase (blaKPC) gene is one of the most widespread carbapenemase genes in the world. However, there are few reports on KPC-producing bacteria in Japan. The aim of this study was therefore to investigate KPC-producing K. pneumoniae in Japan. METHODS: A KPC-2-producingK. pneumoniae strain (KAM260) was isolated from hospital sewage water in Japan in 2018. The complete genome was determined by whole-genome sequencing. Subsequent comparative sequence analysis of the blaKPC-2-carrying plasmid was performed. RESULTS: Klebsiella pneumoniae KAM260, belonging to sequence type 3026 (ST3026), harboured the blaKPC-2 gene in 114.6-kbp plasmid pKAM260_2 with IncFIB(pQIL) and IncFII(K) replicons. pKAM260_2 was highly identical to pKpQIL-like plasmids, which carry blaKPC genes and have spread worldwide. pKAM260_2 had functional conjugation-associated genes and was transferable to Escherichia coli. CONCLUSION: pKAM260_2, the self-transmissible plasmid carrying theblaKPC-2 gene, was detected from hospital sewage water in Japan and was characterised as a pKpQIL-like plasmid. This plasmid needs to be monitored in Japan in the future owing to its high diffusivity.

Draft Genome Sequence of Rhodococcus aetherivorans JCM 14343T, a Bacterium Capable of Degrading Recalcitrant Noncyclic and Cyclic Ethers.

Here, we report the draft genome sequence of Rhodococcus aetherivorans JCM 14343T, which possesses the versatile ability to degrade recalcitrant noncyclic and cyclic ether compounds. The 4.2-Mbp genome of this bacterium contains alkane hydroxylase and propane monooxygenase genes involved in the degradation of noncyclic and cyclic ethers, respectively.

Stimulatory and inhibitory effects of metals on 1,4-dioxane degradation by four different 1,4-dioxane-degrading bacteria.

This study evaluates the effects of various metals on 1,4-dioxane degradation by the following four bacteria: Pseudonocardia sp. D17; Pseudonocardia sp. N23; Mycobacterium sp. D6; and Rhodococcus aetherivorans JCM 14343. Eight transition metals [Co(II), Cu(II), Fe(II), Fe(III), Mn(II), Mo(VI), Ni(II), and Zn(II)] were used as the test metals. Results revealed, for the first time, that metals had not only inhibitory but also stimulatory effects on 1,4-dioxane biodegradation. Cu(II) had the most severe inhibitory effects on 1,4-dioxane degradation by all of the test strains, with significant inhibition at concentrations as low as 0.01-0.1 mg/L. This inhibition was probably caused by cellular toxicity at higher concentrations, and by inhibition of degradative enzymes at lower concentrations. In contrast, Fe(III) enhanced 1,4-dioxane degradation by Mycobacterium sp. D6 and R. aetherivorans JCM 14343 the most, while degradation by the two Pseudonocardia strains was stimulated most notably in the presence of Mn(II), even at concentrations as low as 0.001 mg/L. Enhanced degradation is likely caused by the stimulation of soluble di-iron monooxygenases (SDIMOs) involved in the initial oxidation of 1,4-dioxane. Differences in the stimulatory effects of the tested metals were likely associated with the particular SDIMO types in the test strains.

Development of a Quantitative PCR Assay for Arcobacter spp. and its Application to Environmental Water Samples.

Arcobacter spp. are emerging pathogens associated with gastroenteritis in humans. The objective of this study was to develop a highly sensitive and broadly reactive quantitative PCR (qPCR) assay for Arcobacter spp. and to apply the developed assay to different water sources in the Kathmandu Valley, Nepal. Fifteen samples to be analyzed by next-generation sequencing were collected from 13 shallow dug wells, a deep tube well, and a river in the Kathmandu Valley in August 2015. Among the 86 potential pathogenic bacterial genera identified, Acinetobacter, Pseudomonas, Flavobacterium, and Arcobacter were detected with relatively high abundance in 15, 14, 12, and 8 samples, respectively. A primer pair was designed with maximal nucleotide homologies among Arcobacter spp. by comparing the sequences of 16S rRNA genes. These primers were highly specific to most of the known species of Arcobacter and quantified between 1.0×101 and 6.4×106 copies reaction-1 and sometimes detected as few as 3 copies reaction-1. The qPCR assay was used to quantify Arcobacter spp. in bacterial DNA in not only the above 15 water samples, but also in 33 other samples collected from 15 shallow dug wells, 6 shallow tube wells, 5 stone spouts, 4 deep tube wells, and 3 springs. Thirteen (27%) out of 48 samples tested were positive for Arcobacter spp., with concentrations of 5.3-9.1 log copies 100 mL-1. This qPCR assay represents a powerful new tool to assess the prevalence of Arcobacter spp. in environmental water samples.