Modular Golden Gate Assembly of Linear DNA Templates for Cell-Free Prototyping.

Cell-free transcription and translation (TXTL) systems have emerged as a powerful tool for testing genetic regulatory elements and circuits. Cell-free prototyping can dramatically accelerate the design-build-test-learn cycle of new functions in synthetic biology, in particular when quick-to-assemble linear DNA templates are used. Here, we describe a Golden-Gate-assisted, cloning-free workflow to rapidly produce linear DNA templates for TXTL reactions by assembling transcription units from basic genetic parts of a modular cloning toolbox. Functional DNA templates composed of multiple parts such as promoter, ribosomal binding site (RBS), coding sequence, and terminator are produced in vitro in a one-pot Golden Gate assembly reaction followed by polymerase chain reaction (PCR) amplification. We demonstrate assembly, cell-free testing of promoter and RBS combinations, as well as characterization of a repressor-promoter pair. By eliminating time-consuming transformation and cloning steps in cells and by taking advantage of modular cloning toolboxes, our cell-free prototyping workflow can produce data for large numbers of new assembled constructs within a single day.

Exploring the effects of cellulose sources on silver reduction and the bacterial removal of nanocellulose-based hydrogel beads.

With water access challenged, there is a need to develop efficient and sustainable alternatives for water purification. Here, cellulose nanofibrils (CNFs) isolated from three source materials (softwood, soybean hulls and oat straw) were compared for the generation of hydrogels beads, and compared as support and reducing agent for silver nanoparticles formation. The silver-functionalized hydrogel beads (Ag-CNFs) were characterized, and the surface energy and specific surface area were evaluated. Antimicrobial testing was conducted to assess the efficacy of the Ag-CNFs against E. coli. The results showed that the Ag-CNFs had a higher specific surface area and lower surface energy compared with unmodified CNFs. Softwood-based Ag-CNFs exhibited the highest silver content and specific surface area, while the soybean hull based showed the highest hydrophobic character. The silver-functionalized soybean hull beads (Ag-sbCNF) showed the highest efficacy in reducing the growth of bacteria. Overall, this study highlights the potential of silver-functionalized CNFs hydrogel beads as a promising environmentally friendly and sustainable material for water filtration and disinfection. The findings also suggest that lower surface energy of the Ag-CNFs play an important role in their antimicrobial effect on tested water by enabling shorter retention, providing useful insights into the design of future water filtration materials.

Prevalence and Risk Factors of ß-Lactamase Genes of Extended-Spectrum ß-Lactamases-Producing Escherichia coli From Dairy Farm Environments of Haryana, India.

Presence of extended-spectrum ß-lactamases (ESBL)-producing Enterobacteriaceae in the dairy farm environment and food chain could be a possible interface for the exchange of antimicrobial resistance genes between humans and animals. A total of 600 samples comprised of raw bovine milk, faeces, feed, environmental swabs and water samples from 20 different bovine dairy farms in and around Hisar city, Haryana, India were analysed for presence of ESBL encoding genes. Out of 240 isolates of Escherichia coli obtained, 74 isolates were found to be ESBL producers. Maximum number of ESBL isolates were found from faeces (40.5%) followed by raw milk (37.8%) and environmental swabs (17.5%). Most of the ESBL E. coli isolates were sensitive to chloramphenicol (82.4%) and gentamicin (77.0%) antibiotics. The bla CTX-M gene was found to be most prevalent (52.0%) followed by bla TEM (9.45%) while bla SHV gene alone was not detected in any sample by simplex PCR. However, the co-expression of blaCTX-M + blaTEM (21.6%) and blaCTX-M + blaSHV (4.05%) genes were also observed. The housing system, milking method and the hygienic mangement practices followed at farm level are found to be significant risk factors of ESBL-producing E. coli in dairy farms of Haryana.

Longitudinal genomic surveillance of a UK intensive care unit shows a lack of patient colonisation by multi-drug-resistant Gram-negative bacterial pathogens.

Vulnerable patients in an intensive care unit (ICU) setting are at high risk of infection from bacteria including gut-colonising Escherichia coli and Klebsiella species. Complex ICU procedures often depend on successful antimicrobial treatment, underscoring the importance of understanding the extent of patient colonisation by multi-drug-resistant organisms (MDROs) in large UK ICUs. Previous work on ICUs globally uncovered high rates of colonisation by transmission of MDROs, but the situation in UK ICUs is less understood. Here, we investigated the diversity and antibiotic resistance gene (ARG) carriage of bacteria present in one of the largest UK ICUs at the Queen Elizabeth Hospital Birmingham (QEHB), focusing primarily on E. coli as both a widespread commensal and a globally disseminated multi-drug-resistant pathogen. Samples were taken during highly restrictive coronavirus disease 2019 (COVID-19) control measures from May to December 2021. Whole-genome and metagenomic sequencing were used to detect and report strain-level colonisation of patients, focusing on E. coli sequence types (STs), their colonisation dynamics and antimicrobial resistance gene carriage. We found a lack of multi-drug resistance (MDR) in the QEHB. Only one carbapenemase-producing organism was isolated, a Citrobacter carrying bla KPC-2. There was no evidence supporting the spread of this strain, and there was little evidence overall of nosocomial acquisition or circulation of colonising E. coli. Whilst 22 different E. coli STs were identified, only 1 strain of the pandemic ST131 lineage was isolated. This ST131 strain was non-MDR and was found to be a clade A strain, associated with low levels of antibiotic resistance. Overall, the QEHB ICU had very low levels of pandemic or MDR strains, a result that may be influenced in part by the strict COVID-19 control measures in place at the time. Employing some of these infection prevention and control measures where reasonable in all ICUs might therefore assist in maintaining low levels of nosocomial MDR.

The Combined Use of High Pressure Processing and Lactic Acid Containing Fermentate on Inactivation of Salmonella, Shiga Toxin-producing E. coli and Listeria monocytogenes in Raw Pet Foods.

Raw meat pet foods can pose health risks to pets and humans. High pressure processing (HPP) was used in a previous study to demonstrate its effectiveness in achieving a 5-log reduction of Salmonella, E. coli STEC and L. monocytogenes in commercially available raw pet foods and maintaining the 5-log reduction throughout shelf-life with frozen storage being more effective than refrigerated. L. monocytogenes, being more HPP resistant, could potentially re-grow when stored at refrigeration temperatures and required further optimization. Chicken based raw diet pet food was inoculated with 7-8 log CFU/g cocktails of Salmonella spp., E. coli STEC or L. monocytogenes and stored at 4°C for 24h before the addition of either 0.7% or 1.0% w/v lactic acid fermentate (LAF) and HPP treated at 586 MPa for 2, 3 and 4 min after 24 or 72 h storage at 4 °C. HPP treated products were stored frozen (-10 to -16 °C) up to 21 days with microbiological analyses on days 1, 3, 5, 7, 14 and 21. All HPP and LAF treated samples demonstrated a 5-log reduction of Salmonella spp., E. coli STEC and L. monocytogenes. Samples without LAF and HPP treated after 24h storage at 4°C resulted in an average 4.02 log cfu/g reduction of L. monocytogenes with 2 min HPP hold time while longer HPP hold times at 4 min improved L. monocytogenes reduction by 0.35 log cfu/g. E. coli was found to be more HPP resistant in this study than L. monocytogenes and the addition of LAF had significant impact on the overall pathogen survival during post-HPP storage. Based on qualitative enrichment data for each pathogen, the use of LAF resulted in more complete inactivation compared to samples without LAF. The use of 1% LAF in combination with 586 MPa for 4 min was found to be most effective for the inactivation of Salmonella spp., E. coli STEC and L. monocytogenes. The findings are significant as it provides both formulation and processing controls to ensure the safety of raw diet pet foods.

Targeting threonine deaminase with chiral Au NPs: A novel strategy for E. coli inhibition.

Bacterial infections are becoming a significant threat to global human health due to the growing prevalence of biofilm-related infections and the rise in antibiotic resistance. D/l-cysteine functionalized chiral gold nanoparticles (D/P-Au NPs or L/P-Au NPs) have demonstrated a potent antibacterial effect against E. coli, while the mechanism remains to be elucidated through additional research. Threonine deaminase (TD) is a crucial enzyme involved in branched-chain amino acid (BCAA) biosynthesis in E. coli and is involved in cysteine's antimicrobial effects. This study investigated the interaction between chiral Au NPs (D/P-Au NPs or L/P-Au NPs) and TD as well as its effect on enzyme activity. It demonstrates that chiral Au NPs interact with TD through hydrophobic forces, forming a ground state complex that induces changes in the secondary structure of TD and reduces enzyme activity in a concentration-dependent manner. We found that the exogenous supplementation of isoleucine and valine (2 mg/mL) significantly reduced the antibacterial activity of chiral Au NPs, especially for L/P-Au NPs. The proteomics results indicate that the expression of ilvA and ilvB was down-regulated after L/P-Au NPs treatment, which would interfere with the synthesis of BCAAs. These results demonstrate that chiral Au NPs cause cell death of E. coli partly due to inhibition of TD enzyme activity and the synthesis of branched-chain amino acids.

Cyclomodulins-harboring Escherichia coli isolated from obese and normal-weight subjects induces intestinal dysplasia in a mouse model.

Recently, cyclomodulins have been identified in Escherichia coli (E. coli), which can induce dysplastic damage. This work aimed to determine the dysplastic activity of cyclomodulin-harboring E. coli isolated from CRC patients, obese and normal-weight subjects in a mouse model. Forty-two mice were pretreated with streptomycin, azoxymethane, and dextran sodium sulfate. Mice were infected with E. coli pks + isolated from a CRC patient, with E. coli pks + cif + isolated from obese or normal-weight subjects, or with E. coli HB101. The presence of cyclomodulin-harboring E. coli in the feces, weight loss, changes in fecal consistency, and the presence of blood in the feces were monitored and used to assess the disease activity index (DAI). After 62 days, the mice were sacrificed to evaluate the presence of intestinal polyps and dysplastic damage by histologic sections. Cyclomodulin-harboring E. coli colonized the mice; these mice exhibited weight loss and watery diarrhea, and isolated normal-weight E. coli had a higher DAI. Polyps were observed in mice infected with cyclomodulin-harboring E. coli in the ileum but to a greater extent in obese isolates. E. coli isolated from CRC showed more significant endothelial damage associated with dysplasia in the ileum in equal proportions from obese and normal-weight isolates. In conclusion, E. coli harboring cyclomodulins isolated from CRC, obesity, or normal weight can cause dysplastic damage in the ileum of mice and may be a risk factor for CRC development.

Effect of H2S and cysteine homeostasis disturbance on ciprofloxacin sensitivity of Escherichia coli in cystine-free and cystine-fed minimal medium.

Endogenous H2S has been proposed to be a universal defense mechanism against different antibiotics. Here, we studied the role of H2S transiently generated during ciprofloxacin (CF) treatment in M9 minimal medium with sulfate or produced by E. coli when fed with cystine. The cysM and mstA mutants did not produce H2S, while gshA generated more H2S in response to ciprofloxacin in cystine-free media. All mutants showed a reduced ability to maintain cysteine homeostasis under these conditions. We found no relationship between H2S generation, cysteine concentration and sensitivity to ciprofloxacin. Excess cysteine, which occurred during E. coli growth in cystine-fed media, triggered continuous H2S production, accelerated glutathione synthesis and cysteine export. This was accompanied by a twofold increase in ciprofloxacin tolerance in all strains except gshA, whose sensitivity increased 5-8-fold at high CF doses, indicating the importance of GSH in restoring the intracellular redox situation during growth in cystine-fed media.

Unveiling the potent antimicrobial photodynamic therapy in Gram-positive and Gram-negative bacteria - Water remediation with monocharged chlorins.

Water pollution is a significant concern worldwide, and it includes contaminants such as antibiotic-resistant pathogens. Antimicrobial photodynamic therapy (aPDT) offers a non-invasive and non-toxic alternative for the inactivation of these microorganisms. So, this study reports the synthesis, structural characterisation, photophysical properties, and aPDT efficacy of cationic free-base and zinc(II) chlorin (Chl) derivatives bearing N,N-dimethylpyrrolydinium groups (H2Chl 1a and ZnChl 1b). The aPDT assays were performed against two bacterial models: Staphylococcus aureus (Gram-(+)) and Escherichia coli (Gram-(-)). The H2Chl 1a and ZnChl 1b distinct's solubility profile, coupled with their ability to generate singlet oxygen (1O2) under light exposure, (H2Chl 1a, Ð¤Δ = 0.58 < TPP, Ð¤Δ = 0.65 < ZnChl 1b, Ð¤Δ = 0.83) opens up their potential application as photosensitizers (PS) in aPDT. The effectiveness of H2Chl 1a and ZnChl 1b at 1.0 and 5.0 µM in aPDT against S. aureus and E. coli at 500 W m-2 (total exposure time: 60-120 min) showed a viability reduction >6.0 log10 CFU mL-1. Additionally, KI was used as a coadjuvant to potentiate the photoinactivation of E. coli, reaching the method's detection limit (>4.0 log10 RLU). As most of the PS developed to inactivate Gram-negative bacteria are cationic with three or more charges, the fact that the H2Chl 1a and ZnChl 1b with only one cationic charge photoinactivate E. coli at low concentrations and with a reduced light dose, it is an importing discovery that deserves further exploration. These monocharged chlorin dyes have the potential for water remediation.

Biosynthesis of novel non-proteinogenic amino acids ß-hydroxyenduracididine and ß-methylphenylalanine in Escherichia coli.

Non-proteinogenic amino acids (npAAs) are valuable building blocks for the development of advanced pharmaceuticals and agrochemicals. The surge in interest in their synthesis is primarily due to the potential to enhance and diversify existing bioactive molecules. This can be achieved by altering these bioactive molecules to improve their effectiveness, reducing resistance compared to their natural counterparts or generating molecules with novel functions. Traditional production of npAAs in native hosts requires specialized conditions and complex cultivation media. Furthermore, these compounds are often found in organisms that challenge genetic manipulation. Thus, the recombinant production of these npAAs in a model organism like Escherichia coli paves the way for groundbreaking advancements in synthetic biology. Two synthetic operons, comprising of five heterologous proteins were genomically integrated into E. coli for the synthesis of npAAs ß-methylphenylalanine (BmePhe), ß-hydroxyenduracididine (BhEnd), and enduracididine (End). Proteomic and metabolomic analysis confirmed production of these compounds in E. coli for the first time. Interestingly, we discovered that the exogenous addition of pathway precursors to the E. coli system enhanced the yield of BmePhe by 2.5 times, whereas it concurrently attenuated the production of BhEnd and End, signifying a selective precursor-dependent yield enhancement. The synthetic biology landscape is broadened in this study by expanding the repertoire of amino acids beyond the conventional set of 22 standard proteinogenic amino acids. The biosynthesized npAAs, End, BhEnd, and BmePhe hold promise for engineering proteins with modified functions, integrating into novel metabolites and/or enhancing biological stability and activity. Additionally, these amino acids' biological production and subsequent purification present an alternative to traditional chemical synthesis methods, paving a direct pathway for pharmacological evaluation.

Isolation and evaluation of the pathogenicity of a hybrid shiga toxin-producing and Enterotoxigenic Escherichia coli in pigs.

BACKGROUND: Porcine pathogenic Escherichia coli (E. coli), the globally recognized important pathogen, causes significant economic loss in the field. Enterotoxigenic E. coli (ETEC) causes porcine neonatal and post-weaning diarrhea (PWD), frequently carrying F4 adhesin, F18 adhesin, Heat-Stable toxin (ST), and Heat-Labile toxin (LT). Shiga Toxin-Producing E. coli (STEC) produces F18 adhesin and Shiga toxin type 2e (stx2e), majorly leading to systemic endothelial cell damage and edema disease. In this study, hemolytic pathogenic hybrid STEC/ETEC strains carrying ST and LT genes of ETEC and the Stx2e gene of STEC isolated from pigs with PWD in Taiwan were identified. The pathogenicity of a Taiwan hybrid STEC/ETEC strain was evaluated by oral inoculation in post-weaning pigs. RESULTS: Next generation sequencing and multilocus sequence typing of two hybrid Taiwan porcine STEC/ETEC isolates indicated that these two isolates were closely related to the ST88 porcine hybrid STEC/ETEC isolated from pigs with watery diarrhea. Furthermore, the two hybrid Taiwan porcine STEC/ETEC isolates also displayed combinations of multiple resistance genes encoding mechanisms for target modification and antibiotic inactivation. Animal experiments confirmed that the Taiwan hybrid STEC/ETEC could cause watery diarrhea in post-weaning pigs with no signs of edema disease and minimal histopathological lesions. CONCLUSION: To the best of the authors' knowledge, the present study is the first study demonstrating intestinal pathogenicity of the hybrid STEC/ETEC in pigs. The result suggests that the hybrid STEC/ETEC should be considered as a new emerging pathogen and a new target for vaccine development.

Isolation and evaluation of bacteriophage cocktail for the control of colistin-resistant Escherichia coli.

The frequent emergence of colistin-resistant E. coli worldwide drives the exploration of alternative therapies, and bacteriophages (phages) have emerged as promising candidates to tackle this challenge. In this study, three E. coli phages were isolated, screened, and evaluated against 96 colistin-resistant strains obtained from diverse sources. The combined recognition rate for these strains was 43.6%, while individually it ranged from 17.0% to 24.5%. Notably, among the tested phages (FJ3-79, SD1-92L, and FJ4-63), FJ4-63 demonstrated exceptional characteristics in regulating host population dynamics upon infection by exhibiting a shorter latent period (20 min) and a larger burst size (95.99 ± 3.61 PFU/cell). Furthermore, it exhibited relative stability at pH 3-11 and below 60°C. Transmission electron microscopy and genomic analysis classified phage FJ4-63 belongs to the Dhakavirus genus within the Straboviridae family. Its genome comprised a linear double-stranded DNA measuring 169,669 bp (containing 272 coding sequences) with a GC content of 39.76%, of which 93 (34.2%) had known functions, and the remaining 177 were annotated as hypothetical proteins. Additionally, two tRNAs were recognized, possess the "holin-endolysin" lytic system, and no resistance or virulence genes were detected. The phylogenetic tree and average nucleotide identity (ANI) analysis revealed that phage FJ4-63 exhibited the highest similarity to Escherichia phage C6 (679410.1), indicating a consistent close relationship within the same branch. The cocktail comprising three phages exhibits enhanced in vitro bactericidal efficacy compared to a single phage. At high doses with MOI = 100, it rapidly and completely eradicates bacteria within 1 h while significantly reducing bacterial biofilms. All this evidence suggests that lytic phages offer an effective solution for clinical treatment, with a phage cocktail demonstrating greater potential in the alternative management of colistin-resistant E. coli infections.

Differential Selection for Survival and for Growth in Adaptive Laboratory Evolution Experiments With Benzalkonium Chloride.

Biocides are used to control microorganisms across different applications, but emerging resistance may pose risks for those applications. Resistance to biocides has commonly been studied using adaptive laboratory evolution (ALE) experiments with growth at subinhibitory concentrations linked to serial subculturing. It has been shown recently that Escherichia coli adapts to repeated lethal stress imposed by the biocide benzalkonium chloride (BAC) by increased survival (i.e., tolerance) and not by evolving the ability to grow at increased concentrations (i.e., resistance). Here, we investigate the contributions of evolution for tolerance as opposed to resistance for the outcome of ALE experiments with E. coli exposed to BAC. We find that BAC concentrations close to the half maximal effective concentration (EC50, 4.36 µg mL-1) show initial killing (~40%) before the population resumes growth. This indicates that cells face a two-fold selection pressure: for increased survival and for increased growth. To disentangle the effects of both selection pressures, we conducted two ALE experiments: (i) one with initial killing and continued stress close to the EC50 during growth and (ii) another with initial killing and no stress during growth. Phenotypic characterization of adapted populations showed that growth at higher BAC concentrations was only selected for when BAC was present during growth. Whole genome sequencing revealed distinct differences in mutated genes across treatments. Treatments selecting for survival-only led to mutations in genes for metabolic regulation (cyaA) and cellular structure (flagella fliJ), while treatments selecting for growth and survival led to mutations in genes related to stress response (hslO and tufA). Our results demonstrate that serial subculture ALE experiments with an antimicrobial at subinhibitory concentrations can select for increased growth and survival. This finding has implications for the design of ALE experiments to assess resistance risks of antimicrobials in different scenarios such as disinfection, preservation, and environmental pollution.

Electrochemical and optical biosensors for the detection of E. Coli.

E. coli is a common pathogenic microorganism responsible for numerous food and waterborne illnesses. Traditional detection methods often require long, multi-step processes and specialized equipment. Electrochemical and optical biosensors offer promising alternatives due to their high sensitivity, selectivity, and real-time monitoring capabilities. Recent advancements in sensor development focus on various techniques for detecting E. coli, including optical (fluorescence, colorimetric analysis, surface-enhanced Raman spectroscopy, surface plasmon resonance, localized surface plasmon resonance, chemiluminescence) and electrochemical (amperometric, voltammetry, impedance, potentiometric). Herein, the latest advancements in optical and electrochemical biosensors created for identifying E. coli with an emphasis on surface modifications employing nanomaterials and biomolecules are outlined in this review. Electrochemical biosensors exploit the unique electrochemical properties of E. coli or its specific biomolecules to generate a measurable signal. In contrast, optical biosensors rely on interactions between E. coli and optical elements to generate a detectable response. Moreover, optical detection has been exploited in portable devices such as smart phones and paper-based sensors. Different types of electrodes, nanoparticles, antibodies, aptamers, and fluorescence-based systems have been employed to enhance the sensitivity and specificity of these biosensors. Integrating nanotechnology and biorecognition (which bind to a specific region of the E. coli) elements has enabled the development of portable and miniaturized devices for on-site and point-of-care (POC) applications. These biosensors have demonstrated high sensitivity and offer low detection limits for E. coli detection. The convergence of electrochemical and optical technologies promises excellent opportunities to revolutionize E. coli detection, improving food safety and public health.

Manufacturing of the highly active thermophile PETases PHL7 and PHL7mut3 using Escherichia coli.

BACKGROUND: The global plastic waste crisis requires combined recycling strategies. One approach, enzymatic degradation of PET waste into monomers, followed by re-polymerization, offers a circular economy solution. However, challenges remain in producing sufficient amounts of highly active PET-degrading enzymes without costly downstream processes. RESULTS: Using the growth-decoupled enGenes eX-press V2 E. coli strain, pH, induction strength and feed rate were varied in a factorial-based optimization approach, to find the best-suited production conditions for the PHL7 enzyme. This led to a 40% increase in activity of the fermentation supernatant. Optimization of the expression construct resulted in a further 4-fold activity gain. Finally, the identified improvements were applied to the production of the more active and temperature stable enzyme variant, PHL7mut3. The unpurified fermentation supernatant of the PHL7mut3 fermentation was able to completely degrade our PET film sample after 16 h of incubation at 70 °C at an enzyme loading of only 0.32 mg enzyme per g of PET. CONCLUSIONS: In this research, we present an optimized process for the extracellular production of thermophile and highly active PETases PHL7 and PHL7mut3, eliminating the need for costly purification steps. These advancements support large-scale enzymatic recycling, contributing to solving the global plastic waste crisis.

ChiA: a major player in the virulence of Crohn's disease-associated adherent and invasive Escherichia coli (AIEC).

We investigated the role of ChiA and its associated polymorphisms in the interaction between Crohn's disease (CD)-associated adherent-invasive Escherichia coli (AIEC) and intestinal mucosa. We observed a higher abundance of chiA among the metagenome of CD patients compared to healthy subjects. In dextran sulfate sodium-induced colitis mice model, AIEC-LF82∆chiA colonization was reduced in ileal, colonic and fecal samples compared to wild-type LF82. The binding of ChiA to recombinant human CHI3L1 or mucus was higher with the pathogenic polymorphism. The strength of ChiA-mucin interaction was 300-fold stronger than ChiA-rhCHI3L1. ChiA was able to degrade mucin to promote its growth and enabled LF82 to get closer to epithelial cells. The pathogenic polymorphism of ChiA had a stronger impact on mucus degradation than on the binding capability of AIEC to adhere to the intestinal epithelium. We observed that ChiA could favor an efficient bacterial invasion of intestinal crypts, and that ChiA, especially its pathogenic polymorphism, gives LF82 an advantage to uptake within Peyer's patches, macrophages and mesenteric lymph nodes. All together, these data support the role of ChiA in the virulence of AIEC and show that it could be a promising target to reduce AIEC colonization in patients with CD.

Bacterial Photodynamic Inactivation: Eradication of Staphylococcus aureus and Escherichia coli Mediated by Pyridinium-Pyrazolyl Zinc(II) Phthalocyanines.

Antimicrobial resistance remains an enduring global health issue, manifested when microorganisms, such as bacteria, lack responsiveness to antimicrobial treatments. Photodynamic inactivation (PDI) of microorganisms arises as a noninvasive, nontoxic, and repeatable alternative for the inactivation of a broad range of pathogens. So, this study reports the synthesis, structural characterization, and photophysical properties of a new tetra-ß-substituted pyridinium-pyrazolyl zinc(II) phthalocyanine (ZnPc 1a) that was compared with two previously described pyridinium-pyrazolyl ZnPcs 2a and 3a. The PDI efficacy of these three ZnPcs (1a-3a) against a drug-resistant Gram-positive bacterium (as Staphylococcus aureus) and a Gram-negative bacterium (as Escherichia coli) is also reported. The PDI efficacy toward these bacteria was examined with ZnPcs 1a-3a in the 5.0-10.0 µM range using a white light source with an irradiance of 150 mW/cm2. All ZnPcs displayed a significant PDI activity against S. aureus, with reductions superior to 3 Log CFU/mL. Increasing the treatment time, the E. coli was inactivated until the detection limit of the method (>6.3 Log CFU/mL) using the quaternized ZnPcs 1a-3a (10.0 µM, 120 min) being the inactivation time was reduced when added the KI for ZnPcs 1a and 3a. These findings demonstrate the effective PDI performance of pyridinium-pyrazolyl group-bearing PSs, indicating their potential use as a versatile antimicrobial agent for managing infections induced by Gram-negative and Gram-positive bacteria.

Sucralose and caffeine as chemical indicators of domestic wastewater contamination in the Laurentian Great Lakes Basin.

Surface waters within the basin of the Laurentian Great Lakes are impacted by microbial contamination from municipal wastewater and agricultural runoff, as well as from other sources. In particular, microbial contamination of drinking water is an ongoing problem within many Indigenous communities located in the basin. However, it is difficult to identify the sources of microbial contamination using the traditional monitoring approaches with fecal indicator bacteria, such as total coliforms and Escherichia coli (E. coli). In this study, we evaluated whether surface waters in the basin are contaminated with fecal bacteria of human origin using chemical indicators of domestic wastewater (i.e., caffeine and sucralose) and with Bacteroidales 16S rRNA markers. Study areas included the Grand River watershed within the Lake Erie basin and three nearshore locations within the Great Lakes basin. Two of these sites are sources of drinking water for Indigenous communities. We assessed whether there were relationships between the concentrations of fecal indicator microorganisms and chemical indicators of domestic wastewater at selected study locations. Analysis of genetic markers indicated that about 30% of the Bacteroidales bacteria present at a site in the Grand River were of human fecal origin and the balance were of bovine or general animal origin. The presence of caffeine and sucralose in surface waters indicated that there was upstream contamination by domestic wastewater. However, in the drinking water treatment plant operated by Six Nations of the Grand River, the levels of these chemical indicators and fecal bacteria were reduced by the advanced water treatment technologies. The concentrations of sucralose and caffeine collectively were strongly correlated with the levels of total coliforms in samples from the Grand River (R2 = 0.75) and with levels of E. coli in samples from the Great Lakes basin (R2 = 0.97), but there appeared to be an upper threshold for this relationship. These data indicate that analysis of caffeine and sucralose and genetic markers for strains of Bacteroidales fecal bacteria may be useful tools for identifying the sources of microbiological contamination of surface waters and drinking water.

A One Health exploration of antimicrobial resistance in Escherichia coli originated from urban and rural lakes ecosystem.

Antimicrobial resistance (AMR) has become one of the most serious threats to One Health. Aquatic environments are an ideal non-clinical AMR reservoir and can act as a key battlefront for tackling the AMR. However, AMR data using the One Health approach remain scarce in aquatic environments worldwide. Here, we extensively assessed AMR in Escherichia coli isolated from urban and rural lake ecosystems using the One Health perspective. A total of 162 E. coli isolates obtained from lakes were tested against 25 antimicrobials using an in-vitro antimicrobial susceptibility testing method. A low (2%) to moderate (45%) drug resistance rate was found for all antimicrobials used in human/veterinary medicine or animal/plant agriculture. However, <80% E. coli isolates exhibited multidrug resistance (MDR) phenotype to highly important (amikacin, gentamicin, trimethoprim) or critically important (amoxicillin, ampicillin, colistin) drugs of both human and veterinary medicine. Of concern, >50% of E. coli isolates exhibited MDR to drugs used as last-resorts (chloramphenicol, colistin) or as frontline (nitrofurantoin, sulfamethoxazole, ampicillin, gentamicin) against E. coli infections. In conclusion, the presence of MDR E. coli strains in urban or rural lake ecosystems highlights their possible role as AMR reservoirs with potential One Health risks.

Antibacterial activity and mechanism of Stevia extract against antibiotic-resistant Escherichia coli by interfering with the permeability of the cell wall and the membrane.

Natural plant-derived compounds with broad-spectrum antimicrobial activity have become an effective strategy against multidrug-resistant bacteria. The present study was designed to compare the antibacterial activity of six chlorogenic acid (CA) isomers extracted from stevia and investigated the underlying antibacterial mechanisms involved. The results indicated that isochlorogenic acid C (ICAC) exhibited the strongest antibacterial activity against the tested bacteria, especially E. coli, at a 2 mg/mL minimum inhibitory concentration (MIC) and 8 mg/mL minimum bactericidal concentration (MBC). At the MBC, ICAC inhibited 72.66% of the clinical multidrug-resistant strains. Scanning electron microscopy (SEM) revealed that ICAC induced considerable morphological alterations in E. coli ATCC25922 and C4E2. The significant increase in the activity of extracellular alkaline phosphatase (AKP) indicated that ICAC damages the permeability of the bacterial cell wall. Additionally, the intracellular membrane (IM) permeability and the content of lipopolysaccharide (LPS), a main component of the outer membrane (OM), were determined. The significant decrease in LPS content and increased leakage of intracellular proteins and K+ from E. coli indicated that ICAC could induce the exfoliation of OM and disrupt IM permeability, resulting in the loss of barrier function. The uptake of propidium iodide (PI), a compromised cell membrane nucleic acid stain, and confocal laser scanning microscopy (CLSM) further demonstrated that ICAC disrupted IM integrity. Moreover, the bactericidal effect and damage to bacterial microstructural function occurred in a dose-dependent manner. These data demonstrate that ICAC has excellent antibacterial activity and is a promising approach for overcoming the antibiotic resistance of pathogenic bacteria.