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The innate immune system eliminates bloodstream pathogens such as Escherichia coli in part through complement protein deposition and subsequent bacterial death (i.e., "serum killing"). Some E. coli strains have developed mechanisms to resist serum killing, though the extent of variation in serum killing among bloodstream infection (BSI) isolates and the clinical impact of this variation is not well understood. To address this issue, we developed a novel assay that uses flow cytometry to perform high throughput serum bactericidal assays (SBAs) with E. coli BSI isolates (n = 183) to define the proportion of surviving bacteria after exposure to serum. We further determined whether E. coli resistance to serum killing is associated with clinical outcomes (e.g., in-hospital attributable mortality, in-hospital total mortality, septic shock) and bacterial genotype in the corresponding patients with E. coli BSI. Our novel flow cytometry-based SBA performed similarly to a traditional SBA, though with significantly decreased hands-on bench work. Among E. coli BSI isolates, the mean proportion that survived exposure to 25% serum was 0.68 (Standard deviation 0.02, range 0.57–0.93). We did not identify associations between E. coli resistance to serum killing and clinical outcomes in our adjusted models. Together, this study describes a novel flow cytometry-based approach to the bacterial SBA that allowed for high-throughput testing of E. coli BSI isolates and identified high variability in resistance to serum killing among a large set of BSI isolates. [ABSTRACT FROM AUTHOR]

The aim of the present study was to determine if colostrum and the equipment for harvesting and feeding colostrum are sources of fecal ESBL/AmpC-producing Escherichia coli (ESBL/AmpC-E. coli) in calves. Therefore, 15 male calves fed with pooled colostrum on a dairy farm and held individually in an experimental barn, the colostrum pool and the equipment for harvesting and feeding colostrum were sampled and analyzed for the occurrence of ESBL/AmpC-E. coli. The ESBL-AmpC-E. coli suspicious isolates were subjected to whole-genome sequence analysis. Forty-three of 45 fecal samples were tested positive for ESBL/AmpC-E. coli. In the colostrum sample and in the milking pot, we also found ESBL/AmpC-E. coli. All 45 E. coli isolates were ESBL-producers, mainly commensal sequence type (ST) 10, but also human-extraintestinal pathogenic E. coli ST131 and ST117 were found. The clonal identity of six fecal isolates with the ESBL-E. coli isolate from the colostrum and of five fecal isolates with the strain from the milking pot demonstrates that the hygiene of colostrum or the colostrum equipment can play a significant role in the spread of ESBL-E. coli. Effective sanitation procedures for colostrum harvesting and feeding equipment are crucial to reduce the ESBL-E. coli shedding of neonatal dairy calves. [ABSTRACT FROM AUTHOR]

Background: Uropathogenic Escherichia coli (UPEC) activates innate immune response upon invading the urinary tract, whereas UPEC can also enter bladder epithelial cells (BECs) through interactions with fusiform vesicles on cell surfaces and subsequently escape from the vesicles into the cytoplasm to establish intracellular bacterial communities, finally evading the host immune system and leading to recurrent urinary tract infection (RUTI). Tailin Fang II (TLF-II) is a Chinese herbal formulation composed of botanicals that has been clinically proven to be effective in treating urinary tract infection (UTI). However, the underlying therapeutic mechanisms remain poorly understood. Methods: Network pharmacology analysis of TLF-II was conducted. Female Balb/C mice were transurethrally inoculated with UPEC CFT073 strain to establish the UTI mouse model. Levofloxacin was used as a positive control. Mice were randomly divided into four groups: negative control, UTI, TLF-II, and levofloxacin. Histopathological changes in bladder tissues were assessed by evaluating the bladder organ index and performing hematoxylin-eosin staining. The bacterial load in the bladder tissue and urine sample of mice was quantified. Activation of the TLR4-NF-kB pathway was investigated through immunohistochemistry and western blotting. The urinary levels of interleukin (IL)-1b and IL-6 and urine leukocyte counts were monitored. We also determined the protein expressions of markers associated with fusiform vesicles, Rab27b and Galectin-3, and levels of the phosphate transporter protein SLC20A1. Subsequently, the co-localization of Rab27b and SLC20A1 with CFT073 was examined using confocal fluorescence microscopy. Results: Data of network pharmacology analysis suggested that TLF-II could against UTI through multiple targets and pathways associated with innate immunity and inflammation. Additionally, TLF-II significantly attenuated UPEC-induced bladder injury and reduced the bladder bacterial load. Meanwhile, TLF-II inhibited the expression of TLR4 and NF-kB on BECs and decreased the urine levels of IL-1b and IL-6 and urine leukocyte counts. TLF-II reduced SLC20A1 and Galectin-3 expressions and increased Rab27b expression. The co-localization of SLC20A1 and Rab27b with CFT073 was significantly reduced in the TLF-II group. Conclusion: Collectively, innate immunity and bacterial escape from fusiform vesicles play important roles in UPEC-induced bladder infections. Our findings suggest that TLF-II combats UPEC-induced bladder infections by effectively mitigating bladder inflammation and preventing bacterial escape from fusiform vesicles into the cytoplasm. The findings suggest that TLF-II is a promising option for treating UTI and reducing its recurrence. [ABSTRACT FROM AUTHOR]

Background: Bacterial identification can be done using various testing techniques. Molecular techniques are often used to research dangerous diseases, an approach using genetic information on the pathogenic agent. The enterohemorrhagic invasive species Escherichia coli 0157:H7 was identified from the feces of working horses on the island of Sumbawa. Another advance in molecular technology is genome amplification with qPCR which is the gold standard for detecting E. coli. Aim: This study aims to detect and identify the invasive species E. coli 0157:H7 using the gene encoding chuA with the qPCR method sourced from horse feces. Methods: Fresh fecal samples from horses on Sumbawa Island were isolated and identified, then continued with molecular examination using the gene encoding chuA using the qPCR method. Results: qPCR testing in this study showed that six sample isolates that were positive for E. coli 0157:H7 were detected for the presence of the chuA gene, which is a gene coding for an invasive species of E. coli bacteria. The highest to lowest Cq values and Tm from the qPCR results of the sample isolates were 15.98 (4KJ), 14.90 (19KG), 14.6 (3KJ), 13.77 (20KG), 12.56 (5KGB), and 12.20 (6KJ). Tm values are 86.7 (4KJ), 86.69 (3KJ), 86.56 (5KGB), 85.88 (20KGB), 85.81 (19KG), and 85.74 (6KJ). Conclusion: Validation, standardization of the development, and modification of qPCR technology must be carried out to harmonize testing throughout to avoid wrong interpretation of the test results so that the determination of actions to eradicate and control diseases originating from animals in the field does not occur. [ABSTRACT FROM AUTHOR]

Background and Aim: Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires expensive equipment and sample preparation. The rLipL32 protein is conserved and can be used for the production of immunoglobulin G (IgG) anti-rLipL32 antibody, which can be used as a biomarker for leptospirosis diagnosis. This study aimed to produce and characterize an IgG anti-rLipL32 antibody as a biomarker for leptospirosis diagnosis. Materials and Methods: Escherichia coli rLipL32 was cultured and analyzed by PCR and sequencing. Cultures were used for rLipL32 protein expression and purification and the rLipL32 protein was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The rLipL32 protein was used to produce anti-rLipL32 serum and was analyzed by enzyme-linked immunosorbent assay (ELISA). Serum was purified to obtain IgG anti-rLipL32 antibody and characterized by SDS-PAGE and western blotting. Results: PCR was able to amplify the LipL32 gene from E. coli rLipL32, and sequencing analysis showed 99.19% similarity with pathogenic Leptospira. SDS-PAGE analysis showed a 32-kDa band. ELISA results showed an increase in OD in antirLipL32 serum compared to preimmune serum. Western blotting results showed that the IgG anti-rLipL32 antibody was able to bind and cross-reacts with pathogenic Leptospira serovar but not with E. coli or Staphylococcus aureus. Conclusion: IgG anti-rLipL32 antibody has high specificity and sensitivity against Leptospira pathogens. These findings suggest that IgG anti-rLipL32 antibody is a promising biomarker for the diagnosis of leptospirosis. [ABSTRACT FROM AUTHOR]

Genome-scale metabolic models are powerful tools for understanding cellular physiology. Flux balance analysis (FBA), in particular, is an optimization-based approach widely employed for predicting metabolic phenotypes. In model microbes such as Escherichia coli, FBA has been successful at predicting essential genes, i.e. those genes that impair survival when deleted. A central assumption in this approach is that both wild type and deletion strains optimize the same fitness objective. Although the optimality assumption may hold for the wild type metabolic network, deletion strains are not subject to the same evolutionary pressures and knock-out mutants may steer their metabolism to meet other objectives for survival. Here, we present FlowGAT, a hybrid FBA-machine learning strategy for predicting essentiality directly from wild type metabolic phenotypes. The approach is based on graph-structured representation of metabolic fluxes predicted by FBA, where nodes correspond to enzymatic reactions and edges quantify the propagation of metabolite mass flow between a reaction and its neighbours. We integrate this information into a graph neural network that can be trained on knock-out fitness assay data. Comparisons across different model architectures reveal that FlowGAT predictions for E. coli are close to those of FBA for several growth conditions. This suggests that essentiality of enzymatic genes can be predicted by exploiting the inherent network structure of metabolism. Our approach demonstrates the benefits of combining the mechanistic insights afforded by genome-scale models with the ability of deep learning to infer patterns from complex datasets. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is responsible for the spread and persistence of bacterial infections. Surveillance of AMR in healthy individuals is usually not considered, though these individuals serve as reservoirs for continuous disease transmission. Therefore, it is essential to conduct epidemiological surveillance of AMR in healthy individuals to fully understand the dynamics of AMR transmission in Nigeria. Thirteen multidrug-resistant Citrobacter spp., Enterobacter spp., Klebsiella pneumoniae, and Escherichia coli isolated from stool samples of healthy children were subjected to whole genome sequencing (WGS) using Illumina and Oxford nanopore sequencing platforms. A bioinformatics analysis revealed antimicrobial resistance genes such as the pmrB_Y358N gene responsible for colistin resistance detected in E. coli ST219, virulence genes such as senB, and ybtP&Q, and plasmids in the isolates sequenced. All isolates harbored more than three plasmid replicons of either the Col and/or Inc type. Plasmid reconstruction revealed an integrated tetA gene, a toxin production caa gene in two E. coli isolates, and a cusC gene in K. quasivariicola ST3879, which induces neonatal meningitis. The global spread of AMR pathogenic enteric bacteria is of concern, and surveillance should be extended to healthy individuals, especially children. WGS for epidemiological surveillance will improve the detection of AMR pathogens for management and control. [ABSTRACT FROM AUTHOR]

The physiology of different Escherichia coli stains was analyzed for growth with glycolate as a potentially promising sustainable sole source of carbon and energy. Different E. coli strains showed large differences regarding lag phases after provision of glycolate. Whereas E. coli W showed fast adaptation, E. coli BW25113, JM101, and BL21 (DE3) needed extensive time for adaption (up to 30 generations) until the attainable µ max was reached, which, at 30 °C, amounted to 0.20–0.25 h−1 for all strains. The overexpression of genes encoding glycolate degradation did neither overcome the need for adaptation of E. coli BL21 (DE3) nor improve growth of E. coli W. Rather, high level expression of proteins involved in uptake and initial degradation steps had an adverse effect on growth. Overall, the results show a promising capacity of E. coli strains for growth on glycolate. [Display omitted] • E. coli strains differ regarding adaptation duration for growth on glycolate • Adapted E. coli strains showed efficient growth on glycolate • Overexpression of early degradation genes impairs growth on glycolate. [ABSTRACT FROM AUTHOR]

Biofilms in cooling towers represent a common habitat for the human pathogen Legionella pneumophila. Within the biofilm consortium, frequent interactions with protozoa, i.e. amoebae and ciliates, were reported, while nematodes have only recently been considered as potential environmental reservoir for the pathogenic bacteria. This study is the first approach to investigate the biotic interactions between L. pneumophila and bacterial-feeding nematodes in a semi-natural biofilm model. The species were Diploscapter coronatus, Diploscapter pachys, Plectus similis and Plectus sp., which all co-occur with L. pneumophila in the environment. Biofilms derived from cooling towers were either inoculated with mCherry-labeled L. pneumophila solely or in combination with GFP-labeled Escherichia coli. All experiments were conducted in single-species set-ups and multi-species (D. coronatus and P. similis) set-ups, to account for interspecific competition. Bacterial ingestion was assessed after 24 and 96 h as fluorescence patterns in the digestive tract of the nematodes using confocal laser scanning microscopy. L. pneumophila cells were ingested by all nematode species, with D. coronatus having the highest pathogen load. The fluorescence intensity (i.e. bacterial load) varied between compartments within the digestive tract and was independent of incubation time. Bacterial cells accumulated mostly around the cardia and in the intestine, while less cells were found within stoma and pharynx. Interspecific competition changed the pattern, i.e. with incubation of D. coronatus and P. similis in the same biofilm a significantly higher pathogen load occurred in the intestine of D. coronatus than P. similis after 24 h and 96 h. Remarkably, when given a choice between L. pneumophila and E. coli, P. similis was the only nematode species containing both bacteria after incubation for 24 h. None of the other nematode species contained E. coli after 24 h and 96 h incubation, while L. pneumophila was present. This study thus provides the first evidence, that under environmental conditions L. pneumophila is a frequent diet of bacterial-feeding nematodes, highlighting their potential as pathogen vectors or even host in cooling tower habitats., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ortlieb et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: E. coli chemotactic motion in the presence of a chemonutrient field can be studied using wet laboratory experiments or macroscale-level partial differential equations (PDEs) (among others). Bridging experimental measurements and chemotactic Partial Differential Equations requires knowledge of the evolution of all underlying fields, initial and boundary conditions, and often necessitates strong assumptions. In this work, we propose machine learning approaches, along with ideas from the Whitney and Takens embedding theorems, to circumvent these challenges., Results: Machine learning approaches for identifying underlying PDEs were (a) validated through the use of simulation data from established continuum models and (b) used to infer chemotactic PDEs from experimental data. Such data-driven models were surrogates either for the entire chemotactic PDE right-hand-side (black box models), or, in a more targeted fashion, just for the chemotactic term (gray box models). Furthermore, it was demonstrated that a short history of bacterial density may compensate for the missing measurements of the field of chemonutrient concentration. In fact, given reasonable conditions, such a short history of bacterial density measurements could even be used to infer chemonutrient concentration., Conclusion: Data-driven PDEs are an important modeling tool when studying Chemotaxis at the macroscale, as they can learn bacterial motility from various data sources, fidelities (here, computational models, experiments) or coordinate systems. The resulting data-driven PDEs can then be simulated to reproduce/predict computational or experimental bacterial density profile data independent of the coordinate system, approximate meaningful parameters or functional terms, and even possibly estimate the underlying (unmeasured) chemonutrient field evolution., (© 2024. The Author(s).)

Synthetic genetic oscillators can serve as internal clocks within engineered cells to program periodic expression. However, cell-to-cell variability introduces a dispersion in the characteristics of these clocks that drives the population to complete desynchronization. Here, we introduce the optorepressilator, an optically controllable genetic clock that combines the repressilator, a three-node synthetic network in E. coli , with an optogenetic module enabling to reset, delay, or advance its phase using optical inputs. We demonstrate that a population of optorepressilators can be synchronized by transient green light exposure or entrained to oscillate indefinitely by a train of short pulses, through a mechanism reminiscent of natural circadian clocks. Furthermore, we investigate the system's response to detuned external stimuli observing multiple regimes of global synchronization. Integrating experiments and mathematical modeling, we show that the entrainment mechanism is robust and can be understood quantitatively from single cell to population level., Competing Interests: MC, FL, NP, GF, RD No competing interests declared, (© 2024, Cannarsa et al.)

Antimicrobial resistance (AMR), known as the "silent pandemic," is exacerbated by pathogenic bacteria's ability to form biofilms. Marine compounds hold promise for novel antibacterial drug discovery. Two isolates from preliminary saltwater environment screening demonstrated antimicrobial activity and were subsequently identified as Bacillus subtilis MTUA2 and Bacillus velezensis MTUC2. Minimum inhibitory concentrations (MICs), minimum biofilm inhibition concentrations (MBICs) and minimum biofilm eradication concentrations (MBECs) required to prevent and/or disrupt bacterial growth and biofilm formation were established for MRSA, Staphylococcus aureus, Acinetobacter baumannii and Escherichia coli. The metabolic activity within biofilms was determined by the 2,3,5-triphenyltetrazolium chloride assay. Both Bacillus species exhibited unique antimicrobial effects, reducing MRSA and S. aureus planktonic cell growth by 50% and sessile cell growth for S. aureus and E. coli by 50% and 90%, respectively. No effect was observed against A. baumannii. Significant MBIC and MBEC values were achieved, with 99% inhibition and 90% reduction in MRSA and S. aureus biofilms. Additionally, 90% and 50% inhibition was observed in E. coli and A. baumannii biofilms, respectively, with a 50% reduction in E. coli biofilm. These findings suggest that the mode of action employed by B. subtilis MTUA2 and B. velezensis MTUC2 metabolites should be further characterized and could be beneficial if used independently or in combination with other treatments., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Bacterial infections often involve more than one pathogen. While it is well established that polymicrobial infections can impact disease outcomes, we know little about how pathogens interact and affect each other's behaviour and fitness. Here, we used a microscopy approach to explore interactions between Pseudomonas aeruginosa and six human opportunistic pathogens that often co-occur in polymicrobial infections: Acinetobacter baumannii, Burkholderia cenocepacia, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, and Staphylococcus aureus. When following growing microcolonies on agarose pads over time, we observed a broad spectrum of species-specific ecological interactions, ranging from mutualism to antagonism. For example, P. aeruginosa engaged in a mutually beneficial interaction with E. faecium but suffered from antagonism by E. coli. While we found little evidence for active directional growth towards or away from cohabitants, we observed that some pathogens increased growth in double layers in response to competition and that physical forces due to fast colony expansion had a major impact on fitness. Overall, our work provides an atlas of pathogen interactions, highlighting the diversity of potential species dynamics that may occur in polymicrobial infections. We discuss possible mechanisms driving pathogen interactions and offer predictions of how the different ecological interactions could affect virulence., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Many medical devices implanted in patients to mitigate diseases and medical conditions have different types of topographic features. While appropriate textures can promote the integration of host cells and reduce scar tissue formation, some textured implants with inappropriate topographies have been associated with inflammation, bacterial colonization, or even malignant complications. To better understand how surface topography affects host immune response to colonizing bacteria, a protocol was developed to investigate phagocytosis of bacterial cells attached on polydimethylsiloxane (PDMS) surfaces with different square-shaped recessive patterns. The interaction between activated RAW 264.7 macrophages and Escherichia coli in recessive wells was visualized in 3D using multiple fluorescent markers. The results revealed that there is a threshold dimension of topography, below which phagocytosis of attached bacterial cells is significantly impeded. Specifically, under our experimental condition, up to 100-fold reduction in phagocytosis was observed in square-shaped patterns with 5 µm side length and 10 µm depth compared to the flat control and patterns with 10 µm or longer side length. The spacing between wells also showed significant effects; e.g., phagocytosis in the wells further decreased when spacing increased to 50 µm. These results are helpful for understanding how undesired topographies may contribute to bacterial colonization and thus infection and other associated complications. STATEMENT OF SIGNIFICANCE: Surface topography plays an important role in bacteria-material infections and thus the safety of implantable medical devices. Undesired topographic features can cause biofilm formation and related complications. However, how surface topography affects the capability of host immune cells to clear colonizing bacteria is not well understood. In this study, the interaction between macrophage RAW264.7 and colonizing E. coli cells on polydimethylsiloxane (PDMS) with recessive features is investigated. It was discovered that the size of recessive features and the spacing between these features have significant effects on phagocytosis of bacteria by macrophages. These new results are helpful for understanding the complex interaction among host cells, bacteria, and implanted biomaterials, which will help guide the rational design of safer medical devices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. All rights reserved.)

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., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Oxidative stress in combination with acid stress has been shown to inactivate a wide spectrum of microorganisms, including multi-resistant bacteria. This occurs e.g. in phagolysosomes or during treatment by cold atmospheric pressure plasmas (CAP) and possibly depends on the cell membrane. We therefore explored the effects of CAP-generated reactive oxygen and nitrogen species (RONS) on bacterial growth inhibition and membranes in neutral and acidic suspensions. We observed that growth inhibition was most efficient when bacteria were treated by a mix of short and long-lived RONS in an acidic environment. Membrane packing was affected mainly upon contact with short-lived RONS, while also acidity strongly modulated packing. Under these conditions, Gram-negative bacteria displayed large potassium release while SYTOX Green influx remained marginal. Growth inhibition of Gram-negative bacteria correlated well with outer membrane (OM) permeabilization that occurred upon contact with short and/or long-lived RONS in synergy with acidity. In Gram-positive bacteria, CAP impaired membrane potential possibly through pore formation upon contact with short-lived RONS while formation of membrane protein hydroperoxides was probably involved in these effects. In summary, our study provides a wide perspective on understanding inactivation mechanisms of bacteria by RONS in combination with acidity., (© 2024. The Author(s).)

Diverse insects are intimately associated with specific symbiotic bacteria, where host and symbiont are integrated into an almost inseparable biological entity. These symbiotic bacteria usually exhibit host specificity, uncultivability, reduced genome size, and other peculiar traits relevant to their symbiotic lifestyle. How host-symbiont specificity is established at the very beginning of symbiosis is of interest but poorly understood. To gain insight into the evolutionary issue, we adopted an experimental approach using the recently developed evolutionary model of symbiosis between the stinkbug Plautia stali and Escherichia coli . Based on the laboratory evolution of P. stali-E. coli mutualism, we selected Δ cyaA mutant of E. coli as an artificial symbiont of P. stali that has established mutualism by a single mutation. In addition, we selected a natural cultivable symbiont of P. stali of relatively recent evolutionary origin. These artificial and natural symbiotic bacteria of P. stali were experimentally inoculated to symbiont-deprived newborn nymphs of diverse stinkbug species. Strikingly, the mutualistic E. coli was unable to establish infection and support growth and survival of all the stinkbug species except for P. stali , uncovering that host specificity can be established at a very early stage of symbiotic evolution. Meanwhile, the natural symbiont was able to establish infection and support growth and survival of several stinkbug species in addition to P. stali , unveiling that a broader host range of the symbiont has evolved in nature. Based on these findings, we discuss what factors are relevant to the establishment of host specificity in the evolution of symbiosis.IMPORTANCEHow does host-symbiont specificity emerge at the very beginning of symbiosis? This question is difficult to address because it is generally difficult to directly observe the onset of symbiosis. However, recent development of experimental evolutionary approaches to symbiosis has brought about a breakthrough. Here we tackled this evolutionary issue using a symbiotic Escherichia coli created in laboratory and a natural Pantoea symbiont, which are both mutualistic to the stinkbug Plautia stali . We experimentally replaced essential symbiotic bacteria of diverse stinkbugs with the artificial and natural symbionts of P. stali and evaluated whether the symbiotic bacteria, which evolved for a specific host, can establish infection and support the growth and survival of heterospecific hosts. Strikingly, the artificial symbiont showed strict host specificity to P. stali , whereas the natural symbiont was capable of symbiosis with diverse stinkbugs, which provide insight into how host-symbiont specificity can be established at early evolutionary stages of symbiosis., Competing Interests: The authors declare no conflict of interest.

To survive in challenging environments, animals must develop a system to assess food quality and adjust their feeding behavior accordingly. However, the mechanisms that regulate this chronic physiological food evaluation system, which monitors specific nutrients from ingested food and influences food-response behavior, are still not fully understood. Here, we established a low-quality food evaluation assay system and found that heat-killed E. coli (HK- E. coli), a low-sugar food, triggers cellular UPR ER and immune response. This encourages animals to avoid low-quality food. The physiological system for evaluating low-quality food depends on the UPR ER (IRE-1/XBP-1) - Innate immunity (PMK-1/p38 MAPK) axis, particularly its neuronal function, which subsequently regulates feeding behaviors. Moreover, animals can adapt to a low-quality food environment through sugar supplementation, which inhibits the UPR ER -PMK-1 regulated stress response by increasing vitamin C biosynthesis. This study reveals the role of the cellular stress response pathway as physiological food evaluation system for assessing nutritional deficiencies in food, thereby enhancing survival in natural environments., Competing Interests: PL, XL, BQ No competing interests declared, (© 2024, Liu et al.)

Population bottlenecks can impact the rate of adaptation in evolving populations. On the one hand, each bottleneck reduces the genetic variation that fuels adaptation. On the other hand, each founder that survives a bottleneck can undergo more generations and leave more descendants in a resource-limited environment, which allows surviving beneficial mutations to spread more quickly. A theoretical model predicted that the rate of fitness gains should be maximized using ~8-fold dilutions. Here we investigate the impact of repeated bottlenecks on the dynamics of adaptation using numerical simulations and experimental populations of Escherichia coli . Our simulations confirm the model's prediction when populations evolve in a regime where beneficial mutations are rare and waiting times between successful mutations are long. However, more extreme dilutions maximize fitness gains in simulations when beneficial mutations are common and clonal interference prevents most of them from fixing. To examine these predictions, we propagated 48 E. coli populations with 2-, 8-, 100-, and 1000-fold dilutions for 150 days. Adaptation began earlier and fitness gains were greater with 100- and 1000-fold dilutions than with 8-fold dilutions, consistent with the simulations when beneficial mutations are common. However, the selection pressures in the 2-fold treatment were qualitatively different from the other treatments, violating a critical assumption of the model and simulations. Thus, varying the dilution factor during periodic bottlenecks can have multiple effects on the dynamics of adaptation caused by differential losses of diversity, different numbers of generations, and altered selection.

This experiment aimed to explore the protective effects of dietary palygorskite (Pal) supplementation on inflammatory responses and intestinal barrier function of broiler chickens challenged with Escherichia coli (E. coli). A 2 × 2 factorial arrangement was designed to assess the effects of Pal administration (0 or 5 g/kg of feed) and E. coli challenge (E. coli or bacterial culture medium) on broilers in a 21-d feeding trial. Birds were randomly assigned into one of the 4 groups, and each group had 8 replicates with ten birds each. The challenged chickens were orally gavaged with E. coli suspended in Luria-Bertani broth on 14 d of age, while unchallenged birds were administrated with an equivalent amount of culture medium. The sampling was performed at 21 d of age. Compared with the normal birds, an oral E. coli challenge reduced final body weight, and decreased feed intake, weight gain, and feed efficiency during the challenge period (P < 0.05). E. coli challenge promoted colonization of E. coli in cecal content and their translocation to internal organs (heart, liver, and spleen) (P < 0.05). E. coli infection also increased levels of pro-inflammatory cytokines in jejunum and ileum possibly through activating the toll-like receptor-4-mediated signaling pathway (P < 0.05). Moreover, E. coli administration increased intestinal mucosal permeability (higher serum D-lactate level and diamine oxidase activity, and lower intestinal mucosal disaccharidase activities), altered intestinal morphology, and downregulated the gene expression of intestinal tight junction proteins (P < 0.05). In contrast, Pal supplementation enhanced growth performance, inhibited colonization of E. coli, reduced intestinal inflammation, decreased intestinal permeability, restored intestinal morphology, and normalized the expression of genes responsible for inflammatory processes and maintenance of intestinal mucosal barrier (P < 0.05), and most of these beneficial effects resulting from Pal administration were independent of bacterial challenge. The results indicated dietary Pal incorporation was effective in improving growth performance and alleviating inflammation and intestinal mucosal barrier damage in broilers challenged with E. coli., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Chemotaxis-the movement of cells along chemical gradients-leads to collective behaviors when cells coordinate their movements. Here, using Escherichia coli as a model, we demonstrate a distinct type of bacterial collective response in acidic environments containing organic acids. Bacterial populations immersed in such environments collectively condensed into millimeter-sized focal points. Furthermore, this bacterial condensation fostered the formation of small, tightly packed cell aggregates, resembling non-surface-attached biofilms. These cell aggregates were physically displaced by the free-swimming condensing cells, leading to the segregation of the two cell populations. Bacterial condensation relied on feedback between the tendency of these bacteria to neutralize the pH and their chemotactic repulsion from low pH. Sustained cell condensation occurred when the bacteria occupied only part of the acidic environment, either dynamically or due to physical constraints. Such condensed bacterial populations can mitigate acid stress more efficiently, a principle that may be applicable to other stress conditions., (© 2024. The Author(s).)

Enteric pathogens navigate distinct regional microenvironments within the intestine that cue important adaptive behaviors. We investigated the response of Citrobacter rodentium, a model of human pathogenic Escherichia coli infection in mice, to regional gastrointestinal pH. We found that small intestinal pH (4.4-4.8) triggered virulence gene expression and altered cell morphology, supporting initial intestinal attachment, while higher pH, representative of C. rodentium's replicative niches further along the murine intestine, supported pathogen growth. Gastric pH, a key barrier to intestinal colonization, caused significant accumulation of intra-bacterial reactive oxygen species (ROS), inhibiting growth of C. rodentium and related human pathogens. Within-host adaptation increased gastric acid survival, which may be due to a robust acid tolerance response (ATR) induced at colonic pH. However, the intestinal environment changes throughout the course of infection. We found that murine gastric pH decreases postinfection, corresponding to increased serum gastrin levels and altered host expression of acid secretion-related genes. Similar responses following Salmonella infection may indicate a protective host response to limit further pathogen ingestion. Together, we highlight interlinked bacterial and host adaptive pH responses as an important component of host-pathogen coevolution., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Woodward et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Time-lapse microscopy offers a powerful approach for analyzing cellular activity. In particular, this technique is valuable for assessing the behavior of bacterial populations, which can exhibit growth and intercellular interactions in a monolayer. Such time-lapse imaging typically generates large quantities of data, limiting the options for manual investigation. Several image-processing software packages have been developed to facilitate analysis. It can thus be a challenge to identify the software package best suited to a particular research goal. Here, we compare four software packages that support the analysis of 2D time-lapse images of cellular populations: CellProfiler, SuperSegger-Omnipose, DeLTA, and FAST. We compare their performance against benchmarked results on time-lapse observations of Escherichia coli populations. Performance varies across the packages, with each of the four outperforming the others in at least one aspect of the analysis. Not surprisingly, the packages that have been in development for longer showed the strongest performance. We found that deep learning-based approaches to object segmentation outperformed traditional approaches, but the opposite was true for frame-to-frame object tracking. We offer these comparisons, together with insight into usability, computational efficiency, and feature availability, as a guide to researchers seeking image-processing solutions., Importance: Time-lapse microscopy provides a detailed window into the world of bacterial behavior. However, the vast amount of data produced by these techniques is difficult to analyze manually. We have analyzed four software tools designed to process such data and compared their performance, using populations of commonly studied bacterial species as our test subjects. Our findings offer a roadmap to scientists, helping them choose the right tool for their research. This comparison bridges a gap between microbiology and computational analysis, streamlining research efforts., Competing Interests: The authors declare no conflict of interest.

This systematic review aimed at investigating the role that biosecurity can have in preventing or controlling colibacillosis in broiler production. Primary studies with natural or experimental exposure to avian pathogenic Escherichia coli, evaluating any biosecurity measure to prevent or control colibacillosis in broiler chickens with at least one of the following outcomes: feed conversion ratio (FCR), condemnations at slaughter, and mortality due to colibacillosis, were included. A systematic search was carried out in 4 databases according to the Cochrane handbook and reported following the PRISMA 2020 directions. Studies (n = 3,886) were screened in a 2-phase process and data matching the inclusion criteria were extracted. Risk of bias assessment was performed. Four studies reporting biosecurity measures to prevent or control colibacillosis in broiler production were included. In all studies, only disinfection during either the pre-hatching period (n = 3) or the post-hatching period (n = 1) was evaluated as biosecurity measure in broiler production, as well as its effect on FCR (n = 2) and mortality (n = 4) due to colibacillosis. No studies with effects on condemnations at slaughter were found. Due to the heterogeneity of studies in regard to interventions and outcomes, meta-analysis was not carried out. The limited findings of this systematic review do not provide a comprehensive evidence to statistically evaluate the efficacy of biosecurity to prevent or control colibacillosis in broiler production. The scarcity of evidence found suggests that further and deeper investigations on the topic are needed, considering the variety of interventions related to biosecurity., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health 1 . Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells 2 , we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a β-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies., (© 2024. The Author(s).)

Background. The bacterial membrane plays a critical role in the survival of bacteria and the effectiveness of antimicrobial peptides in protecting the host. The lipid constituents of the bacterial membrane are not evenly distributed, and they could be affected by clustering anionic lipids with cationic peptides with multiple positive charges. That could be harmful to bacteria because it prevents lipids from interacting with other molecular components of the cell membrane, disrupts existing natural domains, or creates phase boundary defects between the clustered lipids and the bulk of the membrane. This preliminary quantitative study is aimed at assembling a correlation between antibiotic resistance and bacterial lipid composition in E. coli, based on the function and arrangement of the bilipid coating of the bacterial cell, intimately associated with the path of antimicrobials through membranes. Methods. Fifteen multiresistant E. coli samples are collected from swine with enterocolitis tested for resistance levels using the disc diffusimetric method (Kirby-Bauer disc diffusion). Pathogen identification completed using the API 20E multitest system revealed the E. coli presence in 11 samples. In these samples, bacterial membrane detection of fatty acid methyl esters (FAME) operating a 240 MS Ion Trap (Varian) GC/MS (Agilent Technologies, Santa Clara, CA, USA) was performed, using the MIDI Sherlock recognition software model. Results. Interpreting the descriptive statistical method, the correlation matrix, and regression curves and after ANOVA analysis, we ascertained that the studied E. coli population statistically confirmed different degrees of resistance in most of the samples analyzed in this test. Conclusions. In one case, the methyl-(Z)-11-tetradecenoate acid was observed to have a relationship with the susceptibility evaluation by using the disc diffusimetric method, which has revealed the lowest rate of antimicrobial resistance, so it has importance in further resistance evaluation studies. [ABSTRACT FROM AUTHOR]

Another study investigated fibroblasts on the RPM and reported that fibroblast differentiation is severely impaired after RPM exposure for short periods [[16]]. This Special Issue (SI), "Microgravity and Space Medicine 2.0", comprises research articles on the research areas of gravitational biology, space medicine and molecular oncology. These ground-based facilities are acknowledged by the European Space Agency (ESA) and National Aeronautics and Space Administration (NASA) and used worldwide for µ I g i -experiments on Earth in our laboratories. It covers studies investigating the effects of altered gravity conditions on poorly differentiated follicular thyroid cancer cells and bacteria ( I Escherichia coli i ) during real microgravity (r-µ I g i ) on the International Space Station (ISS) [[1]]. [Extracted from the article]

Colibacillosis, a bacterial disease caused by avian pathogenic E. coli (APEC), is a prevalent condition in the poultry industry, resulting in substantial economic losses annually. Previously, we identified PTEN as a crucial candidate gene that may play a significant role in chicken's immune response to APEC infection. Bioinformatics analysis indicated that the PTEN protein was unstable, hydrophilic and nuclear localization, with multiple putative phosphorylation sites and a high degree of similarity to duck and goose PTEN. Moreover, PTEN exhibited high expression levels in various tissues such as the stomach, cecum, small intestine, spleen, thymus, harderian gland, muscle, cerebrum, cerebellum, lung, and liver in comparison to heart tissue. Overexpression of PTEN resulted in a significant promotion of the expression level of pro-apoptosis genes and inflammatory mediators, as well as the production of NO, with or without APEC infection, which led to cellular injury. Furthermore, overexpression of PTEN was found to regulate the expression levels of autophagy related genes, regardless of APEC infection. Additionally, PTEN was a target gene of gga-miR-20a-5p and regulated by gga-miR-20a-5p upon APEC infection. Taken together, these findings establish a foundation for investigating the biological function of chicken PTEN, providing a potential target for future treatments against APEC infection as well as the breeding of genetically resistant poultry., Competing Interests: DISCLOSURES The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aim: Integrons play an essential role in disseminating drug resistance genes among bacteria. The aim of this study was to determine the prevalence of integrons, and Extended-Spectrum β-Lactamase (ESBL) genes in Escherichia coli (E. coli) isolates collected from patients with urinary tract infection (UTI) referred to teaching hospitals in Ardabil, Iran. Materials and Methods: In this descriptive, cross-sectional study (2017-2018), 163 isolates of E.coli were collected from patients with UTI. The drug susceptibility pattern of these isolates to 12 common antibiotics was investigated using the disk diffusion method based on CLSI guidelines. The prevalence of class 1, 2,3 integrons and ESBL genes was verified by the PCR method. Finally, the genetic variation of isolates was analyzed using the ERIC-PCR method. Results: Of 163 isolates, 138 (84.7%) isolates were multidrug-resistance (MDR) strains. The lowest and highest antibiotic resistance was reported to nitrofurantoin and ampicillin, with a resistance rate of 1.2% and 89.6%, respectively. The incidence of class 1 and 2 integrons was obtained in 39.9% and 14.1% of the isolates, respectively. Class 3 integron was not found in any of the isolates. Based on the ERIC-PCR fingerprinting method, 4 ERIC types were detected. Conclusion: Our study showed that E. coli isolates taken from patients mainly were MDR strain and resistant to many of the common antibiotics used to treat urinary tract infections. Using the correct dose of medication and multidrug therapy would be effective in reducing the incidence of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Urinary tract infections are primarily caused by uropathogenic Escherichia coli (UPEC). In contrast to the intestinal E. coli strains that reside in nutrient-rich gut environment, UPEC encounter distinct niches, for instance human urine, which is an oxygen- and nutrient-limited environment. Alpha-ketoglutarate (KG) is an abundant metabolite in renal proximal tubule cells; and previously we showed that two-component signaling system (TCS) KguS/KguR contributes to UPEC colonization of murine urinary tract by promoting the utilization of KG as a carbon source under anaerobic conditions. However, knowledge about the KguR regulon and its impact on UPEC fitness is lacking. In this work, we analyzed transcriptomic and metabolomic changes caused by kguR deletion under anaerobiosis when KG is present. Our results indicated that 620 genes were differentially expressed in the Δ kguR mutant, as compared to the wild type; of these genes, 513 genes were downregulated and 107 genes were upregulated. Genes with substantial changes in expression involve KG utilization, acid resistance, iron uptake, amino acid metabolism, capsule biosynthesis, sulfur metabolism, among others. In line with the transcriptomics data, several amino acids (glutamate, lysine, etc.) and uridine 5′-diphosphogalactose (involved in capsule biosynthesis) were significantly less abundant in the Δ kguR mutant. We then confirmed that the Δ kguR mutant, indeed, was more sensitive to acid stress than the wild type, presumably due to downregulation of genes belonging to the glutamate-dependent acid resistance system. Furthermore, using gene expression and electrophoretic mobility shift assays (EMSAs), we demonstrate that KguR autoregulates its own expression by binding to the kguSR promoter region. Lastly, we performed a genome-wide search of KguR binding sites, and this search yielded an output of at least 22 potential binding sites. Taken together, our data establish that in the presence of KG, KguR broadly impacts the physiology of UPEC under anaerobiosis. These findings greatly further our understanding of KguS/KguR system as well as UPEC pathobiology. [ABSTRACT FROM AUTHOR]

Bacteria often thrive in surface-attached communities, where they can form biofilms affording them multiple advantages. In this sessile form, fluid flow is a key component of their environments, renewing nutrients and transporting metabolic products and signaling molecules. It also controls colonization patterns and growth rates on surfaces, through bacteria transport, attachment and detachment. However, the current understanding of bacterial growth on surfaces neglects the possibility that bacteria may modulate their division behavior as a response to flow. Here, we employed single-cell imaging in microfluidic experiments to demonstrate that attached Escherichia coli cells can enter a growth arrest state while simultaneously enhancing their adhesion underflow. Despite utilizing clonal populations, we observed a non-uniform response characterized by bistable dynamics, with co-existing subpopulations of non-dividing and actively dividing bacteria. As the proportion of non-dividing bacteria increased with the applied flow rate, it resulted in a reduction in the average growth rate of bacterial populations on flow-exposed surfaces. Dividing bacteria exhibited asymmetric attachment, whereas non-dividing counterparts adhered to the surface via both cell poles. Hence, this phenotypic diversity allows bacterial colonies to combine enhanced attachment with sustained growth, although at a reduced rate, which may be a significant advantage in fluctuating flow conditions., (© 2024. The Author(s).)

Yolk Peritonitis can lead to a rapid decline in egg production, which seriously affects the health of laying hens and the profitability of chicken farms. Escherichia coli (E. coli) is the most common cause of yolk peritonitis in laying hens. In this study, bacterial samples were collected from the ovaries and fallopian tubes of laying hens with suspected yolk peritonitis from a laying farm in Jiangsu Province, and their pathogenicity and drug resistance were investigated. Initially, morphological and biochemical detection methods were employed to isolate and identify the pathogenic bacteria. The results showed that a total of 16 strains of E. coli were isolated from laying hens with yolk peritonitis. Subsequently, the drug resistance and pathogenicity of a randomly selected E. coli strain were analyzed and predicted by genome sequencing technology, and the drug resistance of E. coli was verified by drug sensitivity test and PCR. Finally, the virulence was verified by infection experiment in mice. The study revealed that the egg-yolk peritonitis in laying hens was caused by E. coli infection, and the genome sequencing analysis revealed that the bacteria had multidrug resistance and high virulence. The drug susceptibility testing indicates that E. coli exhibited resistance to aminoglycosides, β-lactam, macrolides, fluoroquinolones, and sulfonamides. In this study, resistance genes including KdpE, aadA5, APH(3 ")-ID, APH(6)-ID, and TEM-1 were identified, and their expression levels varied across different stages of bacterial growth. The results of virulence analysis indicated a mortality rate of 50% in mice infected with E. coli at a concentration of 2.985 × 10 7 CFU/mL. E. coli infection resulted in damage to various tissues and organs in mice, with the intestinal tissue structure being the most severely affected. This study provides a reference for the study of drug resistance mechanisms in E. coli and provides valuable insights into the selection of drugs for the treatment of vitelline peritonitis., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Introduction: Silica-sprayed tubes (SSTs) are often used to transport synovial fluid samples in equine practice. They promote the coagulation of the sample. The objective of the study is to evaluate the effect of SST on bacterial culture., Materials and Methods: The study was divided into two parts: sterile saline (Part A) and synovial fluid (Part B). Four common bacteria associated with equine synovial sepsis were used: Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Three collection tubes were used: STT, plain (no-additives) and brain and heart infusion (BHI) broth. Bacteria were cultured in horse blood agar plates for 48 h. Outcome variables were negative culture, positive culture and total number of colony-forming units (CFUs). Statistical analysis was performed using Mann-Whitney U test, and significance was set at p < 0.05., Results: The total number of agar plates read was 1557 (779 saline; 778 synovial fluid). Total negative cultures were 25/779 on saline and 3/778 on synovial fluid. In broth, maximum growth CFU was achieved after 8 h for both saline and synovial fluid for all bacteria. S. pyogenesand E. coli produced a significantly lower number of CFU when in SST compared to plain or broth after 4 h, whereas S. aureus (American Type Culture Collection [ATCC] and MRSA) only after 24 h., Discussion: Silica-containing tubes reduced bacterial proliferation, whereas the use of a BHI broth provided the highest bacterial load in the sample. The use of SST may have a negative effect on bacterial proliferation in samples obtained from clinical cases., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Bacterial persisters constitute a small fraction of cells that transiently display multidrug tolerance, allowing them to survive antibiotic treatment and to establish a new population upon recovery from the persistent state. Here, we present a protocol to quantify post-antibiotic persister recovery kinetics and physiological states at the single-cell level. We describe steps for sample preparation, technical setup, and data acquisition using spectrophotometry. Our assay allows for the elucidation of genes and mechanisms involved in persister survival. For complete details on the use and execution of this protocol, please refer to Wilmaerts et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Research on Escherichia coli DNA replication paved the groundwork for many breakthrough discoveries with important implications for our understanding of human molecular biology, due to the high level of conservation of key molecular processes involved. To this day, it attracts a lot of attention, partially by virtue of being an important model organism, but also because the understanding of factors influencing replication fidelity might be important for studies on the emergence of antibiotic resistance. Importantly, the wide access to high-resolution single-molecule and live-cell imaging, whole genome sequencing, and cryo-electron microscopy techniques, which were greatly popularized in the last decade, allows us to revisit certain assumptions about the replisomes and offers very detailed insight into how they work. For many parts of the replisome, step-by-step mechanisms have been reconstituted, and some new players identified. This review summarizes the latest developments in the area, focusing on (a) the structure of the replisome and mechanisms of action of its components, (b) organization of replisome transactions and repair, (c) replisome dynamics, and (d) factors influencing the base and sugar fidelity of DNA synthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Bacteria in biofilms secrete potassium ions to attract free swimming cells. However, the basis of chemotaxis to potassium remains poorly understood. Here, using a microfluidic device, we found that Escherichia coli can rapidly accumulate in regions of high potassium concentration on the order of millimoles. Using a bead assay, we measured the dynamic response of individual flagellar motors to stepwise changes in potassium concentration, finding that the response resulted from the chemotaxis signaling pathway. To characterize the chemotactic response to potassium, we measured the dose-response curve and adaptation kinetics via an Förster resonance energy transfer (FRET) assay, finding that the chemotaxis pathway exhibited a sensitive response and fast adaptation to potassium. We further found that the two major chemoreceptors Tar and Tsr respond differently to potassium. Tar receptors exhibit a biphasic response, whereas Tsr receptors respond to potassium as an attractant. These different responses were consistent with the responses of the two receptors to intracellular pH changes. The sensitive response and fast adaptation allow bacteria to sense and localize small changes in potassium concentration. The differential responses of Tar and Tsr receptors to potassium suggest that cells at different growth stages respond differently to potassium and may have different requirements for potassium., Competing Interests: CZ, RZ, JY No competing interests declared, (© 2023, Zhang et al.)

Multi-factor screenings are commonly used in diverse applications in medicine and bioengineering, including optimizing combination drug treatments and microbiome engineering. Despite the advances in high-throughput technologies, large-scale experiments typically remain prohibitively expensive. Here we introduce a machine learning platform, structure-augmented regression (SAR), that exploits the intrinsic structure of each biological system to learn a high-accuracy model with minimal data requirement. Under different environmental perturbations, each biological system exhibits a unique, structured phenotypic response. This structure can be learned based on limited data and once learned, can constrain subsequent quantitative predictions. We demonstrate that SAR requires significantly fewer data comparing to other existing machine-learning methods to achieve a high prediction accuracy, first on simulated data, then on experimental data of various systems and input dimensions. We then show how a learned structure can guide effective design of new experiments. Our approach has implications for predictive control of biological systems and an integration of machine learning prediction and experimental design., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Interspecific competition can hinder populations from evolutionarily adapting to abiotic environments, particularly by reducing population size and niche space; and feedback may arise between competitive ability and evolutionary adaptation. Here we studied populations of two model bacterial species, Escherichia coli and Pseudomonas fluorescens, that evolved in monocultures and cocultures for approximately 2400 generations at three temperatures. The two species showed a reversal in competitive dominance in cocultures along the temperature gradient. Populations from cocultures where they had been competitively dominant showed the same magnitude of fitness gain as those in monocultures. However, competitively inferior populations in cocultures showed limited abiotic adaptation compared with those in monocultures. The inferior populations in cocultures were also more likely to evolve weaker interspecific competitive ability, or go extinct. The possible competitive ability-adaptation feedback may have crucial consequences for population persistence., (© 2024 John Wiley & Sons Ltd.)

Microorganisms, including potential pathogens, can colonise plastic surfaces in aquatic environments. This study investigates the colonisation of plastic pellets by Escherichia coli (E. coli) as a proxy for faecal pathogens in aquatic environments. Plastic pellets from a polluted beach were placed in seawater aquaria spiked with E. coli. Diverse bacteria, primarily from the Proteobacteria phylum, rapidly colonised the pellets within 24 h, with notable species known for plastic or hydrocarbon degradation. Over 26 days, biofilms formed on the plastic surfaces, reaching bacterial populations of up to 6.8·10 5 gene copies (gc) of the 16S rRNA mm -2 . E. coli, was detected in the pellets for up to 7 days using culture methods, exhibiting varying attachment densities regardless of source or environmental factors. The study highlights plastic biofilms as reservoirs for E. coli, contributing to the survival and persistence of faecal bacteria in aquatic systems. These findings deepen our understanding of the risks associated with plastic pollution in marine settings, offering insights into the behaviour of faecal indicators and their implications for water quality assessments, while providing valuable information on potential pathogen dissemination within plastic-associated microbial communities., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Adaptation of avian pathogenic E. coli (APEC) to changing host environments including virulence factors expression is vital for disease progression. FdeC is an autotransporter adhesin that plays a role in uropathogenic Escherichia coli (UPEC) adhesion to epithelial cells. Expression of fdeC is known to be regulated by environmental conditions in UPEC and Shiga toxin-producing E. coli (STEC). The observation in a previous study that an APEC strain IMT5155 in which the fdeC gene was disrupted by a transposon insertion resulted in elevated adhesion to chicken intestinal cells prompted us to further explore the role of fdeC in infection. We found that the fdeC gene prevalence and FdeC variant prevalence differed between APEC and nonpathogenic E. coli genomes. Expression of the fdeC gene was induced at host body temperature, an infection relevant condition. Disruption of fdeC resulted in greater adhesion to CHIC-8E11 cells and increased motility at 42 °C compared to wild type (WT) and higher expression of multiple transporter proteins that increased inorganic ion export. Increased motility may be related to increased inorganic ion export since this resulted in downregulation of YbjN, a protein known to supress motility. Inactivation of fdeC in APEC strain IMT5155 resulted in a weaker immune response in chickens compared to WT in experimental infections. Our findings suggest that FdeC is upregulated in the host and contributes to interactions with the host by down-modulating motility during colonization. A thorough understanding of the regulation and function of FdeC could provide novel insights into E. coli pathogenesis., (© 2024. The Author(s).)

Background: Mammary Pathogenic Escherichia coli (MPEC) is an important pathogen that can escape the attack of the host immune system through biofilm formation and proliferate in the mammary gland continuously, resulting in mastitis in cows and causing enormous economic losses. As an effector of AI-2 quorum sensing, LsrR extensively affects the expression levels of hundreds of genes related to multiple biological processes in model E. coli strain. However, the regulatory role of LsrR in MPEC and whether it is involved in pathogenesis has been seldom reported., Results: In this study, the function of LsrR in strain MPEC5, obtained from a milk sample in dairy cows with mastitis, was investigated by performing high-throughput sequencing (RNA-seq) assays. The results revealed that LsrR down-regulated the transcript levels of fimAICDFGH (encoding Type 1 pili), which have been reported to be associated with biofilm formation process. Biofilm assays confirmed that deletion of lsrR resulted in a significant increase in biofilm formation in vitro. In addition, electrophoretic mobility shift assay (EMSA) provided evidence that LsrR protein could directly bind to the promoter regions of fimAICDFGH in a dose-dependent manner., Conclusions: These results indicate that LsrR protein inhibits the biofilm formation ability of MPEC5 by directly binding to the fimAICDFGH promoter region. This study presents a novel clue for further exploration of the prevention and treatment of MPEC., (© 2024. The Author(s).)

Innate immune response is the first line of host defense against pathogenic microorganisms, and its excessive or insufficient activation is detrimental to the organism. Many individual microRNAs (miRNAs) have emerged as crucial post-transcriptional regulators of immune homeostasis in Drosophila melanogaster . However, the synergistical regulation of miRNAs located within a cluster on the Imd-immune pathway remains obscured. In our study, a genetic screening with 52 transgenic UAS-miRNAs was performed to identify ten miRNAs or miRNA clusters, including the miR310~313 cluster, which may function on Imd-dependent immune responses. The miRNA RT-qPCR analysis showed that the expression of miR-310~313 cluster members exhibited an increase at 6-12 h post E. coli infection. Furthermore, the overexpression of the miR-310~313 cluster impaired the Drosophila survival. And the overexpression of miR-310/311/312 reduced Dpt expression, an indication of Imd pathway induced by Gram-negative bacteria. Conversely, the knockdown of miR-310/311/312 led to increases in Dpt expression. The Luciferase reporter expression assays and RT-qPCR analysis confirmed that miR-310~313 cluster members directly co-targeted and inhibited Imd transcription. These findings reveal that the members of the miR-310~313 cluster synergistically inhibit Imd-dependent immune responses by co-targeting the Imd gene in Drosophila .

Sonodynamic therapy (SDT) is an emerging antibacterial therapy. This work selected hematoporphyrin monomethyl ether (HMME) as the sonosensitizer, and studied the enhanced inhibition effect of Escherichia coli and biofilm by microbubble-mediated cavitation in SDT. Firstly, the influence of microbubble-mediated cavitation effect on different concentrations of HMME (10 µg/ml, 30 µg/ml, 50 µg/ml) was studied. Using 1,3-diphenylisobenzofuran (DPBF) as an indicator, the effect of microbubble-mediated cavitation on the production of reactive oxygen species (ROS) was studied by absorption spectroscopy. Secondly, using agar medium, laser confocal microscopy and scanning electron microscopy, the effect of microbubble-mediated cavitation on the activity and morphology of bacteria was studied. Finally, the inhibitory effect of cavitation combined with SDT on biofilm was evaluated by laser confocal microscopy. The research results indicate that: (1) Microbubble-mediated ultrasound cavitation can significantly increase cavitation intensity and production of ROS. (2) Microbubble-mediated acoustic cavitation can alter the morphological structure of bacteria. (3) It can significantly enhance the inhibition of SDT on the activity of Escherichia coli and its biofilm. Compared with the control group, the addition of microbubbles resulted in an increase in the number of dead bacteria by 61.7 %, 71.6 %, and 76.2 %, respectively. The fluorescence intensity of the biofilm decreased by 27.1 %, 80.3 %, and 98.2 %, respectively. On the basis of adding microbubbles to ensure antibacterial and biofilm inhibition effects, this work studied the influence of cavitation effect in SDT on bacterial structure, providing a foundation for further revealing the intrinsic mechanism of SDT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Chicken and chicken products have been associated with foodborne pathogens such as Salmonella, Campylobacter, and Escherichia coli (E. coli). Poultry comprises an important segment of the agricultural economy (75 million birds processed as of 2019) in West Virginia (WV). The risk of pathogens on processed chickens has risen with the increased popularity of mobile poultry processing units (MPPUs). This study evaluated the microbial safety of broilers processed in a MPPU in WV. This study assessed aerobic plate counts (APCs), E. coli counts and the presence/absence of Salmonella and Campylobacter on 96 broiler carcasses following each MPPU step of scalding, eviscerating, and chilling. Samples were either chilled in ice water only (W) or ice water with 5 ppm chlorine (CW). The highest number of bacteria recovered from carcasses were APCs (4.21 log 10 CFU/mL) and E. coli (3.77 log 10 CFU/mL; P = 0.02). A total reduction of 0.30 (P = 0.10) and 0.63 (P = 0.01) log 10 CFU/mL for APCs and E. coli, respectively, occurred from chilling carcasses in CW. Overall, results show that E. coli, Salmonella, and Campylobacter were significantly (P < 0.05) reduced from the initial scalding to the chilling step. However, Salmonella frequency doubled (15.63-34.38%) after the evisceration step, indicating that washing carcasses after evisceration may be a critical control point in preventing cross-contamination by Salmonella. Proper chilling is also an important microbial mitigation step in MPPU processing. Results indicate that Campylobacter was more resistant to chilling than Salmonella. Campylobacter was not completely inactivated until carcasses were chilled in CW, whereas W was sufficient to reduce Salmonella on carcasses. The results led to the conclusion that although 5 ppm chlorine (Cl 2 ) achieved more bacterial reductions than water alone, the reductions were not always significant (P > 0.05). Further MPPU studies are needed to verify more effective chilling and processing strategies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Klebsiella aerogenes (previously known as Enterobacter aerogenes) is a common opportunistic pathogen that infect the respiratory tract and central nervous system. However, how it interferes the host regulatory mechanism has not been previously described. When C. elegans were exposed to K. aerogenes, they exhibited a shorter lifespan compared to those fed with E. coli OP50. The time required for 50 % of L4 hermaphrodite nematodes to die when exposed to K. aerogenes was approximately 9 days, whereas it was about 18 days when fed with E. coli OP50. The interaction with K. aerogenes also affected the physical activity of C. elegans. Parameters like pharyngeal pumping, head thrashing, body bending, and swimming showed a gradual decline during infection. The expression of serotonin-mediated axon regeneration K. aerogenes infection led to increased levels of reactive oxygen species (ROS) in C. elegans compared to E. coli OP50-fed worms. The nematodes activated antioxidant mechanisms, including the expression of SODs, to counteract elevated ROS levels. The interaction with K. aerogenes activated immune regulatory pathways in C. elegans, including the mTOR signaling pathway downstream player SGK-1. Lifespan regulatory pathways, such as pha-4 and pmk-1, were also affected, likely contributing to the nematode ability to survive in a pathogenic environment. K. aerogenes infection has a detrimental impact on the healthspan and lifespan of C. elegans, affecting physical activity, intestinal health, serotonin regulation, ROS levels, and immune responses. These findings provide insights into the complex interactions between K. aerogenes and host organisms., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objective: To determine the optimum biofilm formation ratio of Gardnerella vaginalis ( G. vaginalis ) in a mixed culture with Escherichia coli ( E. coli )., Methods: G. vaginalis ATCC14018, E. coli ATCC25922, as well as five strains of G. vaginalis were selected from the vaginal sources of patients whose biofilm forming capacity was determined by the Crystal Violet method. The biofilm forming capacity of E. coli in anaerobic and non-anaerobic environments were compared using the identical assay. The Crystal Violet method was also used to determine the biofilm forming capacity of a co-culture of G. vaginalis and E. coli in different ratios. After Live/Dead staining, biofilm thickness was measured using confocal laser scanning microscopy, and biofilm morphology was observed by scanning electron microscopy., Results: The biofilm forming capacity of E. coli under anaerobic environment was similar to that in a 5% CO 2 environment. The biofilm forming capacity of G. vaginalis and E. coli was stronger at 10 6 :10 5 CFU/mL than at other ratios ( P <0.05). Their thicknesses were greater at 10 6 :10 5 CFU/mL than at the other ratios, with the exception of 10 6 :10 2 CFU/mL ( P <0.05), under laser scanning microscopy. Scanning electron microscopy revealed increased biofilm formation at 10 6 :10 5 CFU/mL and 10 6 :10 2 CFU/mL, but no discernible E. coli was observed at 10 6 :10 2 CFU/mL., Conclusion: G. vaginalis and E. coli showed the greatest biofilm forming capacity at a concentration of 10 6 :10 5 CFU/mL at 48 hours and could be used to simulate a mixed infection of bacterial vaginosis and aerobic vaginitis in vitro ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Shang, Bai, Fan, Zhang, Zhao and Liu.)

Importance: Staphylococcus aureus and Escherichia coli contribute to global health challenges by forming biofilms, a key virulence element implicated in the pathogenesis of several infections., Objective: The study examined the efficacy of various generations of cephalosporins against biofilms developed by pathogenic S. aureus and E. coli ., Methods: The development of biofilms by both bacteria was assessed using petri-plate and microplate methods. Biofilm hydrolysis and inhibition were tested using first to fourth generations of cephalosporins, and the effects were analyzed by crystal violet staining and phase contrast microscopy., Results: Both bacterial strains exhibited well-developed biofilms in petri-plate and microplate assays. Cefradine (first generation) showed 76.78% hydrolysis of S. aureus biofilm, while significant hydrolysis (59.86%) of E. coli biofilm was observed by cefipime (fourth generation). Similarly, cefuroxime, cefadroxil, cefepime, and cefradine caused 78.8%, 71.63%, 70.63%, and 70.51% inhibition of the S. aureus biofilms, respectively. In the case of E. coli, maximum biofilm inhibition (66.47%) was again shown by cefepime. All generations of cephalosporins were more effective against S. aureus than E. coli , which was confirmed by phase contrast microscopy., Conclusions and Relevance: Cephalosporins exhibit dual capabilities of hydrolyzing and inhibiting S. aureus and E. coli biofilms. First-generation cephalosporins exhibited the highest inhibitory activity against S. aureus , while the third and fourth generations significantly inhibited E. coli biofilms. This study highlights the importance of tailored antibiotic strategies based on the biofilm characteristics of specific bacterial strains., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Korean Society of Veterinary Science.)

Microbial infections remain a public health problem due to the upsurge of bacterial resistance. In this study, the antibacterial, antibiofilm, and efflux pump inhibitory activities of the stem bark of Acacia macrostachya, an indigenous African medicinal plant, were investigated. In traditional medicine, the plant is used in the treatment of microbial infections and inflammatory conditions. A crude methanol extract obtained by Soxhlet extraction was partitioned by column chromatography to obtain the petroleum ether, ethyl acetate, and methanol fractions. Antibacterial, efflux pump inhibition and antibiofilm formation activities were assessed by the high-throughput spot culture growth inhibition (HT-SPOTi), ethidium bromide accumulation, and the crystal violet retention assay, respectively. The minimum inhibitory concentrations (MICs) of the crude extract and major fractions ranged from 250 to ≥500 μg/mL. At a concentration of 3.9-250 μg/mL, all extracts demonstrated >80% inhibition of biofilm formation in S. aureus. In P. aeruginosa, the EtOAc fraction showed the highest antibiofilm activity (59-69%) while the pet-ether fraction was most active against E. coli biofilms (45-67%). Among the test samples, the crude extract, methanol, and ethyl acetate fractions showed remarkable efflux pump inhibition in S. aureus, E. coli, and P. aeruginosa. At ½ MIC, the methanol fraction demonstrated significant accumulation of EtBr in E. coli having superior efflux inhibition over the standard EPIs: chlorpromazine and verapamil. Tannins, flavonoids, triterpenoids, phytosterols, coumarins, and saponins were identified in preliminary phytochemical studies. Stigmasterol was identified in the EtOAc fraction. This study justifies the use of A. macrostachya in the treatment of infections in traditional medicine and highlights its potential as a source of bioactive compounds that could possibly interact with some resistance mechanisms in bacteria to combat antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Twenty-five samples of urine were collected from patients suffering Urinary Tract Infection (UTI) from hospital in Baghdad, twenty-two bacterial isolated (88%) were identified. From our result showed the E. coli number 5 is the best bacterial virulence factor for producing OMV, Tumor cells (1x104 - 1x106 cells/ml) have been grown in 96 flat well micro-title plates, with a final volume of 200 cells per well. The microplate was gently shackled and covered by sterilized parafilm then incubation of the plates at 37C°, 5% CO2 for 24hrs. Two folded serial dilutions of the desired compound (12.5, 25, 50, 100, 200, 400µ g/mL) were added to the wells after incubation. Triplicates were used per each concentration as well as controls (serum-free medium treated cells). Plates were incubated for specified exposure time (24 hours) at 37C°, 5 percent CO2. Applied 10µ liters of MTT solution to each well after exposure. The absorbance was determined by a wavelength of 575 nm using an ELISA reader. Statistical analysis was performed on the optical density data to calculate the compound concentration required to cause a 50 percent reduction in cell viability for each cell line. The rate of cell growth inhibition (the cytotoxicity percentage) was calculated and plot of % cytotoxicity against sample concentrations assisted to calculate the concentration which exhibited 50% cytotoxicity (IC50). Calculate: cell number: determine cells per milliliter. [ABSTRACT FROM AUTHOR]