Intact and mutated Shigella diguanylate cyclases increase c-di-GMP.

The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger cyclic di-GMP (c di-GMP) signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but five of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the four intact DGCs in a S. flexneri strain, where these four DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these four DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC pseudogenes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.

Molecular characterization of carbapenem and ceftazidime-avibactam-resistant Enterobacterales and horizontal spread of bla NDM-5 gene at a Lebanese medical center.

Introduction: In the battle against multidrug-resistant bacterial infections, ceftazidime- avibactam (CZA) stands as a pivotal defense, particularly against carbapenemresistant (CR) Gram-negative pathogens. However, the rise in resistance against this drug poses a significant threat to its effectiveness, highlighting the critical need for in-depth studies about its resistance mechanisms. Methods: This research focuses on the genomic characterization of CR- and CZA-resistant Escherichia coli (n=26) and Klebsiella pneumoniae (n=34) strains, harboring the blaNDM and/or blaOXA-48-like genes, at a major Lebanese tertiary care medical center, using whole genome sequencing (WGS). Results: Our findings revealed a notable prevalence of blaNDM in all K. pneumoniae strains isolates, with 27 of these also harboring blaOXA-48. On the other hand, E. coli strains predominantly carried the blaNDM-5 gene. Whole genome sequencing (WGS) identified a predominance of ST383 among K. pneumoniae strains, which possessed a multi-replicon IncFIB-IncHI1B plasmid harboring the blaNDM-5. Additionally, various Inc group plasmids in K. pneumoniae across multiple sequence types were found to carry the blaNDM. Similarly, diverse STs of E. coli were observed to carry blaNDM-5 on different plasmids. Discussion: The study underscores NDM carbapenemases as a paramount resistance mechanism in Lebanon,jeopardizing critical last-resort treatments. It also illuminates the role of varied sequence types and mobile genetic elements in the spread of NDM resistance,stressing the urgent need for strategies to mitigate this threat, especially in nosocomial infections.

Florfenicol core-shell composite nanogels as oral administration for efficient treatment of bacterial enteritis.

To reduce the bitterness of florfenicol, avoid its degradation by gastric acid, and enhance its antibacterial activity against Escherichia coli by targeting and slowly releasing drugs at the site of intestinal infection, with pectin as an anion carrier and chitosan oligosaccharides (COS) as a cationic carrier, florfenicol-loaded COS@pectin core nanogels were self-assembled by electrostatic interaction and then encapsulated in sodium carboxymethylcellulose (CMCNa) shell nanogels through the complexation of CMCNa and Ca2+ to prepare florfenicol core-shell composite nanogels in this study. The florfenicol core-shell composite nanogels were investigated for their formula choice, physicochemical characterization, pH-responsive performances, antibacterial activity, therapeutic efficacy, and in vitro and in vivo biosafety studies. The results indicated that the optimized formula was 0.6 g florfenicol, 0.79 g CMCNa, 0.30 g CaCl2, 0.05 g COS, and 0.10 g pectin, respectively. In addition, the mean particle diameter, polydispersity index, zeta potential, loading capacity, and encapsulation efficiency were 124.0 ± 7.2 nm, -22.9 ± 2.5 mV, 0.42 ± 0.03, 43.4 % ± 3.1 %, and 80.5 % ± 3.4 %, respectively. The appearance, lyophilized mass, resolvability, scanning electron microscopy (SEM), transmission electron microscopy (TEM), powder X-ray diffraction (PXRD), and fourier transform infrared (FTIR) showed that the florfenicol core-shell composite nanogels were successfully prepared. Florfenicol core-shell composite nanogels had satisfactory stability, rheology, and pH-responsiveness, which were conducive to avoid degradation by gastric acid and achieve targeted and slow release at intestinal infection sites. More importantly, florfenicol core-shell composite nanogels had excellent antibacterial activity against Escherichia coli, a satisfactory therapeutic effect, and good palatability. In vitro and in vivo biosafety studies suggested the great promise of florfenicol core-shell composite nanogels. Therefore, the prepared florfenicol core-shell composite nanogels may be helpful for the treatment of bacterial enteritis as a biocompatible oral administration.

AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering.

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.

The antioxidant and antimicrobial activity of ethanolic extract in roots, stems, and leaves of three commercial Cymbopogon species.

BACKGROUND: Cymbopogon is a member of the family Poaceae and has been explored for its phytochemicals and bioactivities. Although the antimicrobial activities of Cymbopogon spp. extracts have been extensively studied, comprehensive analyses are required to identify promising compounds for the treatment of antimicrobial resistance. Therefore, this study investigated the antioxidant and antimicrobial properties of Cymbopogon spp. ethanolic extracts in every single organ. METHODS: Ethanolic extracts were obtained from three Indonesian commercial species of Cymbopogon spp., namely Cymbopogon citratus (L.) Rendle, Cymbopogon nardus (DC.) Spatf., and Cymbopogon winterianus Jowitt. The leaf, stem, and root extracts were evaluated via metabolite profiling using gas chromatography-mass spectrometry (GC-MS). In silico and in vitro analyses were used to evaluate the antioxidant and antimicrobial properties of the Cymbopogon spp. ethanolic extracts. In addition, bioactivity was measured using cytotoxicity assays. Antioxidant assays were performed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to determine toxicity to Huh7it-1 cells using a tetrazolium bromide (MTT) assay. Finally, the antimicrobial activity of these extracts was evaluated against Candida albicans, Bacillus subtilis, Staphylococcus aureus, and Escherichia coli using a well diffusion assay. RESULTS: GC-MS analysis revealed 53 metabolites. Of these, 2,5-bis(1,1-dimethylethyl)- phenol (27.87%), alpha-cadinol (26.76%), and 1,2-dimethoxy-4-(1-propenyl)-benzene (20.56%) were the predominant compounds. C. winterianus and C. nardus leaves exhibited the highest antioxidant activity against DPPH and ABTS, respectively. Contrastingly, the MTT assay showed low cytotoxicity. C. nardus leaf extract exhibited the highest antimicrobial activity against E. coli and S. aureus, whereas C. winterianus stem extract showed the highest activity against B. substilis. Furthermore, computational pathway analysis predicted that antimicrobial activity mechanisms were related to antioxidant activity. CONCLUSIONS: These findings demonstrate that the leaves had strong antioxidant activity, whereas both the leaves and stems showed great antimicrobial activity. Furthermore, all Cymbopogon spp. ethanolic extracts showed low toxicity. These findings provide a foundation for future studies that assess the clinical safety of Cymbopogon spp. as novel drug candidates.

Identification of Escherichia coli strains using MALDI-TOF MS combined with long short-term memory neural networks.

The current study aims to develop a new technique for the precise identification of Escherichia coli strains, utilizing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with a long short-term memory (LSTM) neural network. A total of 48 Escherichia coli strains were isolated and cultured on tryptic soy agar medium for 24 hours for the generation of MALDI-TOF MS spectra. Eight hundred MALDI-TOF MS spectra were obtained per strain, resulting in a database of 38,400 spectra. Fifty percent of the data was utilized for LSTM neural network training, with fine-tuned parameters for strain-level identification. The other half served as the test set to assess model performance. Traditional PCA dimension reduction of MALDI-TOF MS spectra indicated 47 out of 48 strains to be unclassifiable. In contrast, the LSTM neural network demonstrated remarkable efficacy. After 20 training epochs, the model achieved a loss value of 0.0524, an accuracy of 0.999, a precision of 0.985, and a recall of 0.982. When tested on the unseen data, the model attained an overall accuracy of 92.24%. The integration of MALDI-TOF MS and LSTM neural network markedly enhances the identification of Escherichia coli strains. This innovative approach offers an effective and accurate tool for MALDI-TOF MS-based strain-level identification, thus expanding the analytical capabilities of microbial diagnostics.

Therapeutic Phage Candidates for Targeting Prevalent Sequence Types of Carbapenem-Resistant Escherichia coli.

Carbapenem-resistant Escherichia coli (CREC) is a global threat to public health; therefore, alternative treatment options are urgently needed. Bacteriophages have emerged as promising candidates for combating CREC infections. This study aimed to investigate the genetic basis of phage sensitivity in CREC by evaluating carbapenem resistance among multidrug-resistant (MDR) E. coli isolated in Daegu, South Korea and analyzing their sequence types (STs) with phage susceptibility spectra. Among the 60 MDR E. coli isolates, 80.4% were identified as CREC, with 77.0% demonstrating resistance to imipenem and 66.6% to meropenem. Moreover, 70 lytic E. coli bacteriophages were isolated from hospital sewage water and evaluated against those 60 E. coli isolates. The phages exhibited lytic activity of 33%-60%, with average titers ranging from 5.6 × 1012 to 2.4 × 1013 PFU/mL (Plaque-Forming Unit). Furthermore, multilocus sequence typing (MLST) analysis of the bacterial isolates revealed 14 distinct STs, mostly belonging to ST131, ST410, and ST648. Notably, the phage susceptibility spectra of ST73, ST13003, ST648, ST2311, ST167, ST405, ST607, ST7962, and ST131 were significantly different. Thus, the isolated phages can effectively lyse CREC isolates, particularly those with clinically dominant STs. Conversely, ST410 exhibited a 14.2%-87.14% susceptibility spectrum, whereas ST1139, ST1487, ST10, and ST206 did not lyse, suggesting the presence of more resistant STs. Future studies are warranted to identify the reasons behind this resistance and address it. Ultimately, this study will aid in developing focused treatments to address these pressing global health issues.

High-efficient preparation of ß-nicotinamide mononucleotides by crude enzymes cascade catalytic reaction.

ß-nicotinamide mononucleotide (ß-NMN) is a key precursor of nicotinamide adenine dinucleotide, and becomes attractive in the nutrition and health care fields, but its enzymatic synthesis is expensive. In this study, a six-enzyme cascade catalytic system was constructed to produce ß-NMN. Using D-ribose and nicotinamide as substrates, the ß-NMN yield reached 97.5 % catalyzed by purified enzymes. Then, after knocking out the genes encoding proteins that consume ß-NMN in E. coli BL21(DE3), the similar ß-NMN yield, 97.2 %, using the crude enzymes could be also obtained. After that, ß-NMN synthesis was performed under increased substrate concentration, and 'modular' crude enzymes cascade catalytic reaction system was proposed to reduce the inhibition of polyphosphate on ribose-phosphate diphosphokinase activity, and the ß-NMN yield reached 78.4 % at 10 mM D-ribose, which is 1.82 times of that in 'one-pot' reaction and represents the highest ß-NMN preparation level with phosphoribosylpyrophosphate as the core reported till now.

Demographic, clinical and molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli bloodstream infections in Central Australia.

We describe the demographics, clinical and molecular epidemiology of extended-spectrum ß-lactamase (ESBL) Escherichia coli bloodstream infections (BSI) in Central Australia. All ESBL-producing E. coli bloodstream isolates from January 2018 to December 2020 were retrospectively identified. Demographic and clinical information was extracted by chart review. Whole-genome sequencing was performed for multi-locus sequence typing, antibiotic-resistance genes, and phylogenetic relationships. We identified 41 non-duplicate episodes of ESBL E. coli BSI. Median age was 55 years (IQR 47-63), 78% were female, 93% were Aboriginal, and half came from a remote community. Infections were predominantly urinary (68%, 28/41). In the 12 months prior, 70% (26/37) of identified patients had been hospitalised and 81% (30/37) prescribed antibiotics. Meropenem and piperacillin-tazobactam susceptibility was maintained in 100% and 95% of isolates, respectively. Co-resistance to non-ß-lactam antibiotics was 32% to gentamicin, 61% to trimethoprim/sulfamethoxazole, and 68% to ciprofloxacin. For sequenced isolates, 41% (16/35) were sequence type 131 (ST131). Mean acquired antibiotic-resistance genes for each isolate was 12.3 (SD 3.1). Four isolates carried an OXA-1 gene. Only non-ST131 isolates carried AmpC and acquired quinolone-resistance genes. There was some evidence of clustering of closely related strains, but no evidence of community or healthcare admission overlap. ESBL rates are rapidly rising in Central Australia, which is a conducive environment for antibiotic resistance development (e.g., overcrowding, socioeconomic disadvantages, high healthcare exposure and high antibiotic use). Future research is required to explore resistance-transmission dynamics in this unique setting.

Non-native Pathway Engineering with CRISPRi for Carbon Dioxide Assimilation and Valued 5-Aminolevulinic Acid Synthesis in Escherichia coli Nissle.

Carbon dioxide emission and acidification during chemical biosynthesis are critical challenges toward microbial cell factories' sustainability and efficiency. Due to its acidophilic traits among workhorse lineages, the probiotic Escherichia coli Nissle (EcN) has emerged as a promising chemical bioproducer. However, EcN lacks a CO2-fixing system. Herein, EcN was equipped with a simultaneous CO2 fixation system and subsequently utilized to produce low-emission 5-aminolevulinic acid (5-ALA). Two different artificial CO2-assimilating pathways were reconstructed: the novel ribose-1,5-bisphosphate (R15P) route and the conventional ribulose-5-phosphate (Ru5P) route. CRISPRi was employed to target the pfkAB and zwf genes in order to redirect the carbon flux. As expected, the CRISPRi design successfully strengthened the CO2 fixation. The CO2-fixing route via R15P resulted in high biomass, while the engineered Ru5P route acquired the highest 5-ALA and suppressed the CO2 release by 77%. CO2 fixation during 5-ALA production in EcN was successfully synchronized through fine-tuning the non-native pathways with CRISPRi.

Sulfonamide resistance evaluation in five animal species and first report of sul4 in companion animals.

Sulfonamides are one of the oldest groups of antibacterial agents with a broad-spectrum, used as first line treatment in bacterial infections. Their widespread use produced a selective pressure on bacteria, as observed by the high incidence of sulfonamides resistance mainly in Gram negative bacteria isolated from animals. In this research, the presence of sulfonamide resistance genes (sul1, sul2, sul3, and sul4) in phenotypically resistant Escherichia coli isolates has been studied. These genes were amplified in isolates recovered from five animal species, with different interactions to humans: cattle, swine, poultry as livestock, and dogs and cats as companion animals. Isolates were collected according to their phenotypic resistance, and the magnetic bead-based Luminex technology was applied to simultaneously detect sul target genes. The frequency of sul genes was highest in swine, among livestock isolates. The sul1 and sul2 were the most frequently sulfonamide resistance genes detected in all phenotypically resistant isolates. Notably, in companion animals, with a closest interaction with human, sul4 gene was detected. To our knowledge, this is the first report of the presence of sul4 gene in E. coli collected from animals, whereas previously the presence of this gene was reported in environmental, municipal wastewater and human clinical isolates. These results highlighted the importance of continuous antimicrobial resistant genes monitoring in animal species, with a special care to companion animals.

Long-term impact of basin-wide wastewater management on faecal pollution levels along the entire Danube River.

The Danube River is, at 2857 km, the second longest river in Europe and the most international river in the world with 19 countries in its catchment. Along the entire river, faecal pollution levels are mainly influenced by point-source emissions from treated and untreated sewage of municipal origin under base-flow conditions. In the past 2 decades, large investments in wastewater collection and treatment infrastructure were made in the European Union (EU) Member States located in the Danube River Basin (DRB). Overall, the share of population equivalents with appropriately biologically treated wastewater (without disinfection) has increased from 69% to more than 85%. The proportion of tertiary treatment has risen from 46 to 73%. In contrast, no comparable improvements of wastewater infrastructure took place in non-EU Member States in the middle and lower DRB, where a substantial amount of untreated wastewater is still directly discharged into the Danube River. Faecal pollution levels along the whole Danube River and the confluence sites of the most important tributaries were monitored during four Danube River expeditions, the Joint Danube Surveys (JDS). During all four surveys, the longitudinal patterns of faecal pollution were highly consistent, with generally lower levels in the upper section and elevated levels and major hotspots in the middle and lower sections of the Danube River. From 2001 to 2019, a significant decrease in faecal pollution levels could be observed in all three sections with average reduction rates between 72 and 86%. Despite this general improvement in microbiological water quality, no such decreases were observed for the highly polluted stretch in Central Serbia. Further improvements in microbiological water quality can be expected for the next decades on the basis of further investments in wastewater infrastructure in the EU Member States, in the middle and lower DRB. In the upper DRB, and due to the high compliance level as regards collection and treatment, improvements can further be achieved by upgrading sewage treatment plants with quaternary treatment steps as well as by preventing combined sewer overflows. The accession of the Western Balkan countries to the EU would also significantly boost investments in wastewater infrastructure and water quality improvements in the middle section of the Danube. Continuing whole-river expeditions such as the Joint Danube Surveys is highly recommended to monitor the developments in water quality in the future.

The Antibacterial Properties of a Reinforced Zinc Oxide Eugenol Combined with Cloisite 5A Nanoclay: An In-Vitro Study.

Pulpotomies and pulpectomies are the most common clinical approach for dental caries in the primary dentition. Reinforced zinc oxide eugenol (ZOE) is an ideal material for filling in the pulp chamber after pulp therapies. The aim of this study was to assess the addition of Cloisite 5A nanoclay material to ZOE and evaluate its antibacterial properties. In this case-control study, the nanoclay nanoparticles were dissolved using a solvent (Eugenol) in different concentrations and their antibacterial properties were assessed using the agar diffusion test and biofilm analysis of Streptococcus mutans (S. mutans), Enterococcus faecalis (E. faecalis), and Escherichia coli (E. coli) in in vitro conditions using the AATCC 100 standards. The diameter of the inhibition zone was measured and assessed statistically using the SPSS software (Version 28, IBM, Chicago, IL, USA) with a significance level of 0.05. The antibacterial properties of the ZOE with nanoclay particles were significantly greater in comparison to the plain ZOE against E. faecalis, S. mutans, and E. coli. The inhibition zone against E. coli under the effect of the ZOE and nanoclay particles combined was significantly higher than that against E. faecalis and S. mutans. The current study showed that the addition of Cloisite 5A nanoclay particles can improve the antibacterial properties of ZOE significantly at certain concentrations.

Molecular Characterization and Antibacterial Resistance Determination of Escherichia coli Isolated from Fresh Raw Mussels and Ready-to-Eat Stuffed Mussels: A Major Public Health Concern.

Our study focused exclusively on analyzing Escherichia coli (E. coli) contamination in fresh raw mussels and ready-to-eat (RTE) stuffed mussels obtained from authorized and regulated facilities. However, it is critical to recognize that such contamination represents a significant public health threat in regions where unauthorized harvesting and sales practices are prevalent. This study aimed to comprehensively assess the prevalence, molecular characteristics, and antibacterial resistance profiles of E. coli in fresh raw mussels and RTE stuffed mussels. E. coli counts in fresh raw mussel samples ranged from 1 to 2.89 log CFU/g before cooking, with a significant reduction observed post-cooking. RTE stuffed mussel samples predominantly exhibited negligible E. coli presence (<1 log CFU/g). A phylogenetic analysis revealed a dominance of phylogroup A, with variations in the distribution observed across different sampling months. Antibacterial resistance was prevalent among the E. coli isolates, notably showing resistance to ampicillin, streptomycin, and cefotaxime. Extended-spectrum ß-lactamase (ESßL) production was rare, with only one positive isolate detected. A variety of antibacterial resistance genes, including tetB and sul1, were identified among the isolates. Notably, virulence factor genes associated with pathogenicity were absent. In light of these findings, it is imperative to maintain rigorous compliance with quality and safety standards at all stages of the mussel production process, encompassing harvesting, processing, cooking, and consumption. Continuous monitoring, implementation of rigorous hygiene protocols, and responsible antibacterial drug use are crucial measures in mitigating food safety risks and combating antibacterial resistance. Stakeholders, including seafood industry players, regulatory agencies, and healthcare professionals, are essential to ensure effective risk mitigation and safeguard public health in the context of seafood consumption.

Monitoring the Bioprotective Potential of Lactiplantibacillus pentosus Culture on Pathogen Survival and the Shelf-Life of Fresh Ready-to-Eat Salads Stored under Modified Atmosphere Packaging.

Globally, fresh vegetables or minimally processed salads have been implicated in several foodborne disease outbreaks. This work studied the effect of Lactiplantibacillus pentosus FMCC-B281 cells (F) and its supernatant (S) on spoilage and on the fate of Listeria monocytogenes and Escherichia coli O157:H7 on fresh-cut ready-to-eat (RTE) salads during storage. Also, Fourier transform infrared (FTIR) and multispectral imaging (MSI) analysis were used as rapid and non-destructive techniques to estimate the microbiological status of the samples. Fresh romaine lettuce, rocket cabbage, and white cabbage were used in the present study and were inoculated with L. pentosus and the two pathogens. The strains were grown at 37 °C for 24 h in MRS and BHI broths, respectively, and then were centrifuged to collect the supernatant and the pellet (cells). Cells (F, ~5 log CFU/g), the supernatant (S), and a control (C, broth) were used to spray the leaves of each fresh vegetable that had been previously contaminated (sprayed) with the pathogen (3 log CFU/g). Subsequently, the salads were packed under modified atmosphere packaging (10%CO2/10%O2/80%N2) and stored at 4 and 10 °C until spoilage. During storage, microbiological counts and pH were monitored in parallel with FTIR and MSI analyses. The results showed that during storage, the population of the pathogens increased for lettuce and rocket independent of the treatment. For cabbage, pathogen populations remained stable throughout storage. Regarding the spoilage microbiota, the Pseudomonas population was lower in the F samples, while no differences in the populations of Enterobacteriaceae and yeasts/molds were observed for the C, F, and S samples stored at 4 °C. According to sensory evaluation, the shelf-life was shorter for the control samples in contrast to the S and F samples, where their shelf-life was elongated by 1-2 days. Initial pH values were ca. 6.0 for the three leafy vegetables. An increase in the pH of ca. 0.5 values was recorded until the end of storage at both temperatures for all cases of leafy vegetables. FTIR and MSI analyses did not satisfactorily lead to the estimation of the microbiological quality of salads. In conclusion, the applied bioprotective strain (L. pentosus) can elongate the shelf-life of the RTE salads without an effect on pathogen growth.

Enhancing slow sand filtration for safe drinking water production: interdisciplinary insights into Schmutzdecke characteristics and filtration performance in mini-scale filters.

The demand for safe drinking water is constantly challenged by increasing biohazards. One widely used solution is implementing indoor-operated slow sand filtration (SSF) as one of the final barriers in water production. SSF has gained popularity due to its low energy consumption and efficient removal of biohazards, especially microorganisms, without using chemicals. SSF involves both physical-chemical and biological removal, particularly in the "Schmutzdecke", which is a biofilm-like layer on the sand bed surface. To achieve the optimal performance of SSF, a systematic understanding of the influence of SSF operating parameters on the Schmutzdecke development and filter filtration performance is required. Our study focused on three operational parameters, i.e., sand material, sand size, and the addition of an inoculum (suspension of matured Schmutzdecke), on the mini-scale filters. The effects of these parameters on the Schmutzdecke development and SSF removal performance were studied by biochemical analyses and 16S amplicon sequencing, together with spiking experiments with Escherichia coli (E. coli) in the mini-scale filters. Our results indicate that the mini-scale filters successfully developed Schmutzdeckes and generated bacterial breakthrough curves efficiently. The sand size and material were found to have an impact on Schmutzdecke's development. The addition of inoculum to new filters did not induce significant changes in the microbial community composition of the Schmutzdecke, but we observed positive effects of faster Schmutzdecke development and better removal performance in some inoculated filters. Our study highlights the value of mini-scale filters for SSF studies, which provide insights into Schmutzdecke microbial ecology and bacterial removal with significantly reduced requirements of materials and effort as compared to larger-scale filters. We found that operational parameters have a greater impact on the Schmutzdecke biochemical characteristics and removal performances than on the microbial community composition. This suggests that Schmutzdecke characteristics may provide more reliable predictors of SSF removal performance, which could help to improve safe drinking water production.

Isolation, Characterization, and Complete Genome Sequence of Escherichia Phage KIT06 Which Infects Nalidixic Acid-Resistant Escherichia coli.

Escherichia coli (E. coli) is one of the most common sources of infection in humans and animals. The emergence of E. coli which acquires resistance to various antibiotics has made treatment difficult. Bacteriophages can be considered promising agents to expand the options for the treatment of antibiotic-resistant bacteria. This study describes the isolation and characterization of Escherichia phage KIT06, which can infect E. coli resistant to the quinolone antibiotic nalidixic acid. Phage virions possess an icosahedral head that is 93 ± 8 nm in diameter and a contractile tail (116 ± 12 nm × 13 ± 5 nm). The phage was found to be stable under various thermal and pH conditions. A one-step growth curve showed that the latent time of the phage was 20 min, with a burst size of 28 particles per infected cell. Phage KIT06 infected 7 of 12 E. coli strains. It inhibited the growth of the host bacterium and nalidixic acid-resistant E. coli. The lipopolysaccharide and outer membrane proteins of E. coli, tsx and btuB, are phage receptors. Phage KIT06 is a new species of the genus Tequatrovirus with a genome of 167,059 bp consisting of 264 open reading frames (ORFs) that encode gene products related to morphogenesis, replication, regulation, and host lysis. The lack of genes encoding integrase or excisionase indicated that this phage was lytic. Thus, KIT06 could potentially be used to treat antibiotic-resistant E. coli using phage therapy. However, further studies are essential to understand its use in combination with other antimicrobial agents and its safe use in such applications.

Essential Oils from Southern Italian Aromatic Plants Synergize with Antibiotics against Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis Cell Growth and Biofilm Formation.

The spread of antibiotic-resistant pathogens has prompted the development of novel approaches to identify molecules that synergize with antibiotics to enhance their efficacy. This study aimed to investigate the effects of ten Essential Oils (EOs) on the activity of nine antibiotics in influencing growth and biofilm formation in Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecalis. The effects of the EOs alone and in combination with antibiotics on both bacterial growth and biofilm formation were analyzed by measuring the MIC values through the broth microdilution method and the crystal violet assay, respectively. All EOs inhibited the growth of E. coli (1.25 ≤ MIC ≤ 5 mg/mL) while the growth of P. aeruginosa and E. faecalis was only affected by EOs from Origanum vulgare, (MIC = 5 mg/mL) and O. vulgare (MIC = 1.25 mg/mL) and Salvia rosmarinus (MIC = 5 mg/mL), respectively. In E. coli, most EOs induced a four- to sixteen-fold reduction in the MIC values of ampicillin, ciprofloxacin, ceftriaxone, gentamicin, and streptomycin, while in E. faecalis such a reduction is observed in combinations of ciprofloxacin with C. nepeta, C. bergamia, C. limon, C. reticulata, and F. vulgare, of gentamicin with O. vulgare, and of tetracycline with C. limon and O. vulgare. A smaller effect was observed in P. aeruginosa, in which only C. bergamia reduced the concentration of tetracycline four-fold. EO-antibiotic combinations also inhibit the biofilm formation. More precisely, all EOs with ciprofloxacin in E. coli, tetracycline in P. aeruginosa, and gentamicin in E. faecalis showed the highest percentage of inhibition. Combinations induce up- and down-methylation of cytosines and adenines compared to EO or antibiotics alone. The study provides evidence about the role of EOs in enhancing the action of antibiotics by influencing key processes involved in resistance mechanisms such as biofilm formation and epigenetic changes. Synergistic interactions should be effectively considered in dealing with pathogenic microorganisms.

A New Guanidine-Core Small-Molecule Compound as a Potential Antimicrobial Agent against Resistant Bacterial Strains.

The guanidine core has been one of the most studied functional groups in medicinal chemistry, and guanylation reactions are powerful tools for synthesizing this kind of compound. In this study, a series of five guanidine-core small molecules were obtained through guanylation reactions. These compounds were then evaluated against three different strains of Escherichia coli, one collection strain from the American Type Culture Collection (ATCC) of E. coli ATCC 35218, and two clinical extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates (ESBL1 and ESBL2). Moreover, three different strains of Pseudomonas aeruginosa were studied, one collection strain of P. aeruginosa ATCC 27853, and two clinical multidrug-resistant isolates (PA24 and PA35). Among Gram-positive strains, three different strains of Staphylococcus aureus, one collection strain of S. aureus ATCC 29213, and two clinical methicillin-resistant S. aureus (MRSA1 and MRSA2) were evaluated. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) experiments were reported, and the drop plate (DP) method was used to determine the number of viable suspended bacteria in a known beaker volume. The results from this assessment suggest that guanidine-core small molecules hold promise as therapeutic alternatives for treating infections caused by clinical Gram-negative and Gram-positive bacteria, highlighting the need for further studies to explore their potential. The results from this assessment suggest that the chemical structure of CAPP4 might serve as the basis for designing more active guanidine-based antimicrobial compounds, highlighting the need for further studies to explore their potential.

Production of Reverse Transcriptase and DNA Polymerase in Bacterial Expression Systems.

DNA amplification and reverse transcription enzymes have proven to be invaluable in fast and reliable diagnostics and research applications because of their processivity, specificity, and robustness. Our study focused on the production of mutant Taq DNA polymerase and mutant M-MLV reverse transcriptase in the expression hosts Vibrio natriegens and Escherichia coli under various expression conditions. We also examined nonspecific extracellular production in V. natriegens. Intracellularly, M-MLV was produced in V. natriegens at the level of 11% of the total cell proteins (TCPs) compared with 16% of TCPs in E. coli. We obtained a soluble protein that accounted for 11% of the enzyme produced in V. natriegens and 22% of the enzyme produced in E. coli. Taq pol was produced intracellularly in V. natriegens at the level of 30% of TCPs compared with 26% of TCPs in E. coli. However, Taq pol was almost non-soluble in E. coli, whereas in V. natriegens, we obtained a soluble protein that accounted for 23% of the produced enzyme. We detected substantial extracellular production of Taq pol. Thus, V. natriegens is a suitable alternative host with the potential for production of recombinant proteins.