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Hospital wastewater is a major hotspot for the spread of antimicrobial resistance (AMR) in aquatic ecosystems. This study aimed to investigate the prevalence of antibiotic resistance genes (ARGs) and their correlation with crAssphage in a hospital wastewater treatment plant (HWWTP) and a receiving canal. Water samples were analyzed for 94 ARGs and crAssphage relative to the 16S rRNA using high-throughput quantitative polymerase chain reaction (HT-qPCR). Subsequently, 7 ARGs and crAssphage were selected and quantified using qPCR. The results showed that the detected genes ranged from 85 to 93 out of 94 genes. The raw wastewater (WW) samples had the highest gene diversity compared to the upstream canal, which had less diversity than downstream samples, as determined by HT-qPCR. The bla GES was the most abundant in WW samples, while qacEΔ1, merA, IS6100, tnpA, and IS26 showed high prevalence throughout the treatment processes. The concentrations of intI1, sul1, bla TEM, bla NDM, bla VIM, tetQ, mcr-1, crAssphage, and 16S rRNA, measured using qPCR, were the highest in WW and significantly reduced in treated water samples. Although some water quality parameters, such as total suspended solids and dissolved oxygen, did not significantly differ before and after treatment, removal efficiency ranged from 0.60 to 3.23 log reduction values (LRV). The highest LRV was observed for the tetQ, whereas the mcr-1 had the lowest of LRV. Strong positive correlations among the absolute concentrations of ARGs and crAssphage were observed (Spearman's rho = 0.6 - 1.0), and biochemical oxygen demand correlated with bla TEM and bla VIM (Spearman's rho = 0.6 and 0.6, respectively). These results indicate that crAssphage and water quality could reflect the distribution of other ARGs throughout the HWWTP. Further studies are needed to underscore the importance of monitoring ARGs and biomarkers such as crAssphage in HWWTPs and their receiving waters to enhance our understanding of ARG distribution., Competing Interests: Declaration of Competing Interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2024. Published by Elsevier Ltd.)

Concerns are rising about the contamination of recreational waters from human and animal waste, along with associated risks to public health. However, existing guidelines for managing pathogens in these environments have not yet fully integrated risk-based pathogen-specific criteria, which, along with recent advancements in indicators and markers, are essential to improve the protection of public health. This study aimed to establish risk-based critical concentration benchmarks for significant enteric pathogens, i.e., norovirus, rotavirus, adenovirus, Cryptosporidium spp., Giardia lamblia, Campylobacter jejuni, Salmonella spp., and Escherichia coli O157:H7. Applying a 0.036 risk benchmark to both marine and freshwater environments, the study identified the lowest critical concentrations for children, who are the most susceptible group. Norovirus, C. jejuni, and Cryptosporidium presented lowest median critical concentrations for virus, bacteria, and protozoa, respectively: 0.74 GC, 1.73 CFU, and 0.39 viable oocysts per 100 mL in freshwater for children. These values were then used to determine minimum sample volumes corresponding to different recovery rates for culture method, digital polymerase chain reaction and quantitative PCR methods. The results indicate that for children, norovirus required the largest sample volumes of freshwater and marine water (52.08 to 178.57 L, based on the 5th percentile with a 10 % recovery rate), reflecting its low critical concentration and high potential for causing illness. In contrast, adenovirus and rotavirus required significantly smaller volumes (approximately 0.24 to 1.33 L). C. jejuni and Cryptosporidium, which required the highest sampling volumes for bacteria and protozoa, needed 1.72 to 11.09 L and 4.17 to 25.51 L, respectively. Additionally, the presented risk-based framework could provide a model for establishing pathogen thresholds, potentially guiding the creation of extensive risk-based criteria for various pathogens in recreational waters, thus aiding public health authorities in decision-making, strengthening pathogen monitoring, and improving water quality testing accuracy for enhanced health protection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa PA2196 is a TetR family transcriptional repressor. In this study, the deletion of the PA2196 gene caused increased expression of the downstream gene curA (PA2197), which encodes for a NADPH-dependent curcumin/dihydrocurcumin reductase. The PA2196 gene was then identified as curR, and a DNA footprinting assay showed that CurR directly bound to the curA promoter at an imperfect 15-bp inverted repeat, 5'-TAGTTGA-C-TGGTCTA-3'. A curA promoter-lacZ fusion assay and site-directed mutagenesis further demonstrated that the identified CurR binding site plays a crucial role in curA repression by CurR. curA transcription was inducible by sodium hypochlorite (NaOCl) and N-ethylmaleimide (NEM) but not by hydrogen peroxide, organic hydroperoxide, or curcumin. The oxidation and alkylation of CurR by NaOCl and NEM, respectively, resulted in the inactivation of its DNA-binding activity, which induced curA expression. Under the tested conditions, the deletion of either curR or curA did not affect the survival of P. aeruginosa under NaOCl stress in the absence or presence of curcumin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The global challenge posed by rising antimicrobial resistance, and the adoption of a One Health approach, has led to the prioritisation of surveillance for antibiotic resistance genes (ARGs) in various environments. Herein lies an information gap, particularly in the context of Thailand, where there is scarce data on ARG prevalence across diverse environmental matrices and throughout different seasons. This study aimed to fill this void, analysing ARG prevalence by high-throughput qPCR in influent (n = 12) and effluent wastewater (n = 12) and river water (n = 12). The study reveals a substantial and largely uniform presence of ARGs across all water sample types (87 % similarity). Intriguingly, no ARGs were exclusive to specific water types, indicating an extensive circulation of resistance determinants across the aquatic environment. The genes intI1, tnpA, and intI3, part of the integrons and mobile genetic elements group, were detected in high relative abundance in both wastewater and river water samples, suggesting widespread pollution of rivers with wastewater. Additional high-prevalence ARGs across all water types included qepA, aadA2, merA, sul1, qacF/H, sul2, aadB, and ereA. More alarmingly, several ARGs (e.g., bla VIM , intI3, mcr-1, mexB, qepA, vanA, and vanB) showed higher relative abundance in effluent and river water than in influents, which suggests malfunctioning or inadequate wastewater treatment works and implicates this as a possible mechanism for environmental contamination. Nine genes (i.e., bla CTX-M , bla VIM , emrD, ermX, intI1, mphA, qepA, vanA, and vanB) were recovered in greater relative abundance during the dry season in river water samples as compared to the wet season, suggesting there are seasonal impacts on the efficacy of wastewater treatment practices and pollution patterns into receiving waters. This study highlights the urgency for more effective measures to reduce antibiotic resistance dissemination in water systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology (WBE) has been implemented globally. However, there remains confusion about the number and frequency of samples to be collected, as well as which types of treatment systems can provide reliable specific details about the virus prevalence in specific areas or communities, enabling prompt management and intervention measures. More research is necessary to fully comprehend the possibility of deploying sentinel locations in sewer networks in larger geographic areas. The present study introduces the first report on wastewater-based surveillance in Gandhinagar City using digital PCR (d-PCR) as a SARS-Cov-2 quantification tool, which describes the viral load from five pumping stations in Gandhinagar from October 2021 to March 2022. Raw wastewater samples (n = 119) were received and analyzed weekly to detect SARS-CoV-2 RNA, 109 of which were positive for N1 or N2 genes. The monthly variation analysis in viral genome copies depicted the highest concentrations in January 2022 and February 2022 (p < 0.05; Wilcoxon signed rank test) coincided with the Omicron wave, which contributed mainly from Vavol and Jaspur pumping stations. Cross-correlation analysis indicated that WBE from five stations in Gandhinagar, i.e., capital city sewer networks, provided two-week lead times to the citywide and statewide active cases (time-series cross-correlation function [CCF]; 0.666 and 0.648, respectively), mainly from individual contributions of the urbanized Kudasan and Vavol stations (CCF; 0.729 and 0.647, respectively). These findings suggest that sewer pumping stations in urbanized neighborhoods can be used as sentinel sites for statewide clinical surveillance and that WBE surveillance using digital PCR can be an efficient monitoring and management tool., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

Recreational exposure to microbial pollution at urban beaches poses a health risk to beachgoers. The accurate quantification of such risks is crucial in managing beaches effectively and establishing warning guidelines. In this study, we employed a quantitative microbial risk assessment (QMRA) framework to assess marine water quality and estimate the risks associated with Vibrio parahaemolyticus, an autochthonous pathogen that causes gastrointestinal illnesses, and enterococci, a traditional fecal bacteria indicator. The microbial contamination levels of V. parahaemolyticus and enterococci were determined from 48 water samples collected at two beaches in Thailand during dry and wet seasons. The accidentally ingested water volumes were obtained through a survey involving 438 respondents. The probability of illness (P ill ) was estimated using dose-response models and Monte Carlo simulation. The results revealed that enterococci posed a higher risk of illness than V. parahaemolyticus at all seven study sites. The median combined gastrointestinal (GI) risk from both bacteria at all sites met the US EPA risk benchmark of 0.036 and the 0.05 benchmark set by the WHO, but the 95th percentile risk data at all sites exceeded the benchmarks. This emphasizes the need for the continuous monitoring and management of microbial pollution at these sites. The site-specific exposure data showed higher estimated risks with increased variations compared to the WHO-referenced values, which highlights the significance of locally measured microbial concentrations and survey exposure data to avoid underestimation. Estimating the risks from recreational exposure to waterborne bacteria can inform beach management policies aimed at reducing public health risks to swimmers. The study findings improve the understanding of the risks associated with water recreation activities at Southeast Asian beaches and offer valuable insights for the development of water quality guidelines, which are crucial for the sustainable development of the blue economy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

NieR is a TetR family transcriptional repressor previously shown to regulate the NaOCl-inducible efflux pump NieAB in Agrobacterium tumefaciens. NieR is an ortholog of Escherichia coli NemR that specifically senses hypochlorite through the redox switch of a reversible sulfenamide bond between C106 and K175. The amino acid sequence of NieR contains only one cysteine. NieR has C104 and R166, which correspond to C106 and K175 of NemR, respectively. The aim of this study was to investigate the redox-sensing mechanism of NieR under NaOCl stress. C104 and R166 were subjected to mutagenesis to determine their roles. Although the substitution of R166 by alanine slightly reduced its DNA-binding activity, NieR retained its repressor function. By contrast, the DNA-binding and repression activities of NieR were completely lost when C104 was replaced by alanine. C104 substitution with serine only partially impaired the repressor function. Mass spectrometry analysis revealed an intermolecular disulfide bond between the C104 residues of NieR monomers. This study demonstrates the engagement of C104 in the mechanism of NaOCl sensing. C104 oxidation induced the formation of a disulfide-linked dimer that was likely to alter conformation, thus abolishing the DNA-binding ability of NieR and derepressing the target genes., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Microbial contamination deteriorates source water quality, posing a severe problem for drinking water suppliers worldwide and addressed by the Water Safety Plan framework to ensure high-quality and reliable drinking water. Microbial source tracking (MST) is used to examine different microbial pollution sources via host-specific intestinal markers for humans and different types of animals. However, the application of MST in tropical surface water catchments that provide raw water for drinking water supplies is limited. We analyzed a set of MST markers, namely, three cultivable bacteriophages and four molecular PCR and qPCR assays, together with 17 microbial and physicochemical parameters, to identify fecal pollution from general, human-, swine-, and cattle-specific sources. Seventy-two river water samples at six sampling sites were collected over 12 sampling events during wet and dry seasons. We found persistent fecal contamination via the general fecal marker GenBac3 (100 % detection; 2.10-5.42 log 10 copies/100 mL), with humans (crAssphage; 74 % detection; 1.62-3.81 log 10 copies/100 mL) and swine (Pig-2-Bac; 25 % detection; 1.92-2.91 log 10 copies/100 mL). Higher contamination levels were observed during the wet season (p < 0.05). The conventional PCR screening used for the general and human markers showed 94.4 % and 69.8 % agreement with the respective qPCR results. Specifically, in the studied watershed, coliphage could be a screening parameter for the crAssphage marker (90.6 % and 73.7 % positive and negative predictive values; Spearman's rank correlation coefficient = 0.66; p < 0.001). The likelihood of detecting the crAssphage marker significantly increased when total and fecal coliforms exceeded 20,000 and 4000 MPN/100 mL, respectively, as Thailand Surface Water Quality Standards, with odds ratios and 95 % confidence intervals of 15.75 (4.43-55.98) and 5.65 (1.39-23.05). Our study confirms the potential benefits of incorporating MST monitoring into water safety plans, supporting the use of this approach to ensure high-quality drinking water supplies worldwide., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kwanrawee Sirikanchana reports financial support was provided by Metropolitan Waterworks Authority. Chatsinee Surasen reports a relationship with Metropolitan Waterworks Authority that includes: employment. Somsak Passananon reports a relationship with Metropolitan Waterworks Authority that includes: employment., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The effects of the sequential subculture in the presence of a driving force on antimicrobial resistance of Stenotrophomonas maltophilia K279a were investigated. Stationary-phase cells were inoculated into the lysogeny broth medium, with and without antibiotic supplementation, and grown until the stationary phase before being subcultured into the same antibiotic-supplemented medium for six consecutive cycles. Thirty colonies from each cycle and treatment condition were selected and their antibiotic susceptibility profiles were determined. The sequential subculture of K279a for a number of cycles reduced susceptibility to diverse classes of antibiotics, including ciprofloxacin, amikacin, gentamicin, ceftazidime, co-trimoxazole, and chloramphenicol, regardless of the antibiotic used. Supplementation with antibiotics that is, ampicillin, kanamycin, ciprofloxacin, and ceftazidime, at sublethal concentrations significantly accelerated the development rate of strains that reduced susceptibility to other antibiotics. The patterns of reduced susceptibility were different depending on the antibiotic used for supplementation. Thus, without gene transfer, antibiotic-resistant strains of S. maltophilia can readily develop, especially after antibiotic treatments. Whole-genome sequence analysis of the selected antibiotic-resistant mutants identified gene mutations that might be responsible for antimicrobial resistance of S. maltophilia .

Wastewater-based epidemiology (WBE) complements the clinical surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants' distribution in populations. Many developed nations have established national and regional WBE systems; however, governance and budget constraints could be obstacles for low- and middle-income countries. An urgent need thus exists to identify hotspots to serve as sentinel sites for WBE. We hypothesized that representative wastewater treatment plants (WWTPs) in two international gateway cities, Bangkok and Phuket, Thailand, could be sentineled for SARS-CoV-2 and its variants to reflect the clinical distribution patterns at city level and serve as early indicators of new variants entering the country. Municipal wastewater samples (n = 132) were collected from eight representative municipal WWTPs in Bangkok and Phuket during 19 sampling events from October 2021 to March 2022, which were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the US CDC N1 and N2 multiplex and variant (Alpha, Delta, and Omicron BA.1 and BA.2) singleplex assays. The variant detection ratios from Bangkok and Phuket followed similar trends to the national clinical testing data, and each variant's viral loads agreed with the daily new cases (3-d moving average). Omicron BA.1 was detected in Phuket wastewater prior to Bangkok, possibly due to Phuket's WWTPs serving tourist communities. We found that the Omicron BA.1 and BA.2 viral loads predominantly drove the SARS-CoV-2 resurgence. We also noted a shifting pattern in the Bangkok WBE from a 22-d early warning in early 2021 to a near real-time pattern in late 2021. The potential application of tourist hotspots for WBE to indicate the arrival of new variants and re-emerging or unprecedented infectious agents could support tourism-dependent economies by complementing the reduced clinical regulations while maintaining public health protection via wastewater surveillance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Glutaredoxins (Grxs), ubiquitous redox enzymes belonging to the thioredoxin family, catalyze the reduction of thiol-disulfide exchange reactions in a glutathione-dependent manner. A Pseudomonas aeruginosa Δ grxD mutant exhibited hypersensitivity to oxidative stress-generating agents, such as paraquat (PQ) and cumene hydroperoxide (CHP). In vitro studies showed that P. aeruginosa GrxD acts as an electron donor for organic hydroperoxide resistance enzyme (Ohr) during CHP degradation. The ectopic expression of iron-sulfur cluster ([Fe-S]) carrier proteins, including ErpA, IscA, and NfuA, complements the function of GrxD in the Δ grxD mutant under PQ toxicity. Constitutively high expression of iscR , nfuA , tpx , and fprB was observed in the Δ grxD mutant. These results suggest that GrxD functions as a [Fe-S] cluster carrier protein involved in [Fe-S] cluster maturation. Moreover, the Δ grxD mutant demonstrates attenuated virulence in a Drosophila melanogaster host model. Altogether, the data shed light on the physiological role of GrxD in oxidative stress protection and virulence of the human pathogen, P. aeruginosa. IMPORTANCE Glutaredoxins (Grxs) are ubiquitous disulfide reductase enzymes. Monothiol Grxs, containing a CXXS motif, play an essential role in iron homeostasis and maturation of [Fe-S] cluster proteins in various organisms. We now establish that the human pathogen Pseudomonas aeruginosa GrxD is crucial for bacterial virulence, maturation of [Fe-S] clusters and facilitation of Ohr enzyme activity. GrxD contains a conserved signature monothiol motif (C 29 GFS), in which C29 is essential for its function in an oxidative stress protection. Our findings reveal the physiological roles of GrxD in oxidative stress protection and virulence of P. aeruginosa.

Pseudomonas aeruginosa gidA , which encodes a putative tRNA-modifying enzyme, is associated with a variety of virulence phenotypes. Here, we demonstrated that P. aeruginosa gidA is responsible for the modifications of uridine in tRNAs in vivo . Loss of gidA was found to have no impact on the mRNA levels of katA and katB , but it decreased KatA and KatB protein levels, resulting in decreased total catalase activity and a hydrogen peroxide-sensitive phenotype. Furthermore, gidA was found to affect flagella-mediated motility and biofilm formation; and it was required for the full virulence of P. aeruginosa in both Caenorhabditis elegans and macrophage models. Together, these observations reveal the posttranscriptional impact of gidA on the oxidative stress response, highlight the complexity of catalase gene expression regulation, and further support the involvement of gidA in the virulence of P. aeruginosa ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Srimahaeak, Thongdee, Chittrakanwong, Atichartpongkul, Jaroensuk, Phatinuwat, Phaonakrop, Jaresitthikunchai, Roytrakul, Mongkolsuk and Fuangthong.)

Water commuting is a major urban transportation method in Thailand. However, urban boat commuters risk exposure to microbially contaminated bioaerosols or splash. We aimed to investigate the microbial community structures, identify bacterial and viral pathogens, and assess the abundance of antimicrobial resistance genes (ARGs) using next-generation sequencing (NGS) at 10 sampling sites along an 18 km transportation boat route in the Saen Saep Canal, which traverses cultural, commercial, and suburban land-based zones. The shotgun metagenomic (Illumina HiSeq) and 16S rRNA gene amplicon (V4 region) (Illumina MiSeq) sequencing platforms revealed diverse microbial clusters aligned with the zones, with explicit segregation between the cultural and suburban sites. The shotgun metagenomic sequencing further identified bacterial and viral pathogens, and ARGs. The predominant bacterial pathogens (>0.5 % relative abundance) were the Burkholderia cepacia complex, Arcobacter butzleri, Burkholderia vietnamiensis, Klebsiella pneumoniae, and the Enterobacter cloacae complex. The viruses (0.28 %-0.67 % abundance in all microbial sequences) comprised mainly vertebrate viruses and bacteriophages, with encephalomyocarditis virus (33.3 %-58.2 % abundance in viral sequences), hepatitis C virus genotype 1, human alphaherpesvirus 1, and human betaherpesvirus 6A among the human viral pathogens. The 15 ARG types contained 611 ARG subtypes, including those resistant to beta-lactam, which was the most diverse and abundant group (206 subtypes; 17.0 %-27.5 %), aminoglycoside (94 subtypes; 9.6 %-15.3 %), tetracycline (80 subtypes; 15.6 %-20.2 %), and macrolide (79 subtypes; 14.5 %-32.1 %). Interestingly, the abundance of ARGs associated with resistance to beta-lactam, trimethoprim, and sulphonamide, as well as A. butzleri and crAssphage, at the cultural sites was significantly different from the other sites (p < 0.05). We demonstrated the benefits of using NGS to deliver insights into microbial communities, and antimicrobial resistance, both of which pose a risk to human health. Using NGS may facilitate microbial risk mitigation and management for urban water commuters and proximal residents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

TriR serves as a repressor for a resistance-nodulation-cell division (RND) efflux pump TriABC involved in triclosan (TCS) resistance in Agrobacterium tumefaciens. The triR gene is transcribed divergently from the triABC operon. TriR specifically bound to the triR-triA intergenic region, at an imperfect 10 bp inverted repeat, 5'-TTGACTAttC-GgtTAGTCAA-3' (TriR box), that was revealed by DNase I footprinting and electrophoretic mobility shift assay. TCS treatment appeared to up-regulate triR and triABC expression, via preventing TriR binding to the triR-triA intergenic region. Promoter-lacZ fusions and β-galactosidase activity assay further demonstrated TriR-mediated repression of triABC and triR autoregulation. Site-directed mutagenesis confirmed the identified TriR box is essential for TriR repression. A. tumefaciens mutant strains disrupting either triR or triA were constructed to determine their biological functions. The triA mutant showed hypersensitivity to TCS and sodium dodecyl sulfate (SDS), whereas the triR mutant was hyper-resistant, compared to wild-type. In addition to TCS and SDS, overproduction of TriABC from a multi-copy plasmid conferred enhanced resistance to a quaternary ammonium compound, benzalkonium chloride. Molecular modelling was able to predict the model of TriR and docking simulations were able to anticipate plausible binding interactions between TriR and TCS ligand., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Stenotrophomonas maltophilia contains an operon comprising mfsB and mfsC, which encode membrane transporters in the major facilitator superfamily (MFS). The results of the topological analysis predicted that both MfsB and MfsC possess 12 transmembrane helices with the N- and C-termini located inside the cells. The deletion of mfsC increased the susceptibility to diamide, a chemical oxidizing agent, but not to antibiotics and oxidative stress-generating substances relative to wild-type K279a. Moreover, no altered phenotype was observed against all tested substances for the ΔmfsB mutant. The results of the expression analysis revealed that the mfsBC expression was significantly induced by exposure to diamide. The diamide-induced gene expression was mediated by DitR, a TetR-type transcriptional regulator encoded by smlt0547. A constitutively high expression of mfsC in the ditR mutant indicated that DitR acts as a transcriptional repressor of mfsBC under physiological conditions. Purified DitR was bound to three sites spanning from position + 21 to -57, corresponding to the putative mfsBC promoter sequence, thereby interfering with the binding of RNA polymerase. The results of electrophoretic mobility shift assays illustrated that the treatment of purified DitR with diamide caused the release of DitR from the mfsBC promoter region, and the diamide sensing mechanism of DitR required two conserved cysteine residues, Cys92 and Cys127. This suggests that exposure to diamide can oxidize DitR through the oxidation of cysteine residues, leading to its release from the promoter, thus allowing mfsBC transcription. Overall, MfsC and DitR play a role in adaptive resistance against the diamide of S. maltophilia., Competing Interests: The authors have declared that no competing interests exist.

Tilapia tilapinevirus, or Tilapia Lake Virus (TiLV), is a RNA virus associated with mass morbidity and mortality in tilapia, leading to severe economic losses for global tilapia aquaculture. In this study, we investigated the persistence of TiLV in water by spiking sterile distilled water (SDW), freshwater collected from rearing fish tanks (FW) and natural pond water (PW) at 27°C as a representative of environmental water conditions with 0.6 ml of stock virus (3.18 × 10 7 viral copies/ml of water). The water samples were filtered through an electronegative charge membrane and quantified using reverse transcriptase quantitative PCR at 0, 3, 5, 7, 10 and 14 days post-inoculation. The results revealed that TiLV RNA in SDW was reduced by 1.34 log 10 in 14 days. A similar approximately 4 log 10 removal of the virus in FW and PW was observed at 3 and 7 days, respectively. Moreover, the infectivity of TiLV was further studied; the virus lost its infectivity in E-11 cells after 1 day in SDW, FW and PW water samples, even though the virus was spiked 10 more times than in the viral persistence study. Taken together, the results could be applied to improving biosecurity practices in tilapia farms by disinfecting or resting reservoir water for at least three to five days prior to stocking tilapia, to limit the spread of TiLV., (© 2022 John Wiley & Sons Ltd.)

Wastewater surveillance for SARS-CoV-2 RNA has been a successful indicator of COVID-19 outbreaks in populations prior to clinical testing. However, this has been mostly conducted in high-income countries, which means there is a dearth of performance investigations in low- and middle-income countries with different socio-economic settings. This study evaluated the applicability of SARS-CoV-2 RNA monitoring in wastewater (n = 132) to inform COVID-19 infection in the city of Bangkok, Thailand using CDC N1 and N2 RT-qPCR assays. Wastewater influents (n = 112) and effluents (n = 20) were collected from 19 centralized wastewater treatment plants (WWTPs) comprising four large, four medium, and 11 small WWTPs during seven sampling events from January to April 2021 prior to the third COVID-19 resurgence that was officially declared in April 2021. The CDC N1 assay showed higher detection rates and mostly lower Ct values than the CDC N2. SARS-CoV-2 RNA was first detected at the first event when new reported cases were low. Increased positive detection rates preceded an increase in the number of newly reported cases and increased over time with the reported infection incidence. Wastewater surveillance (both positive rates and viral loads) showed strongest correlation with daily new COVID-19 cases at 22-24 days lag (Spearman's Rho = 0.85-1.00). Large WWTPs (serving 432,000-580,000 of the population) exhibited similar trends of viral loads and new cases to those from all 19 WWTPs, emphasizing that routine monitoring of the four large WWTPs could provide sufficient information for the city-scale dynamics. Higher sampling frequency at fewer sites, i.e., at the four representative WWTPs, is therefore suggested especially during the subsiding period of the outbreak to indicate the prevalence of COVID-19 infection, acting as an early warning of COVID-19 resurgence., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Public toilets may increase the risk of COVID-19 infection via airborne transmission; however, related research is limited. We aimed to estimate SARS-CoV-2 infection risk through respiratory transmission using a quantitative microbial risk assessment framework by retrieving SARS-CoV-2 concentrations from the swab tests of 251 Thai patients. Three virus-generating scenarios were investigated: an infector breathing, breathing with a cough, and breathing with a sneeze. The infection risk (95th percentile) was as high as 10 -1 with breathing and increased to 1 with a cough or a sneeze. No significant gender differences for toilet users (receptors) were noted. The highest risk scenario, namely breathing with a sneeze, was further evaluated for risk mitigation measures. Mitigation to a lower risk under 10 -3 succeeded only when the infector and the receptor both wore N95 respirators or surgical masks. Ventilation of up to 20 air changes per hour (ACH) did not decrease the risk. However, an extended waiting time of 10 min between an infector and a receptor resulted in approximately 1.0-log 10 further risk reduction when both wore masks with the WHO-recommended 12 ACH. The volume of expelled droplets, virus concentrations, and receptor dwell time were identified as the main contributors to transmission risk.

The Agrobacterium tumefaciens atu4217 gene, which encodes a TetR family transcription regulator, is a repressor of the atu4218-atu4219-atu4220 operon. The Atu4218 and Atu4219 proteins belong to the HlyD family (membrane fusion protein) and the AcrB/AcrD/AcrF family (inner membrane transporter), respectively, and may form an efflux pump. The atu4220 gene encodes a short-chain dehydrogenase. Quantitative real-time PCR analysis showed induction of atu4217 and atu4218 by NaOCl but not by N-ethylmaleimide or reactive oxygen species (ROS) including H 2 O 2 , menadione and cumene hydroperoxide; therefore, the atu4218 and atu4219 were named NaOCl-inducible efflux genes nieA and nieB, respectively. The atu4217 gene, which was named nieR, serves as a repressor of nieA and nieB. DNase I footprinting assays identified 20-bp imperfect inverted repeat (IR, underlined) motifs 5'-TAGATTTAGGATGCAATCTA-3' (box A) and 5'-TAGATTTCACTTGACATCTA-3' (box R) in the intergenic region of the divergent nieA and nieR genes; these motifs were recognized by the NieR protein. Electrophoretic mobility shift assays demonstrated that NieR specifically binds to the 20-bp IR motifs and that NaOCl prevents this NieR-DNA interaction. Promoter-lacZ fusions and mutagenesis of the NieR boxes (A and R) showed a more dominant role for box A than for box R in the repression of the nieA and nieR promoters. However, full repression of either promoter required both operators. The nieR mutant strain exhibited a small colony phenotype and was more sensitive than the wild-type to NaOCl and antibiotics, including ciprofloxacin, nalidixic acid, novobiocin, and tetracycline. By contrast, the nieAB mutant strain showed no phenotype changes under the tested conditions., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The degradation of coastal water quality from fecal pollution poses a health risk to visitors at recreational beaches. Fecal indicator bacteria (FIB) are a proxy for fecal pollution; however the accuracy of their representation of fecal pollution health risks at recreational beaches impacted by non-point sources is disputed due to non-human derivation. This study aimed to investigate the relationship between FIB and a range of culturable and molecular-based microbial source tracking (MST) markers and pathogenic bacteria, and physicochemical parameters and rainfall. Forty-two marine water samples were collected from seven sampling stations during six events at two tourist beaches in Thailand. Both beaches were contaminated with fecal pollution as evident from the GenBac3 marker at 88%-100% detection and up to 8.71 log 10 copies/100 mL. The human-specific MST marker human polyomaviruses JC and BK (HPyVs) at up to 4.33 log 10 copies/100 mL with 92%-94% positive detection indicated that human sewage was likely the main contamination source. CrAssphage showed lower frequencies and concentrations; its correlations with the FIB group (i.e., total coliforms, fecal coliforms, and enterococci) and GenBac3 diminished its use as a human-specific MST marker for coastal water. Human-specific culturable AIM06 and SR14 bacteriophages and general fecal indicator coliphages also showed less sensitivity than the human-specific molecular assays. The applicability of the GenBac3 endpoint PCR assay as a lower-cost prescreening step prior to the GenBac3 qPCR assay was supported by its 100% positive predictive value, but its limited negative predictive values required subsequent qPCR confirmation. Human enteric adenovirus and Vibrio cholerae were not found in any of the samples. The HPyVs related to Vibrio parahaemolyticus, Vibrio vulnificus, and 5-d rainfall records, all of which were more prevalent and concentrated during the wet season. More monitoring is therefore recommended during wet periods. Temporal differences but no spatial differences were observed, suggesting the need for a sentinel site at each beach for routine monitoring. The exceedance of FIB water quality standards did not indicate increased prevalence or concentrations of the HPyVs or Vibrio spp. pathogen group, so the utility of FIB as an indicator of health risks at tropical beaches maybe challenged. Accurate assessment of fecal pollution by incorporating MST markers could lead to developing a more effective water quality monitoring plan to better protect human health risks in tropical recreational beaches., (Copyright © 2021. Published by Elsevier Ltd.)

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are pollutants of worldwide concern that threaten human health and ecosystems. Anthropogenic activities and wastewater could be ARB and ARG pollution sources; however, research on ARG abundance and microbial source tracking (MST) of contamination in tropical marine waters is limited. This study examined spatiotemporal variations of six ARGs (bla NDM , bla TEM , bla VIM , mcr-1, sul1, and tetQ) against the widely used antibiotic groups and a class 1 integron-integrase gene (intI1) at two Thai tropical recreational beaches (n = 41). Correlations between ARGs and sewage-specific MST markers (i.e., crAssphage and human polyomaviruses [HPyVs]) and fecal indicator bacteria (i.e., total coliforms, fecal coliforms, and enterococci) were also investigated. Bla TEM , intI1, sul1, and tetQ were ubiquitous at both beaches (85.4-100% detection rate); intI1 was the most abundant (3-6 orders in log 10 copies/100 mL), followed by bla TEM (2-4 orders), sul1 (2-3 orders), and tetQ (2-4 orders). Bla NDM was found in 7.3% (up to 4 orders), and no mcr-1 was detected. Interestingly, bla VIM was prevalent at one beach (2-5 orders; n = 17), but found in only one sample at the other (4 orders). Temporal, but not spatial, differences were noticed; bla TEM was at higher levels in the wet season. IntI1 correlated with sul1 and tetQ (Spearman's rho = 0.47-0.97), suggesting potential horizontal gene transfer. CrAssphage, but not HPyVs, correlated with intI1, sul1, and tetQ (Spearman's rho = 0.50-0.74). Higher numbers of ARGs tended to co-occur in samples with higher crAssphage concentrations, implying sewage contribution to the marine water, with a persisting ARG background. This study provides insight into the ARG pollution status of tropical coastal waters and suggests crAssphage as a proxy for ARG pollution, which could facilitate effective management policies to minimize ARG dissemination in marine environments., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

A gene encoding the TetR-type transcriptional regulator mfsR is located immediately downstream of mfsQ and is transcribed in the same transcriptional unit. mfsQ encodes a major facilitator superfamily (MFS) efflux transporter contributing to the resistance of Stenotrophomonas maltophilia towards disinfectants belonging to quaternary ammonium compounds (QACs), which include benzalkonium chloride (BAC). Phylogenetic analysis revealed that MfsR is closely related to CgmR, a QAC-responsive transcriptional regulator belonging to the TetR family. MfsR regulated the expression of the mfsQR operon in a QAC-inducible manner. The constitutively high transcript level of mfsQ in an mfsR mutant indicated that MfsR functions as a transcriptional repressor of the mfsQR operon. Electrophoretic mobility shift assays showed that purified MfsR specifically bound to the putative promoter region of mfsQR, and in vitro treatments with QACs led to the release of MfsR from binding complexes. DNase I protection assays revealed that the MfsR binding box comprises inverted palindromic sequences located between motifs -35 and -10 of the putative mfsQR promoter. BAC-induced adaptive protection was abolished in the mfsR mutant and was restored in the complemented mutant. Overall, MfsR is a QACs-sensing regulator that controls the expression of mfsQ. In the absence of QACs, MfsR binds to the box located in the mfsQR promoter and represses its transcription. The presence of QACs derepresses MfsR activity, allowing RNA polymerase binding and transcription of mfsQR. This MfsR-MsfQ system enables S. maltophilia to withstand high levels of QACs., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cytolethal distending toxin (CDT) is a bacterial genotoxin that causes host cell cycle arrest and death. We previously employed a Saccharomyces cerevisiae model with inducible expression of the CDT catalytic subunit from Aggregatibacter actinomycetemcomitans, Aa CdtB, and showed that a wide variety of host factors play a role in facilitating the activity of CdtB. Our observation that a yeast H2B mutant defective in chromatin condensation was partially resistant to CdtB implies that chromatin structure may affect CDT function. In this study, we identified host chromatin regulatory genes required for CdtB cytotoxicity. We found that the deletion of HTZ1 or certain subunits of SWR, INO80, and SIR complexes increased cellular resistance to CdtB. We hypothesized that CdtB may interact with Htz1 or the chromatin, but immunoprecipitation experiments failed to detect physical interaction between CdtB and Htz1 or the chromatin. However, we observed reduced nuclear localization of CdtB in several mutants, suggesting that impaired nuclear translocation may, at least partly, explain the mechanisms of CdtB resistance. In addition, mutations in chromatin regulatory genes induce changes in the global gene expression profile, and these may indirectly affect CdtB toxicity. Our results suggest that decreased expression of endoplasmic reticulum (ER)-Golgi transport-related genes that may be involved in CdtB transport and/or increased expression of DNA repair genes may contribute to CdtB resistance. These results suggest that the functions of chromatin regulators may contribute to the activity of CDT in host cells.

Anthropogenically impacted urban canals represent distinct freshwater ecosystems that could shape microbial communities in underlying sediments; however, knowledge of the relationships between environmental factors and microbial community compositions and their functions in such an environment is limited. This study characterized the microbial community compositions of malodorous canal sediments at six locations along the Saen Saep Canal in Thailand. 16S rRNA gene amplicon sequencing (MiSeq, Illumina) revealed dominant genera classified as fermentative bacteria, methanogens, and sulfate-reducing bacteria (SRB), all of which emphasized anaerobic environments. SRB, as the primary producers of malodorous hydrogen sulfide, accounted for 8.2-30.4% of the total sequences. dsrB gene clone libraries further identified the SRB species. A constrained correspondence analysis demonstrated a spatial pattern of SRB that correlated with physicochemical parameters in which nitrate and sulfate in sediments were the most influencing factors. Overall, a better understanding of the SRB and other related microorganisms in canal sediments can assist in the future implementation of appropriate olfactory abatement and management methodologies in urban canals.

The persistence of Stenotrophomonas maltophilia , especially in hospital environments where disinfectants are used intensively, is one of the important factors that allow this opportunistic pathogen to establish nosocomial infections. In the present study, we illustrated that S .  maltophilia possesses adaptive resistance to the disinfectant benzalkonium chloride (BAC). This BAC adaptation was abolished in the Δ mfsQ mutant, in which a gene encoding an efflux transporter belonging to the major facilitator superfamily (MFS) was deleted. The Δ mfsQ mutant also showed increased susceptibility to BAC and chlorhexidine gluconate compared with a parental wild type. The expression of mfsQ increased upon exposure to quaternary ammonium compounds, including BAC. Thus, the results of this study suggest that mfsQ plays a role in both adaptive and nonadaptive protection of S . maltophilia from the toxicity of the disinfectant BAC.

Talaromyces marneffei is a thermally dimorphic fungus that causes opportunistic systemic mycoses in patients with AIDS or other immunodeficiency syndromes. The purpose of this study was to develop an immunochromatographic strip test (ICT) based on a solid phase sandwich format immunoassay for the detection of T. marneffei antigens in clinical urine specimens. The T. marneffei yeast phase specific monoclonal antibody 4D1 (MAb4D1) conjugated with colloidal gold nanoparticle was used as a specific signal reporter. Galanthus nivalis Agglutinin (GNA) was adsorbed onto nitrocellulose membrane to serve as the test line. Similarly, a control line was created above the test line by immobilization of rabbit anti-mouse IgG. The immobilized GNA served as capturing molecule and as non-immune mediated anti-terminal mannose of T. marneffei antigenic mannoprotein. The MAb4D1-GNA based ICT showed specific binding activity with yeast phase antigen of T. marneffei, and it did not react with other common pathogenic fungal antigens. The limit of detection of this ICT for T. marneffei antigen spiked in normal urine was approximately 0.6 μg/ml. The diagnostic performance of the ICT was validated using 341 urine samples from patents with culture- confirmed T. marneffei infection and from a control group of healthy individuals and patients with other infections in an endemic area. The ICT exhibited 89.47% sensitivity, 100% specificity, and 97.65% accuracy. Our results demonstrate that the urine-based GNA-MAb4D1 based ICT produces a visual result within 30 minutes and that the test is highly specific for the diagnosis of T. marneffei infection. The findings validate the deployment of the ICT for clinical use., Competing Interests: The authors have declared that no competing interests exist.

Rapid economic development has caused industrial expansion into residential communities, leading to higher fecal pollution loads that could be discharged into aquatic environments. However, little is known regarding the potential microbial impact on human health. This study investigated microbial contamination from coastal industrial-residential community areas in nine sampling sites in waterways during three dry events. A general microbial source tracking (MST) marker, GenBac3, was detected in all samples from all three events, indicating continuing fecal pollution in the area, mostly from human sewage contamination. This was shown by the human-specific genetic marker crAssphage (88.9%) and human polyomavirus (HPyVs; 92.6%) detection. Enteric human adenovirus (HAdV40/41) showed three positive results only from residential sites in the first event. No spatial difference was observed for MST markers and traditional fecal indicators (total coliforms and Escherichia coli ) in each event. Still, a significantly lower abundance of GenBac3, HPyVs, and total coliforms in the first sampling event was detected. Spearman's rho analysis indicated a strong correlation among certain pairs of microbial parameters. Multivariate analysis revealed two clusters of samples separated by land use type (industrial vs. residential). According to factor analysis of mixed data, the land use parameter was more associated with physicochemical parameters (i.e., salinity, conductivity, water temperature, and dissolved oxygen). A Quantitative Microbial Risk Assessment (QMRA) was then conducted to estimate the annual infection risks of HAdV40/41 for non-potable water reuse purposes using predicted concentrations from crAssphage and HPyVs. The highest risks (95th percentiles) were ranked by food crop irrigation, aquaculture, and toilet flushing, at 10 -1 , 10 -2 , and 10 -3 per person per year (pppy). Required treatment levels to achieve a 10 -4 pppy annual infection risk were estimated. QMRA-based water treatment scenarios were suggested, including chlorination for toilet flushing reuse and depth filtration prior to chlorination for aquaculture and food crop irrigation. Microbial monitoring combined with a QMRA could provide better insights into fecal pollution patterns and the associated risks, facilitating effective water quality management and appropriate prior treatments for water reuse., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kongprajug, Denpetkul, Chyerochana, Mongkolsuk and Sirikanchana.)

Identifying sewage contamination via microbial source tracking (MST) marker genes has proven useful for effective water quality management worldwide; however, performance evaluations for these marker genes in tropical areas are limited. Therefore, this research evaluated four human-associated MST marker genes (human polyomaviruses (JC and BK viruses [HPyVs]), bacteriophage crAssphage (CPQ&#95;056), Lachnospiraceae Lachno3, and Bacteroides BacV6-21) for tracking sewage pollution in aquatic environments of Thailand. The viral marker genes, HPyV and crAssphage were highly sensitive and specific to sewage from onsite wastewater treatment plants (OWTPs; n = 19), with no cross-detection in 120 composite swine, cattle, chicken, duck, goat, sheep, and buffalo fecal samples. The bacterial marker genes, Lachno3 and BacV6-21, demonstrated high sensitivity but moderate specificity; however, using both markers could improve specificity to >0.80 (max value of 1.00). The most abundant markers in OWTP samples were Lachno3 and BacV6-21 (5.42-8.02 and nondetect-8.05 log 10 copies/100 mL), crAssphage (5.28-7.38 log 10 copies/100 mL), and HPyVs (3.66-6.53 log 10 copies/100 mL), respectively. Due to their increased specificity, the abundance of viral markers were further investigated in environmental waters, in which HPyVs showed greater levels (up to 4.33 log 10 copies/100 mL) and greater detection rates (92.7%) in two coastal beaches (n = 41) than crAssphage (up to 3.51 log 10 copies/100 mL and 56.1%). HPyVs were also found at slightly lower levels (up to 5.10 log 10 copies/100 mL), but at higher detection rates (92.6%), in a freshwater canal (n = 27) than crAssphage (up to 5.21 log 10 copies/100 mL and 88.9%). HPyVs and crAssphage marker genes were identified as highly sensitive and specific for tracking sewage pollution in aquatic environments of Thailand. This study underlines the importance of characterizing and validating MST markers in host groups and environmental waters before including them in a water quality management toolbox., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Objectives: Antibacterial resistance (ABR) is a major global health security threat, with a disproportionate burden on lower-and middle-income countries (LMICs). It is not understood how 'One Health', where human health is co-dependent on animal health and the environment, might impact the burden of ABR in LMICs. Thailand's 2017 "National Strategic Plan on Antimicrobial Resistance" (NSP-AMR) aims to reduce AMR morbidity by 50% through 20% reductions in human and 30% in animal antibacterial use (ABU). There is a need to understand the implications of such a plan within a One Health perspective., Methods: A model of ABU, gut colonisation with extended-spectrum beta-lactamase (ESBL)-producing bacteria and transmission was calibrated using estimates of the prevalence of ESBL-producing bacteria in Thailand. This model was used to project the reduction in human ABR over 20 years (2020-2040) for each One Health driver, including individual transmission rates between humans, animals and the environment, and to estimate the long-term impact of the NSP-AMR intervention., Results: The model predicts that human ABU was the most important factor in reducing the colonisation of humans with resistant bacteria (maximum 65.7-99.7% reduction). The NSP-AMR is projected to reduce human colonisation by 6.0-18.8%, with more ambitious targets (30% reductions in human ABU) increasing this to 8.5-24.9%., Conclusions: Our model provides a simple framework to explain the mechanisms underpinning ABR, suggesting that future interventions targeting the simultaneous reduction of transmission and ABU would help to control ABR more effectively in Thailand., Competing Interests: Declarations of interest: none, (© 2021 The Authors.)

Pseudomonas aeruginosa , a well - known cause of nosocomial infection, is frequently antibiotic resistant and this complicates treatment. Links between oxidative stress responses inducing antibiotic resistance through over - production of RND - type efflux pumps have been reported in P. aeruginosa , but this has not previously been associated with MFS - type efflux pumps. Two MFS efflux pumps encoded by mfs1 and mfs2 were selected for study because they were found to be sodium hypochlorite (NaOCl) inducible. Antibiotic susceptibility testing was used to define the importance of these MFS pumps in antibiotic resistance and proteomics was used to characterize the resistance mechanisms involved. The results revealed that mfs1 is NaOCl inducible whereas mfs2 is NaOCl, N - Ethylmaleimide and t - butyl hydroperoxide inducible. Deletion of mfs1 or mfs2 did not affect antibiotic or paraquat susceptibility. However, over - production of Mfs1 and Mfs2 reduced susceptibility to aminoglycosides, quinolones, and paraquat. Proteomics, gene expression analysis and targeted mutagenesis showed that over - production of the MexXY RND - type efflux pump in a manner dependent upon armZ , but not amgRS , is the cause of reduced antibiotic susceptibility upon over - production of Mfs1 and Mfs2. mexXY operon expression analysis in strains carrying various lengths of mfs1 and mfs2 revealed that at least three transmembrane domains are necessary for mexXY over - expression and decreased antibiotic susceptibility. Over-expression of the MFS-type efflux pump gene tetA (C) did not give the same effect. Changes in paraquat susceptibility were independent of mexXY and armZ suggesting that it is a substrate of Mfs1 and Mfs2. Altogether, this is the first evidence of cascade effects where the over - production of an MFS pump causes over - production of an RND pump, in this case MexXY via increased armZ expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dulyayangkul, Satapoomin, Avison, Charoenlap, Vattanaviboon and Mongkolsuk.)

Capsid integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-one recent peer-reviewed studies employing capsid integrity qPCR for viruses in the last decade (2009-2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 μM; incubation on ice or at room temperature (20 - 25 °C) for 5-120 min; and dye activation using LED or high light (500-800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

A formylglycine-generating enzyme (FGE)-sulfatase-based whole-cell biosensor was genetically improved into a single-copy system by integrating the Sinorhizobium meliloti transcriptional activator ChpR and the chpA promoter-FGE-sulfatase fusion into the Escherichia coli chromosome. The sensitivity was further enhanced through a random mutagenesis of the chpR . The new integrated biosensor offered both a lower detection limit [5 nM chlorpyrifos (CPF)] and fluorescence background. The ready-to-use kit was developed using silica gel for on-field detection. The biosensor kit was stable for 20 days when stored at 4 °C. Moreover, a 1-(1-naphthylmethyl)-piperazine (NMP) efflux pump inhibitor can improve the sensitivity by 57 %.

Reactive chlorine species (RCS), particularly hypochlorous acid (HOCl), are powerful antimicrobial oxidants generated by biological pathways and chemical syntheses. Pseudomonas aeruginosa is an important opportunistic pathogen that has adapted mechanisms for protection and survival in harsh environments, including RCS exposure. Based on previous transcriptomic studies of HOCl exposure in P. aeruginosa , we found that the expression of PA0565 , or rcsA , which encodes an alkyl hydroperoxidase D-like protein, exhibited the highest induction among the RCS-induced genes. In this study, rcsA expression was dominant under HOCl stress and greatly increased under HOCl-related stress conditions. Functional analysis of RcsA showed that the distinguishing core amino acid residues Cys60, Cys63, and His67 were required for the degradation of sodium hypochlorite (NaOCl), suggesting an extended motif in the AhpD family. After allelic exchange mutagenesis in the P. aeruginosa rcsA , the P. aeruginosa rcsA deletion mutant showed significantly decreased HOCl resistance. Ectopic expression of P. aeruginosa rcsA led to significantly increased NaOCl resistance in Escherichia coli Moreover, the pathogenicity of the rcsA mutant decreased dramatically in both Caenorhabditis elegans and Drosophila melanogaster host model systems compared to the wild type (WT). Finally, the Cys60, Cys63, and His67 variants of RcsA were unsuccessful at complementing phenotypes of the rcsA mutant. Overall, our data indicate the importance of P. aeruginosa RcsA in defense against HOCl stress under disinfections and during infections of hosts, which involves the catalytic Cys60, Cys63, and His67 residues. IMPORTANCE Pseudomonas aeruginosa is a common pathogen that is a major cause of serious infections in many hosts. Hypochlorous acid (HOCl) is a potent antimicrobial agent found in household bleach and is a widely used disinfectant. P. aeruginosa has evolved adaptive mechanisms for protection and survival during HOCl exposure. We identified P. aeruginosa rcsA as a HOCl-responsive gene encoding an antioxidant protein that may be involved in HOCl degradation. RcsA has a distinguishing core motif containing functional Cys60, Cys63, and His67 residues. P. aeruginosa rcsA plays an important role in bleach tolerance, with expression of P. aeruginosa rcsA in Escherichia coli also conferring HOCl resistance. Interestingly, RcsA is required for full virulence in worm and fruit fly infection models, indicating a correlation between mechanisms of bleach toxicity and host immunity during infection. This provides new insights into the mechanisms used by P. aeruginosa to persist in harsh environments such as hospitals., (Copyright © 2020 American Society for Microbiology.)

Candida albicans is an opportunistic yeast that can cause life-threatening systemic infection in immunocompromised individuals. During infections, C. albicans has to cope with genotoxic stresses generated by the host immune system. DNA-protein crosslink (DPC), the covalent linkage of proteins with DNA, is one type of DNA damages that can be caused by the host immune response. DPCs are bulky lesions that interfere with the progression of replication and transcription machineries, and hence threaten genomic integrity. Accordingly, either a DPC tolerance mechanism or a DPC repair pathway is essential for C. albicans to maintain genomic stability and survive in the host. Here, we identified Wss1 (weak suppressor of Smt3) in C. albicans (CaWss1) using bioinformatics, genetic complementation, and biochemical studies. We showed that CaWss1 promotes cell survival under genotoxic stress conditions that generate DPCs and that the catalytic metalloprotease domain of CaWss1 is essential for its cellular function. Interactions of CaWss1 with Cdc48 and small ubiquitin-like modifier, although not strictly required, contribute to the function of CaWss1 in the suppression of the growth defects under DPC-inducing conditions. This report is the first investigation of the role of CaWss1 in DPC tolerance in C. albicans., (© 2020 John Wiley & Sons Ltd.)

Bisphenol A (BPA) is one of the most common toxic endocrine disruptors in the environment. A fast, efficient and environmental-friendly method for BPA detoxification is urgently needed. In this study, we show that the enzymatic transformation of BPA into a non-estrogenic BPA sulfate can be performed by the aryl sulfotransferase (ASTB) from Desulfitobacterium hafniense. We developed and compared two Escherichia coli ASTB cell-surface displaying systems using the outer membrane porin F (OprF) and the lipoprotein outer membrane A (Lpp-OmpA) as carriers. The surface localization of both fusion proteins was confirmed by Western blot and flow cytometry analysis as well as the enzymatic activity assay of the outer membrane fractions. Unfortunately, Lpp-OmpA-ASTB cells had an adverse effect on cell growth. In contrast, the OprF-ASTB cell biocatalyst was stable, expressing 70% of enzyme activity for 7 days. It also efficiently sulfated 90% of 5 mM BPA (1 mg/mL) in wastewater within 6 h., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

2,4-Dichlorophenoxyacetic acid (2,4-D) has been widely used as a herbicide for agricultural purposes. Currently, the available methods for detecting 2,4-D require multi-step sample preparations and expensive instruments. The use of a whole cell biosensor is an interesting approach that is straightforward and simple to use. In this study, we constructed a genetic-based Agrobacterium tumefaciens biosensor based on a cadA promoter and cadR regulator from Bradyrhizobium sp. strain HW13 (2,4-D degrader) with a formylglycine generating enzyme (FGE)-sulfatase as the reporter gene. The performance of the biosensor was further improved through direct evolution of the cadR activator. The detection limit of cadR mutants for phenoxyacetic acid herbicides including 2,4-D and 4-Chlorophenoxyacetic acid (4-CPAA) were 1.56 μM (an eight-fold improvement compared to wild-type CadR). The biosensor could detect 2,4-D contamination in environmental samples without encountering interference from other complex compounds. The Agrobacterium biosensor was also stable after storing in a simple Luria-Bertani (LB) medium at 4 °C for 30 days where the activity remained at 82% when exposed to 100 μM of 2,4-D. This novel biosensor, with its high stability under simple storage conditions, exhibits promising potential to be used as an inexpensive and easy-to-use tool to screen for 2,4-D contamination in environmental sources., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The widely used microbial source tracking (MST) technique, quantitative polymerase chain reaction (qPCR), quantifies host-specific gene abundance in polluted water to identify and prioritize contamination sources. This study characterized the effects of a qPCR data analysis using the sample PCR efficiencies (the LinRegPCR model) on gene abundance and compared them with the standard curve-based method (the mixed model). Five qPCR assays were evaluated: the universal GenBac3, human-specific HF183/BFDrev and CPQ&#95;056, swine-specific Pig-2-Bac, and cattle-specific Bac3qPCR assays. The LinRegPCR model increased the low-copy amplification, especially in the HF183/BFDrev assay, thus lowering the specificity to 0.34. Up to 1.41 log 10 copies/g and 0.41 log 10 copies/100 mL differences were observed for composite fecal and sewage samples ( n = 147) by the LinRegPCR approach, corresponding to an 18.2% increase and 6.4% decrease, respectively. Freshwater samples ( n = 48) demonstrated a maximum of 1.95 log 10 copies/100 mL difference between the two models. Identical attributing sources by both models were shown in 54.55% of environmental samples; meanwhile, the LinRegPCR approach improved the inability to identify sources by the mixed model in 29.55% of the samples. This study emphasizes the need for a standardized data analysis protocol for qPCR MST assays for interlaboratory consistency and comparability.

Typically, biological systems are protected from the toxic effect of free radicals by antioxidant defense. Extracts from orchids have been reported to show high levels of exogenous antioxidant activity including Bulbophyllum orchids but so far, there have been no reports on antioxidant enzymes. Therefore, differences in protein expression from leaves and pseudobulbs of Bulbophyllum morphologlorum Kraenzl and Dendrobium Sonia Earsakul were studied using two-dimensional gel electrophoresis and mass spectrometry (LC/MS/MS). Interestingly, the largest group of these stress response proteins were associated with antioxidant defense and temperature stress, including superoxide dismutase (Cu-Zn) and heat shock protein 70. The high expression of this antioxidant enzyme from Bulbophyllum morphologlorum Kraenzl was confirmed by activity staining on native-PAGE, and the two Cu/Zn-SODs isoenzymes were identified as Cu/Zn-SOD 1 and Cu/Zn-SOD 2 by LC/MS/MS. The results suggested that Bulbophyllum orchid can be a potential plant source for medicines and natural antioxidant supplements., Competing Interests: The authors declared that they have no conflict of interest., (© 2020 The Author(s).)

The bacteriophages of E. faecalis strains AIM06 (DSM100702) and SR14 (DSM100701) have previously been validated as human-specific microbial source tracking (MST) markers in Thailand. In this study, their spatial and temporal distribution in a freshwater river was investigated for the first time (n = 48). The abundance of enterococci as a standard microbial water quality parameter was evaluated by both the qPCR detection assay with primers and a hydrolysis probe according to the US EPA Method 1611 and the US EPA Method 1600 membrane filtration culture method. AIM06 and SR14 phages were detected by a double layer agar assay and were present in 87.5% and 81.3% of all samples with a co-presence of 92.9% of phage-positive samples. After spiking the representative phages, the ranges of recovery efficiencies were 57.9-99.6% and 49.6-99.9% (n = 48) for AIM06 and SR14 phages, respectively. The absolute abundance of AIM06 and SR14 phages ranged from 0.25 to 221.94 and from 0.25 to 76.66 PFU/100 mL, respectively. Enterococci DNA copies and CFU were detected in all samples ranging from 3.24 to 6.32 log10 copies/100 mL and 100.00 to 1593 CFU/100 mL, respectively. Enterococci in the qPCR assay also showed a moderate correlation with the culture method. The AIM06 and SR14 phage results indicated continuing human faecal pollution along the river with no significant different levels among stations. Interestingly, the higher levels of enterococci in downstream stations for both the qPCR and culture methods along with the significant correlation with other faecal indicator organisms and non-human MST markers implied non-human faecal pollution. In conclusion, this study provides insightful information that could lead to effective water quality management and public health risk reduction from exposure to faecally-contaminated water., Competing Interests: Declaration of competing interest The author(s) declare(s) that there is no conflict of interest regarding the publication of this article., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Zinc uptake regulator (Zur) is a transcriptional regulator that represses zinc acquisition genes under high zinc conditions. The aim of this study was to identify and investigate the role of Zur-binding motifs (Zur boxes) in the differential regulation of Zur target genes, including the zinT, znuA, znuCB-zur operon, the troCBA operon, and yciC, in Agrobacterium tumefaciens. DNase I footprinting and gel shift assays were performed, confirming that Zur directly binds to 18-bp inverted repeat motifs found in the promoter of these Zur-regulated genes. Furthermore, promoter-lacZ fusions and mutagenesis of the identified Zur boxes were performed to assess the role of each Zur box. A Zur box found in the zinT promoter was required for zinc-dependent repression by Zur. The intergenic region between the znuA gene and the znuCB-zur operon contains two Zur boxes, named A and C, which immediately precede the genes znuA and znuC, respectively. Zur box A, but not Zur box C, was essential for the repression of the znuA promoter. Both Zur boxes A and C were implicated in the repression of the znuC promoter, in which mutation of either box alone was sufficient for full derepression of the znuC promoter. Three Zur boxes named T, M, and Y were identified in the intergenic region between the troCBA operon and the yciC gene. Zur box Y, which immediately precedes yciC, was shown to be responsible for Zur repression of the yciC promoter. In contrast, two Zur boxes, T and M, were essential for the complete repression of the troCBA operon, and full derepression of the troC promoter was exhibited when both Zur boxes were mutated simultaneously. Sequence analysis of the identified Zur boxes revealed a correlation between deviation from the core recognition sequence of the Zur box and the requirement of two Zur boxes for Zur regulation of distinctive promoters.

The occurrence of antibiotic resistance bacteria has become a major threat to public health. We have recently discovered a transcriptional activator that belongs to MarR family, EstR, and an esterase B (EstB) with a newly proposed de-arenethiolase activity from Sphingobium sp. SM42. De-arenethiolase activity involves the removal of the small aromatic side chain of cephalosporin antibiotics as an excellent leaving group by the enzymatic CS bond cleavage. Here, we report the regulation of estB through EstR as an activator in response to a third generation cephalosporin, cefoperazone, antibiotic. Cefoperazone induced the expression of estB in wild type Sphingobium sp., but not in the estR knockout strain, and the induction was restored in the complemented strain. Moreover, we revealed the importance of EstB localization in periplasm. Since EsB has the ability to inactivate selected β-lactam antibiotics in vitro, it is possible that the enzyme works at the periplasmic space of Gram negative bacteria similar to β-lactamases. EstB was genetically engineered by incorporating NlpA binding motif, or OmpA signal sequence, or SpyTag-SpyCatcher to the estB gene to mobilize it to different compartments of periplasm; inner membrane, outer membrane, and periplasmic space, respectively. Surprisingly, we found that Sphingobium sp. SM42 and E. coli expressing EstB at the periplasm were more sensitive to cefoperazone. The possible drug enhancement mechanism by enzyme was proposed. This work might lead to a novel strategy to tackle antibiotic resistance problem., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

An understanding of microbial communities present in anaerobic bioreactors can strongly facilitate the development of approaches to control undesirable microorganisms, such as sulfate-reducing bacteria (SRB), in the system. In this study, overall microbial communities present in anaerobic bioreactors from seven industrial wastewater treatment plants (including food, pulp and paper industries) were investigated using 16S rRNA gene amplicon sequencing (MiSeq, Illumina). The dominant methanogens identified in the anaerobic bioreactors treating industrial wastewater were Methanobacterium and Methanosaeta ; Methanospirillum was a predominant methanogen in the anaerobic sludge digester. Hydrogenotrophic and acetoclastic methanogens were detected at similar relative abundances in the anaerobic covered lagoons treating starch wastewater, whereas hydrogenotrophic methanogens were the predominant methanogens present in the sludge digester. SRB communities were further investigated using dsrB gene clone libraries. The results indicated the presence of SRB, such as uncultured Desulfobulbus sp., Syntrophobacter fumaroxidans , Syntrophorhabdus sp. PtaB.Bin027, and Desulfovibrio fructosivarans JJ. Incomplete-oxidizing SRB were the predominant SRB in all of the anaerobic bioreactors treating wastewater. In contrast, similar relative abundances of complete and incomplete-oxidizing SRB were observed in the sludge digester. The results of this study can further facilitate the development of SRB-controlling strategies to improve the efficiency of wastewater treatment.

Pinpointing water pollution sources using host-specific gastrointestinal microbes, known as microbial source tracking (MST), have significant benefits for countries with water quality management issues related to pollution. A recently discovered bacteriophage, crAssphage, shows promise as a human-specific MST marker. However, loss of genetic materials during the recovery and the detection processes could alter the ability to measure virus quantities in a water sample. This study determined the crAssphage recovery efficiencies in water sources, including seawater, freshwater, and influent and effluent from a wastewater treatment plant, by spiking natural crAssphage concentrates prior to DNA extraction and quantitative PCR analysis. The results showed that river and seawater with no or low crAssphage background experienced no recovery loss. Evaluating recovery efficiencies in samples with high crAssphage backgrounds posed a challenge due to the inability to prepare high crAssphage titers. This study highlights the importance of intra-laboratory assessment of recovery efficiency in environmental samples for retrieving absolute crAssphage quantification with correction of bias among water samples and increase in data accuracy. PRACTITIONER POINTS: In laboratory assessment of recovery efficiency is crucial for bias correction and data accuracy for absolute crAssphage quantification in water samples. No loss in crAssphage recovery was observed in river and seawater that contained no or low crAssphage backgrounds. Inability to prepare high crAssphage spike concentrations remains the major limitation for evaluating recovery in samples with high crAssphage backgrounds. The results underline the importance of evaluating method recovery in real environmental samples that reflect actual matrix effect. Absolute crAssphage quantification, as human-specific pollution marker, could be used for prioritizing water quality restoration and area-based management plan., (© 2019 Water Environment Federation.)

A late detection of pathogenic microorganisms in food and drinking water has a high potential to cause adverse health impacts in those who have ingested the pathogens. For this reason there is intense interest in developing precise, rapid and sensitive assays that can detect multiple foodborne pathogens. Such assays would be valuable components in the campaign to minimize foodborne illness. Here, we discuss the emerging types of assays based on gold nanoparticles (GNPs) for rapidly diagnosing single or multiple foodborne pathogen infections. Colorimetric and lateral flow assays based on GNPs may be read by the human eye. Refractometric sensors based on a shift in the position of a plasmon resonance absorption peak can be read by the new generation of inexpensive optical spectrometers. Surface-enhanced Raman spectroscopy and the quartz microbalance require slightly more sophisticated equipment but can be very sensitive. A wide range of electrochemical techniques are also under development. Given the range of options provided by GNPs, we confidently expect that some, or all, of these technologies will eventually enter routine use for detecting pathogens in food. This article is categorized under: Diagnostic Tools > Biosensing., (© 2019 Wiley Periodicals, Inc.)

The tRNA (m 1 G37) methyltransferase TrmD catalyzes m 1 G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa ( Pa TrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of Pa TrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as Pa TrmD substrates. Analysis of steady-state kinetics with S -adenosyl-l-methionine (SAM) and tRNA Leu(GAG) showed that Pa TrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNA Leu(GAG) bind to Pa TrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM ( K i = 0.41 ± 0.07 µM) and uncompetitive for tRNA ( K i = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric Pa TrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into Pa TrmD as a potential target for the development of antibiotics., (© 2019 Jaroensuk et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The Agrobacterium tumefaciens ActSR two-component regulatory system is a member of a homologous group of global redox-responsive regulatory systems that adjust the expression of energy-consuming and energy-supplying metabolic pathways in order to maintain cellular redox balance. In this study, the transcriptional organization of the hrpB-actSR locus was determined and the effect of actSR system inactivation on stress resistance was investigated. It was found that hrpB is transcribed as a monocistronic mRNA and actS is transcribed along with actR as a bicistronic mRNA, while actR is also transcribed as a monocistronic message. Each message is initiated from a separate promoter. Inactivation of actR resulted in decreased resistance to membrane stress (sodium dodecyl sulfate), acid stress (pH 5.5), iron starvation (bipyridyl) and iron excess (FeCl 3 ), and antibiotic stress (tetracycline and ciprofloxacin). Resistance to oxidative stress in the form of organic peroxide (cumene hydroperoxide) increased, while resistance to inorganic peroxide (H 2 O 2 ) decreased. An actR insertion mutant displayed reduced catalase activity, even though transcription of katA and catE remained unchanged. Complementation of the actR inactivation mutant with plasmid-encoded actR or overexpression of hemH , encoding ferrochelatase, restored wild-type catalase activity and H 2 O 2 resistance levels. Gel mobility shift and hemH promoter -lacZ fusion results indicated that ActR is a positive regulator of hemH that binds directly to the hemH promoter region. Thus, inactivation of the A. tumefaciens ActSR system affects resistance to multiple stresses, including reduced resistance to H 2 O 2 resulting from a reduction in catalase activity due to reduced expression of hemH .

Tilapia lake virus (TiLV) is an emerging virus associated with high fish mortality and economic losses. This study investigates the virucidal effects of the following disinfectants (active ingredients) on TiLV: 2.5 ppm iodine, 10 ppm sodium hypochlorite (NaOCl), 300 ppm hydrogen peroxide (H 2 O 2 ), 80 ppm formalin and 5,000 ppm (0.5%) Virkon ® . Factors that affect the disinfectants' efficacy, including temperature, contact time and soiling (organic matter) interference, were examined under conditions mimicking natural aquaculture practices. TiLV inactivation of higher than 5 log 10 TCID 50  ml -1 was achieved after 10 min and at 28°C for all disinfectants except formalin; similar inactivation levels were reached by NaOCl and Virkon ® at 10 min and 4°C. Extended exposure to formalin from 10 to 60 min at 28°C rendered more than 5 log 10 inactivation. Increasing synthetic organic matter in the water to mimic soiling interference reduced the efficacy of NaOCl, iodine and H 2 O 2 when tested at 10 min and 28°C; however, Virkon ® still achieved more than 5 log 10 inactivation. This study demonstrates that most common disinfectants effectively reduced viral loads to minimum levels. To limit the spread of TiLV in aquaculture farms and related facilities, the appropriate use of such disinfectants should therefore be promoted and implemented., (© 2019 John Wiley & Sons Ltd.)

Cellular response to oxidative stress is a crucial mechanism that promotes the survival of Pseudomonas aeruginosa during infection. However, the translational regulation of oxidative stress response remains largely unknown. Here, we reveal a tRNA modification-mediated translational response to H2O2 in P. aeruginosa. We demonstrated that the P. aeruginosa trmB gene encodes a tRNA guanine (46)-N7-methyltransferase that catalyzes the formation of m7G46 in the tRNA variable loop. Twenty-three tRNA substrates of TrmB with a guanosine residue at position 46 were identified, including 11 novel tRNA substrates. We showed that loss of trmB had a strong negative effect on the translation of Phe- and Asp-enriched mRNAs. The trmB-mediated m7G modification modulated the expression of the catalase genes katA and katB, which are enriched with Phe/Asp codons at the translational level. In response to H2O2 exposure, the level of m7G modification increased, consistent with the increased translation efficiency of Phe- and Asp-enriched mRNAs. Inactivation of trmB led to decreased KatA and KatB protein abundance and decreased catalase activity, resulting in H2O2-sensitive phenotype. Taken together, our observations reveal a novel role of m7G46 tRNA modification in oxidative stress response through translational regulation of Phe- and Asp-enriched genes, such as katA and katB., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The expression of the Agrobacterium tumefaciens emrAB operon, which encodes a membrane fusion protein and an inner membrane protein, is inducible by various flavonoids, including apigenin, genistein, luteolin, naringenin, and quercetin. Among these flavonoids, quercetin is the best inducer, followed by genistein. The emrR gene is divergently transcribed from the emrAB operon. The EmrR protein, which belongs to the TetR transcriptional regulator family, negatively regulates the expression of emrAB and of itself. Electrophoretic mobility shift assays and DNase I footprinting showed that EmrR binds directly at two EmrR-binding sites in the emrR-emrAB intergenic region and that quercetin inhibits the DNA-binding activity of EmrR. Promoter-lacZ fusion analyses and 5' rapid amplification of cDNA ends were performed to map the emrR and emrAB promoters. Compared with the wild-type strain, the emrA mutant strain exhibited similar levels of resistance to the tested antibiotics. In contrast, disruption of emrR conferred protection against nalidixic acid and novobiocin, but it rendered A. tumefaciens sensitive to tetracycline and erythromycin. The emrR mutation also destabilized the outer membrane of A. tumefaciens, resulting in increased sensitivity to SDS and low pH. These findings demonstrate that proper regulation of emrR-emrAB is required for free-living A. tumefaciens to survive in deleterious environments in which toxic compounds are present. Nonetheless, A. tumefaciens strains that lack emrR or emrA still have the ability to cause tumors when infecting Nicotiana benthamiana plants.

Iron-sulfur ([Fe-S]) cluster proteins have essential functions in many biological processes. [Fe-S] homeostasis is crucial for bacterial survival under a wide range of environmental conditions. IscR is a global transcriptional regulator in Pseudomonas aeruginosa; it has been shown to regulate genes involved in [Fe-S] cluster biosynthesis, iron homeostasis, resistance to oxidants, and pathogenicity. Many aspects of the IscR transcriptional regulatory mechanism differ from those of other well-studied systems. This study demonstrates the mechanisms of IscR Type-1 binding to its target sites that mediate the repression of gene expression at the isc operon, nfuA, and tpx. The analysis of IscR binding to multiple binding sites in the promoter region of the isc operon reveals that IscR first binds to the high-affinity site B followed by binding to the low-affinity site A. The results of in vitro IscR binding assays and in vivo analysis of IscR-mediated repression of gene expression support the role of site B as the primary site, while site A has only a minor role in the efficiency of IscR repression of gene expression. Ligation of an [Fe-S] cluster to IscR is required for the binding of IscR to target sites and in vivo repression and stress-induced gene expression. Analysis of Type-1 sites in many bacteria, including P. aeruginosa, indicates that the first and the last three AT-rich bases were among the most highly conserved bases within all analyzed Type-1 sites. Herein, we first propose the putative sequence of P. aeruginosa IscR Type-1 binding motif as 5'AWWSSYRMNNWWWTNNNWSGGNYWW3'. This can benefit further studies in the identification of novel genes under the IscR regulon and the regulatory mechanism model of P. aeruginosa IscR as it contributes to the roles of an [Fe-S] cluster in several biologically important cellular activities., Competing Interests: The authors have declared that no competing interests exist.