Removal and monitoring of residual nucleic acids from core streptavidin inclusion bodies for increased refolding yield.

Commercial production of recombinant streptavidin (SAV) using soluble expression route is cost-prohibitive, resulting from its inherent toxicity toward commercially available Escherichia coli hosts (such as BL21) and low productivity of existing manufacturing processes. Quality challenges can also result from binding of streptavidin in the host cells. One way to overcome these challenges is to allow formation of inclusion bodies (IBs). Nevertheless, carried-over cellular contaminants during IBs preparation can hinder protein refolding and application of SAV in nucleic acid-based applications. Hence, removing associated contaminants in recombinant IBs is imperative for maximum product outcomes. In this study, the IBs isolation method from our group was improved to remove residual DNA found in refolded core SAV (cSAV). The improvements were attained by incorporating quantitative real-time polymerase chain reactions (qPCR) for residual DNA monitoring. We attained 99 % cellular DNA removal from cSAV IBs via additional wash and sonication steps, and the addition of benzonase nuclease during lysis. A 10 % increment of cSAV refolding yield (72 %) and 83 % reduction of residual DNA from refolding of 1 mg cSAV IBs were observed under extensive sonication. Refolding of cSAV was not affected and its activity was not compromised. The optimized process reported here highlights the importance of obtaining cSAV IBs with minimal contaminants prior to refolding to increase product yield, and the usefulness of the qPCR method to monitor nucleic acid removed from each step of the process.

Golden Gate-Assisted Gene Doctoring for Streamlined and Efficient Recombineering in Bacteria.

Gene Doctoring is a genetic modification technique for E. coli and related bacteria, in which the Red-recombinase from bacteriophage λ mediates chromosomal integration of a fragment of DNA by homologous recombination (known as recombineering). In contrast to the traditional recombineering method, the integrated fragment for Gene Doctoring is supplied on a donor plasmid rather than as a linear DNA. This protects the DNA from degradation, facilitates transformation, and ensures multiple copies are present per cell, increasing the efficiency and making the technique particularly suitable for strains that are difficult to modify. Production of the donor plasmid has, until recently, relied on traditional cloning techniques that are inflexible, tedious, and inefficient. This protocol describes a procedure for Gene Doctoring combined with Golden Gate assembly of a donor plasmid, using a custom-designed plasmid backbone, for rapid and simple production of complex, multi-part assemblies. Insertion of a gene for superfolder green fluorescent protein, with selection by tetracycline resistance, into E. coli strain MG1655 is used as an example but in principle the method can be tailored for virtually any modification in a wide range of bacteria.

Application of MinION Long-Read Sequencer for Semi-targeted Detection of Foodborne Pathogens.

Foodborne pathogens remain a serious health issue in developed and developing countries. Safeness of food products has been assured for years with culture-based microbiological methods; however, these present several limitations such as turnaround time and extensive hands-on work, which have been typically address taking advantage of DNA-based methods such as real-time PCR (qPCR). These, and other similar techniques, are targeted assays, meaning that they are directed for the specific detection of one specific microbe. Even though reliable, this approach suffers from an important limitation that unless specific assays are design for every single pathogen potentially present, foods may be considered erroneously safe. To address this problem, next-generation sequencing (NGS) can be used as this is a nontargeted method; thus it has the capacity to detect every potential threat present. In this chapter, a protocol for the simultaneous detection and preliminary serotyping of Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Escherichia coli O157:H7 is described.

Corrigendum: Dihydromyricetin improves growth performance, immunity, and intestinal functions in weaned pigs challenged by enterotoxigenic Escherichia coli.

[This corrects the article DOI: 10.3389/fvets.2024.1421871.].

Uropathogenic Escherichia coli causes significant urothelial damage in an ex vivo porcine bladder model, with no protective effect observed from cranberry or D-mannose.

Urinary tract infections (UTIs), primarily caused by uropathogenic Escherichia coli (UPEC), have an unclear impact on bladder mucosal physiology. This study investigates UPEC's effects on the urothelium and lamina propria using an ex vivo porcine bladder model. Bladder mucosal strips were analysed for contractile responses to acetylcholine, serotonin, and neurokinin A. Given rising antibiotic resistance, non-antibiotic agents such as cranberry and D-mannose were also evaluated for their potential to prevent UPEC-induced damage. The findings of the current study revealed that UPEC significantly compromised urothelial integrity, barrier function, and permeability, with loss of urothelial cells, uroplakins, and tight junction protein ZO-1 expression. Additionally, infected bladders exhibited a markedly enhanced contractile response to serotonin compared to uninfected controls. Notably, neither cranberry nor D-mannose offered protection against UPEC-mediated damage or mitigated the heightened serotonin-induced contractility. This study provides novel insights into how UPEC disrupts bladder cell biology and highlights the possible involvement of serotonin in the pathophysiology of UTIs.

Genomic evidence of Escherichia coli gut population diversity translocation in leukemia patients.

Escherichia coli, a commensal species of the human gut, is an opportunistic pathogen that can reach extra-intestinal compartments, including the bloodstream and the bladder, among others. In non-immunosuppressed patients, purifying or neutral evolution of E. coli populations has been reported in the gut. Conversely, it has been suggested that when migrating to extra-intestinal compartments, E. coli genomes undergo diversifying selection as supported by strong evidence for adaptation. The level of genomic polymorphism and the size of the populations translocating from gut to extra-intestinal compartments is largely unknown. To gain insights into the pathophysiology of these translocations, we investigated the level of polymorphism and the evolutionary forces acting on the genomes of 77 E. coli isolated from various compartments in three immunosuppressed patients. Each patient had a unique strain, which was a mutator in one case. In all instances, we observed that translocation encompasses much of the genomic diversity present in the gut. The same signature of selection, whether purifying or diversifying, and as anticipated, neutral for mutator isolates, was observed in both the gut and bloodstream. Additionally, we found a limited number of non-specific mutations among compartments for non-mutator isolates. In all cases, urine isolates were dominated by neutral selection. These findings indicate that substantial proportions of populations are undergoing translocation and that they present a complex compartment-specific pattern of selection at the patient level.IMPORTANCEIt has been suggested that intra and extra-intestinal compartments differentially constrain the evolution of E. coli strains. Whether host particular conditions, such as immunosuppression, could affect the strain evolutionary trajectories remains understudied. We found that, in immunosuppressed patients, large fractions of E. coli gut populations are translocating with variable modifications of the signature of selection for commensal and pathogenic isolates according to the compartment and/or the patient. Such multiple site sampling should be performed in large cohorts of patients to gain a better understanding of E. coli extra-intestinal diseases.

Estrogen-stimulated uropathogenic E. coli mediate enhanced neutrophil responses.

Urinary tract infection (UTI) is one of the most common bacterial infections worldwide and the most common cause is uropathogenic Escherichia coli (UPEC). Current research is mostly focused on how UPEC affects host factors, whereas the effect of host factors on UPEC is less studied. Our previous studies have shown that estrogen alters UPEC virulence. However, the effect of this altered UPEC virulence on neutrophils is unknown. The aim of the present study was to investigate how the altered UPEC virulence mediated by estrogen modulates neutrophil responses. We found that estradiol-stimulated CFT073 increased neutrophil phagocytosis, NETs formation and intracellular ROS production. We observed that the total ROS production from neutrophils was reduced by estradiol-stimulated CFT073. We also found that estradiol-stimulated CFT073 induced less cytotoxicity in neutrophils. Additionally, we found that several cytokines and chemokines like IL-8, IL-1ß, CXCL6, MCP-1 and MCP-4 were increased upon estradiol-stimulated CFT073 infection. In conclusion, this study demonstrates that the estrogen-mediated alterations to UPEC virulence modulates neutrophil responses, most likely in a host-beneficial manner.

Truncated M13 phage for smart detection of E. coli under dark field.

BACKGROUND: The urgent need for affordable and rapid detection methodologies for foodborne pathogens, particularly Escherichia coli (E. coli), highlights the importance of developing efficient and widely accessible diagnostic systems. Dark field microscopy, although effective, requires specific isolation of the target bacteria which can be hindered by the high cost of producing specialized antibodies. Alternatively, M13 bacteriophage, which naturally targets E. coli, offers a cost-efficient option with well-established techniques for its display and modification. Nevertheless, its filamentous structure with a large length-diameter ratio contributes to nonspecific binding and low separation efficiency, posing significant challenges. Consequently, refining M13 phage methodologies and their integration with advanced microscopy techniques stands as a critical pathway to improve detection specificity and efficiency in food safety diagnostics. METHODS: We employed a dual-plasmid strategy to generate a truncated M13 phage (tM13). This engineered tM13 incorporates two key genetic modifications: a partial mutation at the N-terminus of pIII and biotinylation at the hydrophobic end of pVIII. These alterations enable efficient attachment of tM13 to diverse E. coli strains, facilitating rapid magnetic separation. For detection, we additionally implemented a convolutional neural network (CNN)-based algorithm for precise identification and quantification of bacterial cells using dark field microscopy. RESULTS: The results obtained from spike-in and clinical sample analyses demonstrated the accuracy, high sensitivity (with a detection limit of 10 CFU/µL), and time-saving nature (30 min) of our tM13-based immunomagnetic enrichment approach combined with AI-enabled analytics, thereby supporting its potential to facilitate the identification of diverse E. coli strains in complex samples. CONCLUSION: The study established a rapid and accurate detection strategy for E. coli utilizing truncated M13 phages as capture probes, along with a dark field microscopy detection platform that integrates an image processing model and convolutional neural network.

Predominance of enterotoxigenic Escherichia coli among ESBL/plasmid-mediated AmpC-producing strains isolated from diarrheic foals: a public health concern.

BACKGROUND: The upsurge of diarrheagenic E. coli pathotypes carrying extended-spectrum beta-lactamases (ESBLs)/plasmid-mediated AmpC ß-lactamase (pAmpC) among animals constitutes an emerging threat for humans and animals. This study investigated the burden of ESBL-/pAmpC-producing diarrheagenic E. coli among diarrheic foals and its potential public health implications. Rectal swabs were collected from 80 diarrheic foals. These swabs were processed to isolate and identify ESBL/pAmpC-producing E. coli using a selective culture medium, biochemical tests, phenotypic identification, and molecular identification of ESBL- and pAmpC-encoding genes. Moreover, all ESBL-/pAmpC-producing E. coli isolates were examined for different virulence genes related to diarrheagenic E. coli pathotypes. RESULTS: Out of 80 examined foals, 26 (32.5%) were confirmed as ESBL-/pAmpC-producing E. coli, of which 14 (17.5%) animals carried only ESBL-producing E. coli, whereas 12 (15%) animals possessed ESBL-pAmpC-producing E. coli. The only detected diarrheagenic pathotype was enterotoxigenic, encoded by the heat-stable enterotoxin gene (ST) with a prevalence rate of 80.8% (21/26). The ST gene was further characterized where STa, STb, and STa + STb were found in one, four, and 16 strains, respectively. Moreover, all enterotoxigenic E. coli (ETEC) isolates exhibited a multidrug-resistance pattern. The phylogenetic analysis of 3 obtained partial STb sequences revealed high genetic relatedness to ETEC isolates retrieved from humans, conferring such sequences' public health significance. CONCLUSIONS: These findings highlight that diarrheic foals could serve as a potential reservoir for multidrug-resistant ESBL-/pAmpC-producing enterotoxigenic E. coli.

Prevalence and molecular characteristics of carbapenem-resistant Escherichia coli isolated from dogs in South Korea.

IMPORTANCE: Carbapenem-resistant Enterobacteriaceae are emerging as a global public health risk. Therefore, assessing the prevalence of carbapenem-resistant Escherichia coli (CRE) in both humans and animals is important. OBJECTIVE: We aimed to ascertain the occurrence and characteristics of CRE isolated from companion animals, dogs and cats. METHODS: E. coli strains were tested for antimicrobial susceptibility using the broth microdilution technique. Antimicrobial resistance genes were detected by polymerase chain reaction and sequencing analysis. The molecular characteristics of CRE were determined using multi-locus sequence typing, replicon typing, and pulsed-field gel electrophoresis (PFGE). RESULTS: In total, 13 CRE isolates (0.13%) were identified from dogs possessing blaNDM-5 along with ß-lactamase genes, mostly blaCMY-2 (92.2%) and blaTEM-1 (53.8%). The commonly observed mutations were S83L and D87N in gyrA, S80I in parC, and S458A in parE. CRE carried non-beta-lactam resistance genes, with the majority being tet(B) (100%), sul (84.6%), and aac(3)-II (53.8%). Nine different PFGE patterns (P1-P9), IncX3-type plasmids (69.2%), and ST410 (84.6%) were predominantly detected. CONCLUSIONS AND RELEVANCE: This investigation provides significant insight into the prevalence and molecular characteristics of blaNDM-5-carrying E. coli in dogs. The co-existence of blaNDM-5 and other antimicrobial resistance genes in E. coli potentially poses severe health hazards to humans.

Comparison of liquid and filter sampling techniques for recovery of Bacillus spores and Escherichia coli from environmental water.

Historically, detecting water contamination has involved collecting and directly analyzing liquid samples, but recent advances in filter sampling methods offer numerous potential advantages. Emerging technologies, including environmental DNA (eDNA) samplers, could be used for remote microbial contamination sampling, but work is needed to determine if target microorganisms can be recovered from filters at comparable levels to traditional sampling methods. In this study, Escherichia coli and a surrogate for Bacillus anthracis spores were sampled from synthetic stormwater and quantified using both direct liquid and filter methods, and dwell time tests compared microorganism persistence in water and on filters. At nearly all tested timepoints, the recoveries of spores from membrane filters were within 0.5 log10 colony forming units per sample (CFU/sample) compared to the liquid-only samples, suggesting that the use of filter sampling is a feasible alternative to liquid-based sampling, and samples were held for up to 4 weeks without significant sample degradation. Recoveries for E. coli remained relatively consistent for ∼3 days in phosphate buffered saline (PBS), in synthetic stormwater, and on membrane filters, but decreases in recoveries were observed for samples held for >3 days. These results indicate that emerging water sampling technologies, which reduce logistical burdens and offer potential cost savings, can be leveraged to characterize biological contamination in water matrices with multiple types of microbiological agents.

Severe Autoimmune Hemolytic Anemia Following Pneumococcal Vaccination in an Infant With Underlying Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency: Report of a Case and Review of the Literature.

Autoimmune hemolytic anemia (AIHA) and glucose-6-phosphate dehydrogenase (G6PD) deficiency are two distinct causes of hemolysis in children and a combination of both diseases is considered rare, especially in early infancy. We present such a rare case of severe AIHA in an infant with G6PD deficiency in the setting of Escherichia coli urinary tract infection and recent pneumococcal vaccination history, with the goal of analyzing potential links between them, examining the causative role of vaccines, and reviewing available literature.

Molecular characterization of common zoonotic protozoan parasites and bacteria causing diarrhea in dairy calves in Ningxia Hui Autonomous Region, China.

Diarrhea caused by zoonotic pathogens is one of the most common diseases in dairy calves, threatening the health of young animals. Humans are also at risk, in particular children. To explore the pathogens causing diarrhea in dairy calves, the present study applied PCR-based sequencing tools to investigate the occurrence and molecular characteristics of three parasites (Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi) and three bacterial pathogens (Escherichia coli, Clostridium perfringens, and Salmonella spp.) in 343 fecal samples of diarrheic dairy calves from five farms in Lingwu County, Ningxia Hui Autonomous Region, China. The total positive rate of these pathogens in diarrheic dairy calves was 91.0% (312/343; 95% CI, 87.9-94.0), with C. perfringens (61.5%, 211/343; 95% CI, 56.3-66.7) being the dominant one. Co-infection with two to five pathogens was found in 67.3% (231/343; 95% CI, 62.4-72.3) of investigated samples. There were significant differences (p < 0.05) in the positive rates of Cryptosporidium spp. and diarrheagenic E. coli among farms, age groups, and seasons. Two Cryptosporidium species (C. parvum and C. bovis) and five gp60 subtypes of C. parvum (IIdA15G1, IIdA20G1, IIdA19G1, IIdA14G1, and a novel IIdA13G1) were identified. Two assemblages (assemblage E and zoonotic assemblage A) of G. duodenalis and six ITS genotypes of E. bieneusi (J, Henan-IV, EbpC, I, EbpA, and ESH-01) were observed. Four virulence genes (eaeA, stx1, stx2, and st) of diarrheagenic E. coli and one toxin type (type A) of C. perfringens were detected. Our study enriches our knowledge on the characteristics and zoonotic potential of diarrhea-related pathogens in dairy calves.

Title: Caractérisation moléculaire des protozoaires parasites zoonotiques courants et des bactéries responsables de diarrhée chez les veaux laitiers dans la région autonome Hui du Ningxia, en Chine. Abstract: La diarrhée causée par des agents pathogènes zoonotiques est l'une des maladies les plus courantes chez les veaux laitiers, menaçant la santé des jeunes animaux. Ceci est également un risque pour la santé humaine, en particulier les enfants. Pour explorer les agents pathogènes responsables de la diarrhée chez les veaux laitiers, cette étude a utilisé des outils de séquençage basés sur la PCR pour étudier l'occurrence et les caractères moléculaires de trois parasites (Cryptosporidium spp., Giardia duodenalis et Enterocytozoon bieneusi) et de trois agents pathogènes bactériens (Escherichia coli, Clostridium perfringens et Salmonella spp.) dans 343 échantillons fécaux de veaux laitiers diarrhéiques provenant de cinq fermes du comté de Lingwu, région autonome Hui du Ningxia, en Chine. Le taux total positif de ces pathogènes chez les veaux laitiers diarrhéiques était de 91,0 % (312/343; IC à 95 %, 87,9­94,0), et C. perfringens (61,5 %, 211/343; IC à 95 %, 56,3­66,7) était le plus répandu. Une co-infection avec deux à cinq pathogènes a été trouvée dans 67,3 % (231/343; IC à 95 %, 62,4­72,3) des échantillons étudiés. Il y avait des différences significatives (p < 0,05) dans les taux positifs de Cryptosporidium spp. et d'E. coli diarrhéogènes entre les fermes, les groupes d'âge et les saisons. Deux espèces de Cryptosporidium (C. parvum et C. bovis) et cinq sous-types de gp60 de C. parvum (IIdA15G1, IIdA20G1, IIdA19G1, IIdA14G1 et un nouveau, IIdA13G1) ont été identifiés. Deux assemblages (assemblage E et assemblage zoonotique A) de G. duodenalis et six génotypes ITS d'E. bieneusi (J, Henan-IV, EbpC, I, EbpA et ESH-01) ont été observés. Quatre gènes de virulence (eaeA, stx1, stx2 et st) d'E. coli diarrhéogènes et un type de toxine (type A) de C. perfringens ont été détectés. Notre étude enrichit les connaissances sur les caractères et le potentiel zoonotique des agents pathogènes liés à la diarrhée chez les veaux laitiers.

Microbial production of adipic acid from 6-hydroxyhexanoic acid for biocatalytic upcycling of polycaprolactone.

To valorize waste polycaprolactone (PCL), one of the most widely used biodegradable plastics, into a value-added chemical, we upcycled 6-hydroxyhexanoic acid (6-HHA), the sole monomer of PCL, into adipic acid (AA) using a microbial method. Recombinant Escherichia coli strains expressing chnD (6-HHA dehydrogenase) and chnE (6-oxohexanoic acid dehydrogenase) genes from three bacteria were constructed, and all these strains successfully produced AA from 6-HHA. Among these, the E. coli strain harboring ChnDE genes from Acinetobacter strain SE19 (E. coli [pKK-AcChn]) showed the highest AA-producing ability. To increase the AA production titer, we optimized the culture temperature of this strain in flask culture and performed fed-batch fermentation in a 5 L bioreactor. After the fed-batch fermentation, the AA production titer increased to 15.6 g/L. As 6-HHA is a monomer of PCL, our results provide the groundwork for the development of a biocatalytic upcycling method of PCL.

Effects of monoglyceride blend on systemic and intestinal immune responses, and gut health of weaned pigs experimentally infected with a pathogenic Escherichia coli.

BACKGROUND: Monoglycerides have emerged as a promising alternative to conventional practices due to their biological activities, including antimicrobial properties. However, few studies have assessed the efficacy of monoglyceride blend on weaned pigs and their impacts on performance, immune response, and gut health using a disease challenge model. Therefore, this study aimed to investigate the effects of dietary monoglycerides of short- and medium-chain fatty acids on the immunity and gut health of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli F18. RESULTS: Pigs supplemented with high-dose zinc oxide (ZNO) had greater (P < 0.05) growth performance than other treatments, but no difference was observed in average daily feed intake between ZNO and monoglycerides groups during the post-challenge period. Pigs in ZNO and antibiotic groups had lower (P < 0.05) severity of diarrhea than control, but the severity of diarrhea was not different between antibiotic and monoglycerides groups. Pigs fed with monoglycerides or ZNO had lower (P < 0.05) serum haptoglobin on d 2 or 5 post-inoculation than control. Pigs in ZNO had greater (P < 0.05) goblet cell numbers per villus, villus area and height, and villus height:crypt depth ratio (VH:CD) in duodenum on d 5 post-inoculation than pigs in other treatments. Pigs supplemented with monoglycerides, ZNO, or antibiotics had reduced (P < 0.05) ileal crypt depth compared with control on d 5 post-inoculation, contributing to the increase (P = 0.06) in VH:CD. Consistently, pigs in ZNO expressed the lowest (P < 0.05) TNFa, IL6, IL10, IL12, IL1A, IL1B, and PTGS2 in ileal mucosa on d 5 post-inoculation, and no difference was observed in the expression of those genes between ZNO and monoglycerides. Supplementation of ZNO and antibiotic had significant impacts on metabolic pathways in the serum compared with control, particularly on carbohydrate and amino acid metabolism, while limited impacts on serum metabolites were observed in monoglycerides group when compared with control. CONCLUSIONS: The results suggest that supplementation of monoglyceride blend may enhance disease resistance of weaned pigs by alleviating the severity of diarrhea and mitigating intestinal and systemic inflammation, although the effectiveness may not be comparable to high-dose zinc oxide.

Avian pathogenic Escherichia coli (APEC): current insights and future challenges.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in avian species, and new investigations have implicated APEC as a possible foodborne zoonotic pathogen. This review analyzes APEC's pathogenic and virulence features, assesses the zoonotic potential, provides an update on antibiotic resistance and vaccine research efforts, and outlines alternate management approaches. Aside from established virulence factors, various additional components, including 2-component systems (TCS), adhesins, secretion systems (SS), invasions, iron acquisition systems, quorum sensing systems (QS), transcriptional regulators (TR), toxins, and genes linked with metabolism, contribute to APEC pathogenesis. APEC may spread to diverse species of birds in all business sectors and can infect birds of varying ages. However, younger birds experience more severe sickness than mature ones, probably due to their developing immune systems, and stress factors such as vaccination, Mycoplasma Infections, poor housing circumstances, respiratory viruses, and other risk factors for secondary infections can all make APEC both primary and secondary pathogens. Understanding these factors will help in generating new and effective treatments. Moreover, APEC O145 was the most prevalent serotype recently reported in all of China. Thus, the APEC's zoonotic potential should not be underrated. Furthermore, it has already been noted that APEC is resistant to almost all antibiotic classes, including carbapenems. A robust vaccine capable of protecting against multiple APEC serotypes is urgently needed. Alternative medications, particularly virulence inhibitors, can provide a special method with a decreased likelihood of acquiring resistance.

Improving the growth and intestinal colonization of Escherichia coli Nissle 1917 by strengthening its oligopeptides importation ability.

Escherichia coli Nissle 1917 (EcN), the probiotic featured with well-established safety in different host, is emerging as a favored chassis for the construction of engineered probiotics for disease treatment. However, limited by the low intestinal colonization ability of EcN, repeated administration is required to maximize the health benefits of the EcN-derived engineered probiotics. Here, using fecal metabolites as "metabolites pool", we developed a metabolomic strategy to characterize the comprehensive metabolic profile of EcN. Compared with Prevotella copri DSM 18205 (P. copri), one of the dominant microbes in gut flora, EcN exhibited minor growth advantage under the fecal metabolites-containing condition for its lower metabolic capability towards fecal metabolites. Further study indicated that EcN lacked the ability to import the oligopeptides containing more than two amino acids. The shortage of oligopeptides-derived amino acids might limit the growth of EcN by restricting its purine metabolism. Assisted with the bioinformatic and qRT-PCR analyses, we identified a tripeptides-specific importer Pc-OPT in P. copri, which was mainly distributed in genera Prevotella and Bacteroides. Overexpression of Pc-OPT improved the tripeptides importation of EcN and promoted its growth and intestinal colonization. Notably, 16S rRNA gene amplicon sequencing results indicated that strengthening the oligopeptides importation ability of EcN might promote its intestinal colonization by adjusting the gut microbial composition. Our study reveals that the growth and intestinal colonization of EcN is limited by its insufficient oligopeptides importation and paves road for promoting the efficacy of the EcN-derived synthetic probiotics by improving their intestinal colonization ability.

Secretion of the human parathyroid hormone through a microcin type I secretion system in Escherichia coli.

BACKGROUND: Gram negative bacteria possess different secretion systems to export proteins to the extracellular medium. The simplest one, type I secretion system (T1SS), forms a channel across the cell envelope to export proteins in a single step. Peptides secreted by the T1SSs comprise a group of antibiotics, called class II microcins, which carry an amino terminal secretion domain that is processed concomitantly with export. Mature microcins range in size from 60 to 90 amino acids and differ in their sequences. Microcin T1SSs show a high versatility in relation to the peptides they are able to secrete, being mainly limited by the length of the substrates. Different bioactive peptides unrelated to bacteriocins could be secreted by microcin V (MccV) T1SS, while retaining their biological activity. RESULTS: In this work heterologous secretion of two variants of human parathyroid hormone (PTH) by MccV T1SS was evaluated. PTH is a bioactive peptide of 84 amino acids (PTH84), which is involved in the maintenance of bone homeostasis. Currently, a drug corresponding to the active fraction of the hormone, which resides in its first 34 amino acids (PTH34), is commercially produced as a recombinant peptide in Escherichia coli. However, research continues to improve this recombinant production. Here, gene fusions encoding hybrid peptides composed of the MccV secretion domain attached to each hormone variant were constructed and expressed in the presence of microcin T1SS in E. coli cells. Both PTH peptides (PTH34 and PTH84) were recovered from the culture supernatants and could be confirmed to lack the MccV secretion domain, i.e. microcin T1SS efficiently recognised, processed and secreted both PTH variants. Furthermore, the secreted peptides were stable in the extracellular medium unlike their unprocessed counterparts present in the intracellular space. CONCLUSION: The successful secretion of PTH variants using MccV T1SS could be considered as a new alternative for their production, since they would be recovered directly from the extracellular space without additional sequences. Furthermore, it would be a new example revealing the potential of microcin type I secretion systems to be conceived as a novel strategy for the production of recombinant peptides in E. coli.

Effects of dietary Lactobacillus postbiotics and bacitracin on the modulation of mucosa-associated microbiota and pattern recognition receptors affecting immunocompetence of jejunal mucosa in pigs challenged with enterotoxigenic F18+ Escherichia coli.

BACKGROUND: Enterotoxigenic Escherichia coli (E. coli) is a threat to humans and animals that causes intestinal disorders. Antimicrobial resistance has urged alternatives, including Lactobacillus postbiotics, to mitigate the effects of enterotoxigenic E. coli. METHODS: Forty-eight newly weaned pigs were allotted to NC: no challenge/no supplement; PC: F18+ E. coli challenge/no supplement; ATB: F18+ E. coli challenge/bacitracin; and LPB: F18+ E. coli challenge/postbiotics and fed diets for 28 d. On d 7, pigs were orally inoculated with F18+ E. coli. At d 28, the mucosa-associated microbiota, immune and oxidative stress status, intestinal morphology, the gene expression of pattern recognition receptors (PRR), and intestinal barrier function were measured. Data were analyzed using the MIXED procedure in SAS 9.4. RESULTS: PC increased (P < 0.05) Helicobacter mastomyrinus whereas reduced (P < 0.05) Prevotella copri and P. stercorea compared to NC. The LPB increased (P < 0.05) P. stercorea and Dialister succinatiphilus compared with PC. The ATB increased (P < 0.05) Propionibacterium acnes, Corynebacterium glutamicum, and Sphingomonas pseudosanguinis compared to PC. The PC tended to reduce (P = 0.054) PGLYRP4 and increased (P < 0.05) TLR4, CD14, MDA, and crypt cell proliferation compared with NC. The ATB reduced (P < 0.05) NOD1 compared with PC. The LPB increased (P < 0.05) PGLYRP4, and interferon-γ and reduced (P < 0.05) NOD1 compared with PC. The ATB and LPB reduced (P < 0.05) TNF-α and MDA compared with PC. CONCLUSIONS: The F18+ E. coli challenge compromised intestinal health. Bacitracin increased beneficial bacteria showing a trend towards increasing the intestinal barrier function, possibly by reducing the expression of PRR genes. Lactobacillus postbiotics enhanced the immunocompetence of nursery pigs by increasing the expression of interferon-γ and PGLYRP4, and by reducing TLR4, NOD1, and CD14.

Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria.

While highly effective at killing Gram-positive bacteria, auranofin lacks significant activity against Gram-negative species for reasons that largely remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the low susceptibility of the Gram-negative model organism Escherichia coli to auranofin when compared to the Gram-positive model organism Bacillus subtilis. The proteome response of E. coli exposed to auranofin suggests a combination of inactivation of thiol-containing enzymes and the induction of systemic oxidative stress. Susceptibility tests in E. coli mutants lacking proteins upregulated upon auranofin treatment suggested that none of them are directly involved in E. coli's high tolerance to auranofin. E. coli cells lacking the efflux pump component TolC were more sensitive to auranofin treatment, but not to an extent that would fully explain the observed difference in susceptibility of Gram-positive and Gram-negative organisms. We thus tested whether E. coli's thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from B. subtilis, which was not the case. However, E. coli strains lacking the low-molecular-weight thiol glutathione, but not glutathione reductase, showed a high susceptibility to auranofin. Bacterial cells expressing the genetically encoded redox probe roGFP2 allowed us to observe the oxidation of cellular protein thiols in situ. Based on our findings, we hypothesize that auranofin leads to a global disturbance in the cellular thiol redox homeostasis in bacteria, but Gram-negative bacteria are inherently more resistant due to the presence of drug export systems and high cellular concentrations of glutathione.IMPORTANCEAuranofin is an FDA-approved drug for the treatment of rheumatoid arthritis. However, it has also high antibacterial activity, in particular against Gram-positive organisms. In the current antibiotics crisis, this would make it an ideal candidate for drug repurposing. However, its much lower activity against Gram-negative organisms prevents its broad-spectrum application. Here we show that, on the level of the presumed target, there is no difference in susceptibility between Gram-negative and Gram-positive species: thioredoxin reductases from both Escherichia coli and Bacillus subtilis are equally inhibited by auranofin. In both species, auranofin treatment leads to oxidative protein modification on a systemic level, as monitored by proteomics and the genetically encoded redox probe roGFP2. The single largest contributor to E. coli's relative resistance to auranofin seems to be the low-molecular-weight thiol glutathione, which is absent in B. subtilis and other Gram-positive species.