Antimicrobial and therapeutic potentials of the blackberry extracts against Escherichia coli infection in male albino rats.

BACKGROUND: Blackberries have garnered attention recently due to their high concentration of bioactive components like anthocyanin and their health advantages. Therefore, this study aims to determine the bioactive profile, antioxidant and antibacterial effects of blackberry extracts (BBEs). Then, evaluate the protective effect of BBEs (20%, 30% and 40%) in a rat model of 2 mL of 4-8 × 106 Escherichia coli ATTC 25922 strain colony-forming unit mL-1 oral infection on the seventh day of the experiment. RESULTS: Rats were divided into six groups: G1: control (C-: normal or negative group), G2: (C+: infected or positive group), G3: infected-treated group by 20% BBE, G4: infected-treated group by 30% BBE, G5: infected-treated group by 40% of BBE and G6: infected-treated group by Gentamicin. The results showed that BBE had a high content of total phenolic compounds, flavonoid, anthocyanin contents, and different vitamins (vitamins A, E and C), reaching 450, 186, 58.83 mg 100 g-1, 2.68, 2.14 and 107.46 mg 100g-1 fresh weight, respectively, which showed great antioxidant and antibacterial effects. Therefore, liver enzymes, kidney function and lipid profiles were significantly higher in the infected group than in the control or infected-treated groups. Furthermore, BBE ameliorated inflammation of the intestine and hepatocyte damage compared to the infected control group. CONCLUSION: These results suggest that consistent intake of BBE might alleviate hepatic inflammation and the gut microbiota in ways that could significantly impact human health. © 2024 Society of Chemical Industry.

Bacterial Opsonization Changes Adhesion Interactions between Endothelial Cells and Neutrophils in a Model of Experimental Septicemia.

The influence of non-opsonized and opsonized S. aureus 2879M and E. coli 321 strains on the total strength of interaction between the endothelial cell and neutrophil during the docking process was studied using in vitro model of experimental septicemia. We observed a decrease in the force and work of adhesion between receptors of neutrophils and endothelial cells under the influence of non-opsonized strains and further decrease in the affinity of single interactions between cells under the influence of opsonized S. aureus, which was compensated by an increase in the number of contacts, as well as an increase in the force of adhesion under the influence of opsonized E. coli compared to non-opsonized bacteria, which remained below the control level, while adhesion work reaches the control level. Thus, opsonization of S. aureus aggravates the "immunological uncoupling" between neutrophils and endothelial cells, while opsonization of E. coli reduces the pathological effect compared to non-opsonized bacteria.

Host-derived CEACAM-laden vesicles engage enterotoxigenic E. coli for elimination and toxin neutralization.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections.

FhlA is a Formate Binding Protein.

Escherichia coli uses glycolysis and mixed acid fermentation and produces formate as by product. One system E. coli uses for formate oxidation is formate hydrogen lyase complex (FHL). The expression of the FHL complex is dependent on formate and regulated by the transcriptional regulator FhlA. The structure of FhlA is composed of three domains. The N-terminal domain is putatively responsible for formate binding and FhlA oligomerization as a tetramer, the central portion of FhlA contains a AAA+ domain that hydrolyzes ATP, and the C-terminal domain binds DNA. Formate enhances FhlA-mediated expression of FHL; however, FhlA direct interaction with formate has never been demonstrated. Formate-protein interactions are challenging to assess, due to the small and ubiquitous nature of the molecule. Here, we have developed three techniques to assess formate-protein interaction. We use these techniques to confirm that FhlA binds formate in the N-terminal domain in vitro, and that this interaction is partially dependent on residues E183 and E363, consistent with previous reports. This study is a proof of concept that these techniques can be used to assess other formate-protein interactions.

Investigating Urinary Pathogens and Their Antibiotic Resistance: A Cross-Sectional Urine Culture Study.

INTRODUCTION: Urinary tract infections (UTIs) are among the most prevalent infectious diseases. Females are more affected than males. The primary culprit is Escherichia coli. Multiple research investigations have documented widespread antimicrobial resistance in uropathogens, sparking global concerns, especially regarding the rise of multidrug resistance (MDR). METHODOLOGY: This cross-sectional study was conducted from December 2023 to March 2024. A non-probability purposive sampling technique was employed to select participants, and informed consent was obtained from them. Data were extracted from the culture and sensitivity reports of these patients. The collected data were meticulously entered into IBM SPSS Statistics for Windows, Version 21 (IBM Corp., Armonk, NY). The findings were then presented using a blend of percentages and numerical figures, offering a clear and concise representation of the data. RESULTS: Our study of 313 participants showed a higher prevalence of UTIs in females (219, 70%) compared to males (94, 30%). E. coli and Citrobacter were the predominant pathogens, with E. coli and Citrobacter more common in females, while Enterobacter and Staphylococcus were more prevalent in males. Antibiogram analysis revealed sensitivities to specific drugs like nitrofurantoin and meropenem, while resistance was observed against others, including polymyxin B and ampicillin. These findings stress the need for tailored UTI treatment approaches. CONCLUSIONS: In conclusion, our research highlights a concerning trend of escalating antibiotic resistance among Pakistani patients with UTIs. Tobramycin B, ticarcillin-clavulanic acid, ampicillin, and clotrimazole exhibited the highest resistance rates, while imipenem, meropenem, nitrofurantoin, sulfonamides, and tigecycline demonstrated notable sensitivity. These findings emphasize the urgent need for the exploration of alternative treatment options to combat rising resistance levels effectively.

Occurrence of blaOXA-116 Carbapenemase in Escherichia coli ST2519 of Clinical Origin: A Report from Northeast India.

Carbapenem-resistant Escherichia coli pose a significant threat to global public health due to the dearth of available treatment options, resulting in infections with high mortality and morbidity. The study aimed to investigate the mechanism of carbapenem resistance in a carbapenem non-susceptible E. coli isolate recovered from an urinary tract infection patient admitted to a tertiary referral hospital, through whole-genome sequencing using Illumina NovaSeq 6000 platform. Carbapenemase production followed by antibiotic susceptibility testing were performed following Clinical Laboratory Standard Institute guidelines. Polymerase chain reaction targeting carbapenemase genes was performed followed by an investigation of horizontal transferability. The Center for Genomic Epidemiology database was used to analyze the sequenced data. ST2519 E. coli BJD_EC1808 with a genome size of 5.8 Mb harbored Col440I plasmid and a chromosomally located blaOXA-116 gene with an IS18 element upstream, along with multiple antibiotic resistance genes conferring clinical resistance toward beta-lactams, aminoglycosides, amphenicols, sulfonamides, tetracyclines, trimethoprim, rifampin, macrolide, and streptogramin antibiotics and antiseptics. E. coli ST2519 harboring blaOXA-116 associated with a mobile genetic element exhibiting carbapenem resistance is a public health threat due to its limiting effect on the therapeutic usage of carbapenem and their dissemination into carbapenem non-susceptible phenotypes will contribute to carbapenem resistance burden and, therefore, warrants urgent monitoring and clinical intervention.

Antimicrobial non-porous surfaces: a comparison of the standards ISO 22196:2011 and the recently published ISO 7581:2023.

The application of antimicrobial surfaces requires the proof of their effectivity by in vitro methods in laboratories. One of the most well-known test methods is ISO 22196:2011, which represents a simple and inexpensive protocol by applying the bacterial suspension with known volume and concentration covered under a polyethylene film on the surfaces. The incubation is then done under defined humidity conditions for 24 h. Another approach for testing of non-porous surfaces is the newly published ISO 7581:2023. A "dry test" is achieved through spreading and drying 1 µL of a bacterial suspension on the surface. In this study, low alloyed carbon steel, polyethylene terephthalate (PET), and glass specimens were tested uncoated (reference) and coated with zinc according to both ISOs to compare and to evaluate the advantages and disadvantages of each one of them. Although ISO 7581:2023 allows a more realistic test environment than ISO 22196:2011, the reproducibility of the results is not given due to the low application volume. In addition, not all bacterial strains are equally suitable for this testing type. Individual adaptations to the protocols, including incubation conditions (time, temperature, or relative humidity), testing strains and volume, seem necessary to generate conditions that simulate the final application. Nevertheless, both ISOs, if used correctly, provide a good basis for estimating the antimicrobial efficacy of non-porous surfaces.

Influence of Kidney Environment Parameters on Antibiotic Efficacy Against Uropathogenic Escherichia coli.

BACKGROUND AND OBJECTIVE: Urinary tract infections (UTIs) are common infections affecting the urinary system, predominantly caused by bacterial pathogens, with Escherichia coli being the most frequent pathogen. Infections of the kidney (eg, pyelonephritis) are severe and challenging to treat, due to the specific tissue microenvironment. In this study, the influence of different parameters mimicking the kidney environment on the effectiveness of antibiotics prescribed for pyelonephritis on the growth of uropathogenic strains was analyzed. METHODS: To investigate the influence of different factors mimicking the kidney environment, we tested the effect of different kidney-representative concentrations of sodium chloride and urea, and different pH values on the efficacy of ertapenem, levofloxacin, and ceftriaxone. The effectiveness was assessed by determining the minimal inhibitory concentrations (MICs) against various E. coli strains. KEY FINDINGS AND LIMITATIONS: The study revealed that pH significantly influences the MIC values of levofloxacin. Acidification of the pH led to an increase of the MIC values, while an alkaline pH had the opposite effect. The influence of sodium chloride and urea concentrations was strain and antibiotic specific. Since three different antibiotics were tested in this study, further research with additional antibiotics is warranted. CONCLUSIONS AND CLINICAL IMPLICATIONS: These results suggest that the physicochemical conditions within the kidney can substantially influence the success of antibiotic therapy for pyelonephritis. Therefore, it is crucial for clinicians to consider these factors when selecting and dosing antibiotics. Further research is needed to evaluate a broader range of antibiotics and additional environmental parameters, to develop a more comprehensive understanding of how the kidney environment affects antimicrobial activity. This knowledge will be vital in optimizing treatment strategies for pyelonephritis, ultimately improving patient outcomes. PATIENT SUMMARY: The physicochemical conditions within the kidney influence the success of antibiotic therapy for pyelonephritis. Our findings are vital in optimizing treatment strategies and will ultimately improve patient outcomes.

Coupling enterotoxigenic Escherichia coli heat-stable peptide toxin with 8-arm PEG enhances immunogenicity.

Enterotoxigenic Escherichia coli (ETEC) strains, which produce the heat-stable enterotoxin (ST) either alone or in combination with the heat-labile enterotoxin, contribute to the bulk of the burden of child diarrheal disease in resource-limited countries and are associated with mortality. Developing an effective vaccine targeting ST presents challenges due to its potent enterotoxicity, non-immunogenicity, and the risk of autoimmune reaction stemming from its structural similarity to the human endogenous ligands, guanylin, and uroguanylin. This study aimed to assess a novel synthetic vaccine carrier platform employing a single chemical coupling step for making human ST (STh) immunogenic. Specifically, the method involved cross-linking STh to an 8-arm N-hydroxysuccinimide (NHS) ester-activated PEG cross-linker. A conjugate of STh with 8-arm structure was prepared, and its formation was confirmed through immunoblotting analysis. The impact of conjugation on STh epitopes was assessed using ELISAs with polyclonal and monoclonal antibodies targeting various epitopes of STh. Immunization of mice with the conjugate induced the production of anti-STh antibodies, exhibiting neutralizing activity against STh.

Stress response in Escherichia coli following sublethal phenalene-1-one mediated antimicrobial photodynamic therapy: an RNA-Seq study.

Since the molecular mechanisms behind adaptation and the bacterial stress response toward antimicrobial photodynamic therapy (aPDT) are not entirely clear yet, the aim of the present study was to investigate the transcriptomic stress response in Escherichia coli after sublethal treatment with aPDT using RNA sequencing (RNA-Seq). Planktonic cultures of stationary phase E. coli were treated with aPDT using a sublethal dose of the photosensitizer SAPYR. After treatment, RNA was extracted, and RNA-Seq was performed on the Illumina NextSeq 500. Differentially expressed genes were analyzed and validated by qRT-PCR. Furthermore, expression of specific stress response proteins was investigated using Western blot analysis.The analysis of the differential gene expression following pathway enrichment analysis revealed a considerable number of genes and pathways significantly up- or down-regulated in E. coli after sublethal treatment with aPDT. Expression of 1018 genes was up-regulated and of 648 genes was down-regulated after sublethal treatment with aPDT as compared to irradiated controls. Analysis of differentially expressed genes and significantly de-regulated pathways showed regulation of genes involved in oxidative stress response and bacterial membrane damage. In conclusion, the results show a transcriptomic stress response in E. coli upon exposure to aPDT using SAPYR and give an insight into potential molecular mechanisms that may result in development of adaptation.

Synthesis of a Phosphoethanolamine Cellulose Mimetic and Evaluation of Its Unanticipated Biofilm Modulating Properties.

When coordinating and adhering to a surface, microorganisms produce a biofilm matrix consisting of extracellular DNA, lipids, proteins, and polysaccharides that are intrinsic to the survival of bacterial communities. Indeed, bacteria produce a variety of structurally diverse polysaccharides that play integral roles in the emergence and maintenance of biofilms by providing structural rigidity, adhesion, and protection from environmental stressors. While the roles that polysaccharides play in biofilm dynamics have been described for several bacterial species, the difficulty in isolating homogeneous material has resulted in few structures being elucidated. Recently, Cegelski and co-workers discovered that uropathogenic Escherichia coli (UPEC) secrete a chemically modified cellulose called phosphoethanolamine cellulose (pEtN cellulose) that plays a vital role in biofilm assembly. However, limited chemical tools exist to further examine the functional role of this polysaccharide across bacterial species. To address this critical need, we hypothesized that we could design and synthesize an unnatural glycopolymer to mimic the structure of pEtN cellulose. Herein, we describe the synthesis and evaluation of a pEtN cellulose glycomimetic which was generated using ring-opening metathesis polymerization. Surprisingly, the synthetic polymers behave counter to native pEtN cellulose in that the synthetic polymers repress biofilm formation in E. coli laboratory strain 11775T and UPEC strain 700415 with longer glycopolymers displaying greater repression. To evaluate the mechanism of action, changes in biofilm and cell morphology were visualized using high resolution field-emission gun scanning electron microscopy which further revealed changes in cell surface appendages. Our results suggest synthetic pEtN cellulose glycopolymers act as an antiadhesive and inhibit biofilm formation across E. coli strains, highlighting a potential new inroad to the development of bioinspired, biofilm-modulating materials.

Anti-microbial, anti-biofilm, and efflux pump inhibitory effects of ellagic acid-bonded magnetic nanoparticles against Escherichia coli isolates.

The spread of microbial resistance is a threat to public health. In this study, the anti-microbial, anti-biofilm, and efflux pump inhibitory effects of ellagic acid-loaded magnetic nanoparticles (Fe3O4NPs@EA) against beta-lactamase producing Escherichia coli isolates have been investigated. The effects of Fe3O4 NPs@EA on the growth inhibition of E. coli isolates were determined by disc diffusion method and determining the minimum inhibitory concentration was done using broth micro-dilution method. The anti-biofilm effect of nanoparticles was investigated using the microplate method. The efflux pump inhibitory effect of nanoparticles was investigated using cart-wheel method and by investigating the effect of nanoparticles on acrB and tolC genes expression levels. Fe3O4 NPs@EA showed anti-bacterial effects against test bacteria, and the MIC of these nanoparticles varied from 0.19 to 1.56 mg/mL. These nanoparticles caused a 43-62% reduction in biofilm formation of test bacteria compared to control. Furthermore, efflux pump inhibitory effect of these nanoparticles was confirmed at a concentration of 1/8 MIC, and the expression of acrB and tolC genes decreased in bacteria treated with 1/4 MIC Fe3O4 NPs@EA. According to the results, the use of nanoparticles containing ellagic acid can provide a basis for the development of new treatments against drug-resistant E. coli. This substance may improve the concentration of antibiotics in the bacterial cell and increase their effectiveness by inhibiting the efflux in E. coli isolates.

Molecular detection of avian pathogens in poultry red mite (Dermanyssus gallinae) in Algerian layer farms as a potential predictive tool.

The poultry red mite Dermanyssus gallinae is a hematophagous ectoparasite of layer hens. Infestations with poultry red mites pose an increasing threat to the egg production industry, causing serious problems to animal health and welfare, directly or indirectly as a vector of several infectious agents. In this study, we aimed to investigate common avian pathogens in mites. The mite samples were collected from 58 poultry farms in 7 regions accounting for more than 70 % of the national egg production in Algeria. The presence of 13 avian pathogens was detected using DNA and RNA samples from mites collected. Results revealed significant associations between PRM and potential pathogens such as Escherichia coli, Salmonella enterica, fowlpox virus, and gallid herpesvirus 1. Pathogen detection in Dermanyssus gallinae could serve as an early diagnostic or a risk analysis tool for infectious diseases in poultry farms, facilitating effective disease management strategies. Despite further research being necessary to address uncertainties, such a strategy could be used to enhance the integrated management of poultry health.

Efficient synthesis of 5-hydroxytryptophan in Escherichia coli by bifunctional utilization of whey powder as a substrate for cell growth and inducer production.

5-Hydroxytryptophan (5-HTP), a precursor of the neurotransmitter serotonin in mammals, has demonstrated efficacy in treating various diseases such as depression, fibromyalgia and obesity. However, conventional biosynthesis methods of 5-HTP are limited by low yield and high reagent and process costs. In this study, the strain C1T7-S337A/F318Y with optimized promoter distribution was obtained, and the 5-HTP yield was 60.30 % higher than that of the initial strain. An efficient fermentation process for 5-HTP synthesis was developed using strain C1T7-S337A/F318Y with whey powder as a substrate for cell growth and inducer production. Shake flask fermentation experiments yielded 1.302 g/L 5-HTP from 2.0 g/L L-tryptophan (L-Trp), surpassing the whole-cell biocatalysis by 42.86 %. Scale-up to a 5 L fermenter further increased the yield to 1.649 g/L. This fermentation strategy substantially slashed reagent cost by 95.39 %, providing a more economically viable and environmentally sustainable route for industrial biosynthesis of 5-HTP. Moreover, it contributes to the broader utilization of whey powder in various industries.

Protective effect of dihydromyricetin on intestinal epithelium in weaned pigs upon enterotoxigenic Escherichia coli challenge.

Dihydromyricetin (DMY), a natural flavonoid compound, are believed to prevent inflammatory response, dealing with pathogens and repairing the intestinal barrier. The objective of this study was to investigate whether DMY supplementation could attenuate intestinal damage in the context of enterotoxigenic Escherichia coli K88 (ETEC F4+) infection. After weaning, different litters of pigs were randomly assigned to one of the following treatments: (1) non-challenged control (CON, fed with basal diet); (2) ETEC-challenged control (ECON, fed with basal diet); and (3) ETEC challenge + DMY treatment (EDMY, fed with basal diet plus 300 mg kg-1 DMY). We observed a significant reduction in fecal Escherichia coli shedding and diarrhea incidence, but an increase in ADG in pigs of EDMY group compared to the pigs of ECON group. Relative to the pigs of ECON group, dietary DMY treatment decreased (P < 0.05) concentrations of the serum D-xylose, D-lactate and diamine oxidase (DAO), but increased the abundance of zonula occludens-1 (ZO-1) in the jejunum of pigs. In addition, DMY also decreased (P < 0.05) the number of S-phase cells and the percentage of total apoptotic epithelial cells of jejunal epithelium in pigs of the EDMY group compared to the pigs of the ECON group. Furthermore, DMY decreased the mRNA expression levels of critical immune-associated genes TLR4, NFκB, Caspase3, Caspase9, IL-1ß, IL-6, TNF-α and the protein p-NFκB and p-IκBα expressions of intestinal epithelium in pigs of the EDMY group compared to the pigs of the ECON group. Compared to the ECON group, DMY elevated (P < 0.05) the expression levels of ß-defensins PBD1, PBD2, PBD3, PBD129, as well as the abundance of secreted IgA in intestinal mucosae of the EDMY group. Thus, our results indicate that DMY may relieve intestinal integrity damage due to Escherichia coli F4.

Genome characterization of a multi-drug resistant Escherichia coli strain, L1PEag1, isolated from commercial cape gooseberry fruits (Physalis peruviana L.).

Introduction: Foodborne infections, which are frequently linked to bacterial contamination, are a serious concern to public health on a global scale. Whether agricultural farming practices help spread genes linked to antibiotic resistance in bacteria associated with humans or animals is a controversial question. Methods: This study applied a long-read Oxford Nanopore MinION-based sequencing to obtain the complete genome sequence of a multi-drug resistant Escherichia coli strain (L1PEag1), isolated from commercial cape gooseberry fruits (Physalis peruviana L.) in Ecuador. Using different genome analysis tools, the serotype, Multi Locus Sequence Typing (MLST), virulence genes, and antimicrobial resistance (AMR) genes of the L1PEag1 isolate were determined. Additionally, in vitro assays were performed to demonstrate functional genes. Results: The complete genome sequence of the L1PEag1 isolate was assembled into a circular chromosome of 4825.722 Kbp and one plasmid of 3.561 Kbp. The L1PEag1 isolate belongs to the B2 phylogroup, sequence type ST1170, and O1:H4 serotype based on in silico genome analysis. The genome contains 4,473 genes, 88 tRNA, 8 5S rRNA, 7 16S rRNA, and 7 23S rRNA. The average GC content is 50.58%. The specific annotation consisted of 4,439 and 3,723 genes annotated with KEEG and COG respectively, 3 intact prophage regions, 23 genomic islands (GIs), and 4 insertion sequences (ISs) of the ISAs1 and IS630 families. The L1PEag1 isolate carries 25 virulence genes, and 4 perfect and 51 strict antibiotic resistant gene (ARG) regions based on VirulenceFinder and RGI annotation. Besides, the in vitro antibiotic profile indicated resistance to kanamycin (K30), azithromycin (AZM15), clindamycin (DA2), novobiocin (NV30), amikacin (AMK30), and other antibiotics. The L1PEag1 isolate was predicted as a human pathogen, matching 464 protein families (0.934 likelihood). Conclusion: Our work emphasizes the necessity of monitoring environmental antibiotic resistance, particularly in commercial settings to contribute to develop early mitigation techniques for dealing with resistance diffusion.

Potential of bacteriophage phT4A as a biocontrol agent against Escherichia coli in food matrices.

Escherichia coli is one of the most prevalent foodborne pathogens, frequently found in meat and dairy products. Current decontamination methods are often associated with changes in organoleptic characteristics, nutrient loss, and potentially harmful side effects. Furthermore, despite the array of available methods, foodborne outbreaks still frequently occur. For this reason, bacteriophages (or simply phages) emerged as a natural alternative for the biocontrol of bacterial contamination in food without altering their organoleptic properties. In this study, the potential of phage phT4A was assessed in the biocontrol of E. coli in liquid (milk) and solid (ham) food matrices. Firstly, as foods have different pH and temperature values, the influence of these parameters on phage phT4A viability was also assessed to develop an effective protocol. Phage phT4A proved to be stable for long storage periods at pH 7-8 (56 days) and temperatures of 4-37 °C (21 days). Before application of phages to inactivate pathogenic bacteria in food, previous assays were carried out in Tryptic Soy Broth (TSB) to study the dynamics of phage-bacteria interaction. Then, the antibacterial potential of phage phT4A was evaluated in the two food matrices at different temperatures (4, 10 and 25 °C). This phage was more efficient at 25 °C in all tested matrices (maximum inactivation of 6.6, 3.9 and 1.8 log CFU/mL in TSB, milk and ham, respectively) than at 10 °C (maximum decrease of 4.7, 2.1 and 1.0 log CFU/mL in TSB, milk and ham, respectively) and 4 °C (maximum reduction of 2.6 and 0.7 log CFU/mL in TSB and milk, respectively). However, the decrease of temperature from 25 °C to 10 and 4 °C prevented bacterial regrowth. The results suggest that during phage treatment, a balance between an incubation temperature that provide effective results in terms of bacterial inactivation by the phages and at the same time prevents or delays bacterial regrowth, is needed. The application of phage phT4A at a temperature of 10 °C can be an effective strategy in terms of bacterial inactivation, delaying bacterial regrowth and also reducing energy costs.

The multifaceted role of TolA protein in promoting survival, biofilm formation and virulence of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) can spread beyond the intestines and cause systemic infections, leading to various clinical manifestations, including airsacculitis, pericarditis, perihepatitis and colisepticemia. The mechanisms facilitating this extraintestinal infections are not fully understood. In this study, we investigate how the tolA gene affects APEC virulence by encoding a protein involved in maintaining outer membrane integrity. We constructed a tolA deletion mutant of APEC strain E058 and evaluated its growth and survival in various environments, including in vitro cultures and in vivo infection models in chickens. We found that the motility-defective ΔtolA mutant exhibits reduced biofilm formation ability and weakened resistance to the environmental stresses, suggesting an important role for TolA in APEC's survival. The lack of tolA gene affects the bacterial ability to resist the host's immune system, such as complement-mediated serum killing or phagocytosis, as shown by the serum killing and macrophage phagocytosis assays. Additionally, in vivo infection studies using chickens demonstrated that the ΔtolA mutant displayed attenuated virulence, evidenced by reduced mortality and lower tissue bacterial burden. Reverse transcription quantitative real-time PCR (RT-qPCR) analysis revealed that inactivation of tolA led to downregulation of virulence genes associated with serum resistance (traT) and flagellar biosynthesis (fliR). Taken together, our findings demonstrate the multifaceted role of TolA protein in promoting the survival, immune evasion, biofilm formation, and virulence of APEC E058. This suggests that targeting TolA could potentially offer new strategies for combating APEC infections.

Isolation, Identification, and Characterization of L-asparaginase-Producing Human Commensal Bacterial Strains: A Promising Next-Gen Probiotics.

L-asparaginase is an FDA-approved drug for treating blood cancer, but its inherent antigenicity and L-glutaminase activity are associated with hypersensitivity and organ toxicity. Extracellularly produced glutaminase-free L-asparaginase from human commensal bacteria may be a good alternative to reduce the side effects of therapeutic L-asparaginase. Here, we report the isolation and characterization of fourteen L-asparaginase-producing bacterial strains belonging to the genera Acinetobacter, Escherichia, Klebsiella, and Pseudomonas from human stool and saliva samples. To the best of our knowledge, this is the first report of L-asparaginase-producing human commensal bacterial strains isolated from healthy individuals. L-asparaginase produced by fecal and salivary isolates exhibited significantly higher activity (3.64 to 16.96 U/ml) toward L-asparagine than L-glutamine. Interestingly, L-asparaginase from fecal isolates, Escherichia coli strains 3F1 and 3F2 and salivary isolate Klebsiella pneumoniae 3S3, exhibited no L-glutaminase activity. These isolates were also sensitive to all tested antibiotics. Additionally, these three isolates demonstrated tolerance to pH 3.0 (≥ 88% survival) and 0.3% bile (≥ 95% survival), indicating their potential as probiotics. Among these isolates, L-asparaginase from the highest-producing K. pneumoniae 3S3 strain was found to be a homodimer, with native and subunit molecular weights of 110 kDa and 55 kDa, respectively. The purified enzyme can be further explored for its antitumor and immunomodulatory properties. Overall, future research can be expanded to include the use of a pool of human commensal bacteria as genuine and alternative sources of L-asparaginase for effective cancer treatments and cutting-edge next-generation probiotics.

Investigating bottom phase extraction from aqueous two-phase systems for detecting bacteria using the lateral-flow immunoassay.

Lateral-flow immunoassays (LFAs) can be used to diagnose urinary tract infections caused by Escherichia coli (E. coli) at the point of care. Unfortunately, urine samples containing dilute concentrations of E. coli can yield false negative results on LFAs. Our laboratory was first to implement aqueous two-phase systems (ATPSs) to preconcentrate samples into smaller volumes prior to their application on LFAs. This is achieved by manipulating the ratio of the volume of the top phase to that of the bottom phase (volume ratio; VR) and concentrating biomarkers in the bottom phase which, when applied to LFAs in fixed volumes, leads to corresponding improvements in sensitivity. This work is the first demonstration that the same LOD can be achieved irrespective of the VR when the entire bottom phase is added to LFAs. A custom 3D-printed device was also developed to decrease liquid handling steps. Across different VRs expected from patient urine variability, this diagnostic workflow successfully detected E. coli concentrations down to 2 × 105 colony-forming units (cfu) mL-1 in synthetic urine, demonstrating consistent 10-fold improvements in sensitivity compared to trials conducted without ATPS preconcentration. This method successfully addresses the variability of patient samples while remaining easy to use at the point of care.