Avaliação das boas práticas de fabricação e da contaminação de preparações cárneas de restaurantes institucionais e comerciais na cidade de Americana, SP / Evaluation of good manufacturing practices and contamination in meat preparations from institutional and commercial restaurants in the city of Americana/SP

Diante do ritmo acelerado da vida contemporânea, observa-se um aumento na tendência dos indivíduos em optar por realizar suas refeições fora de casa. A carne, reconhecida como um componente essencial na alimentação dos brasileiros, está suscetível à contaminação pois apresenta ambiente favorável à proliferação de microrganismos patogênicos. Fazendo-se necessária uma análise de contaminação pós-produção afim de evitar Doenças Transmitidas por Alimentos. No presente estudo objetivouse avaliar as boas práticas de fabricação e contaminação de preparações de carne bovina assada, de restaurantes particulares e institucionalizados no município de Americana-SP. Amostras de carne prontas para o consumo foram obtidas de seis estabelecimentos comerciais e seis institucionais. Durante a coleta, foram verificadas as temperaturas e realizadas análises de conformidades com a RDC n° 275, de 2002. As amostras foram examinadas para detectar a presença ou ausência de E. coli e coliformes termotolerantes a 45° C. Para a análise foi realizada a técnica de tubos múltiplos para quantificar a totalidade dos coliformes. Observou-se que, conforme estipulado pela Resolução n°43 de 2015, nenhuma das amostras oriundas de restaurantes comerciais, e a maioria das provenientes de restaurantes institucionais, atingiram as temperaturas requeridas. No que concerne à identificação de E. coli através de testes microbiológicos, foi constatado que seis amostras de restaurantes comerciais e quatro de restaurantes institucionais testaram positivo para a presença deste microrganismo. Conclui-se que as amostras de restaurantes comerciais apresentaram níveis de contaminação superiores em comparação com as amostras de restaurantes institucionais.

Given the fast-paced rhythm of contemporary life, there is an increase in individuals choosing to have their meals outside the home. Meat, recognized as an essential component in the Brazilian diet, is susceptible to contamination as it provides a favorable environment for the proliferation of pathogenic microorganisms. It is necessary to conduct post-production contamination analysis to prevent Foodborne Diseases. This study aimed to evaluate the good manufacturing practices and contamination of roasted beef preparations from private and institutional restaurants in the city of Americana-SP. Samples of ready-to-eat meat were obtained from six commercial establishments and six institutional ones. During collection, temperatures were checked, and conformity analyses were conducted according to RDC No. 275, 2002. The samples were examined for the presence or absence of E. coli and thermotolerant coliforms at 45°C using the multiple tube technique to quantify the total coliforms. It was observed that, as stipulated by Resolution No. 43, 2015, none of the samples from commercial restaurants and the majority from institutional restaurants reached the required temperatures. Regarding the identification of E. coli through microbiological tests, it was found that six samples from commercial restaurants and four from institutional ones tested positive for the presence of this microorganism. It is concluded that samples from commercial restaurants showed higher contamination levels compared to institutional restaurant samples.

A dual-channel electrochemical biosensor enables concurrent detection of pathogens and antibiotic resistance.

Diarrheagenic E. coli infections, commonly treated with ß-lactam antibiotics, contribute to antibiotic resistance - a pressing public health concern. Rapid monitoring of pathogen antibiotic resistance is vital to combat antimicrobial spread. Current bacterial diagnosis methods identify pathogens or determine antibiotic resistance separately, necessitating multiple assays. There is an urgent need for tools that simultaneously identify infectious agents and their antibiotic resistance at the point of care (POC). We developed an integrated electrochemical chip-based biosensor for detecting enteropathogenic E. coli (EPEC), a major neonatal diarrheal pathogen, using an antibody against a virulence marker, termed EspB, and the ß-lactam resistance marker, ß-lactamase. A dual-channel microfabricated chip, bio-functionalized with a specific EspB monoclonal antibody, and nitrocefin, a ß -lactamase substrate was utilized. The chip facilitated electrochemical impedance spectroscopy (EIS)-based detection of EspB antigen and EspB-expressing bacteria. For ß-lactam resistance profiling, a second channel enabled differential-pulse voltammetric (DPV) measurement of hydrolyzed nitrocefin. EIS-based detection of EspB antigen was calibrated (LOD: 4.3 ng/mL ±1 and LOQ: 13.0 ng/mL ±3) as well as DPV-based detection of the antibiotic resistance marker, ß-lactamase (LOD: 3.6 ng/mL ±1.65 and LOQ: 10 ng/mL ±4). The integrated EIS and DPV biosensor was employed for the simultaneous detection of EspB-expressing and ß-lactamase-producing bacteria. The combined readout from both channels allowed the distinction between antibiotic-resistant and -sensitive pathogenic bacteria. The integrated electrochemical biosensor successfully achieved simultaneous, rapid detection of double positive EspB- and ß-lactamase-expressing bacteria. Such distinction enabled by a portable device within a short assay time and a simplified sample preparation, may be highly valuable in mitigating the spread of AMR. This new diagnostic tool holds promise for the development of POC devices in clinical diagnosis.

Deletion of yghZ in Escherichia coli promotes growth in presence of furfural with xylose as carbon source.

Thermo-acidic pretreatment of lignocellulosic biomass is required to make it amenable to microbial metabolism and results in generation of furfural due to breakdown of pentose sugars. Furfural is toxic to microbial metabolism and results in reduced microbial productivity and increased production costs. This study asks if deletion of yghZ gene which encodes a NADPH-dependent aldehyde reductase enzyme results in improved furfural tolerance in Escherichia coli host. The ∆yghZ strain-SSK201-was tested for tolerance to furfural in presence of 5% xylose as a carbon source in AM1 minimal medium. At 96 h and in presence of 1.0 g/L furfural, the culture harboring strain SSK201 displayed 4.5-fold higher biomass, 2-fold lower furfural concentration and 15.75-fold higher specific growth rate (µ) as compared to the parent strain SSK42. The furfural tolerance advantage of SSK201 was retained when the carbon source was switched to glucose in AM1 medium and was lost in rich LB medium. The findings have potential to be scaled up to a hydrolysate culture medium, which contains furan inhibitors and lack nutritionally rich components, under bioreactor cultivation and observe growth advantage of the ∆yghZ host. It harbors potential to generate robust industrial strains which can convert lignocellulosic carbon into metabolites of interest in a cost-efficient manner.

Functional evaluation of Bacillus licheniformis PF9 for its potential in controlling enterotoxigenic Escherichia coli in weaned piglets.

During the bacterial selection, isolate PF9 demonstrated tolerance to low pH and high bile salt and an ability to extend the lifespan of Caenorhabditis elegans infected with enterotoxigenic Escherichia coli (ETEC; P < 0.05). Thirty-two weaned piglets susceptible to ETEC F4 were randomly allocated to four treatments as follows: 1) non-challenged negative control group (NNC; basal diet and piglets gavaged with phosphate-buffered saline), 2) negative control group (NC; basal diet and piglets challenged with ETEC F4, 3 × 107 CFU per pig), 3) positive control (PC; basal diet + 80 mg·kg-1 of avilamycin and piglets challenged with ETEC F4), and 4) probiotic candidate (PF9; control basal diet + 2.5 × 109 CFU·kg-1 diet of B. licheniformis PF9 and piglets challenged with ETEC F4). The infection of ETEC F4 decreased average daily gain and gain:feed in the NC group when compared to the NNC group (P < 0.05). The inoculation of ETEC F4 induced severe diarrhea at 3 h postinoculum (hpi), 36, 40 hpi in the NC group when compared to the NNC group (P < 0.05). The supplementation of B. licheniformis PF9 significantly relieved diarrhea severity at 3 hpi when compared to the NC group (P < 0.05). The inoculation of ETEC F4 reduced duodenal, jejunal, and ileal villus height (VH) in the NC group when compared to the NNC group. A significant (P < 0.05) decrease was detected in the duodenal VH in the PC and NNC groups. Moreover, the NNC group had a reduced relative mRNA level of Na+-glucose cotransporter 1 (SGLT1) when compared to the NC group (P < 0.05). Compared to the NC and NNC groups, the supplementation of B. licheniformis PF9 increased the relative mRNA levels of aminopeptidase N, occludin, zonula occludens-1, and SGLT1 (P < 0.05). The supplementation of B. licheniformis PF9 also significantly increased the relative mRNA level of excitatory amino acid transporter 1 when compared to the NC group (P < 0.05). Piglets supplemented with B. licheniformis PF9 showed lower relative abundance of Bacteroidetes in the colon than piglets from the NNC group (P < 0.05). The NNC group had a higher relative abundance of Firmicutes in the ileum than all the challenged piglets (P < 0.05); however, a lower relative abundance of Proteobacteria in the ileum and colon was observed in the NC group (P < 0.05). This study provides evidence that B. licheniformis PF9 has the potential to improve the gut health of piglets under challenging conditions.

High-risk Escherichia coli clones that cause neonatal meningitis and association with recrudescent infection.

Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974-2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.

Multivariate modular metabolic engineering and medium optimization for vitamin B12 production by Escherichia coli.

Vitamin B12 is a complex compound synthesized by microorganisms. The industrial production of vitamin B12 relies on specific microbial fermentation processes. E. coli has been utilized as a host for the de novo biosynthesis of vitamin B12, incorporating approximately 30 heterologous genes. However, a metabolic imbalance in the intricate pathway significantly limits vitamin B12 production. In this study, we employed multivariate modular metabolic engineering to enhance vitamin B12 production in E. coli by manipulating two modules comprising a total of 10 genes within the vitamin B12 biosynthetic pathway. These two modules were integrated into the chromosome of a chassis cell, regulated by T7, J23119, and J23106 promoters to achieve combinatorial pathway optimization. The highest vitamin B12 titer was attained by engineering the two modules controlled by J23119 and T7 promoters. The inclusion of yeast powder to the fermentation medium increased the vitamin B12 titer to 1.52 mg/L. This enhancement was attributed to the effect of yeast powder on elevating the oxygen transfer rate and augmenting the strain's isopropyl-ß-d-1-thiogalactopyranoside (IPTG) tolerance. Ultimately, vitamin B12 titer of 2.89 mg/L was achieved through scaled-up fermentation in a 5-liter fermenter. The strategies reported herein will expedite the development of industry-scale vitamin B12 production utilizing E. coli.

Tools and methods for high-throughput single-cell imaging with the mother machine.

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge. To fill this gap, we implemented our own software, MM3, as a plugin for the multidimensional image viewer napari. napari-MM3 is a complete and modular image analysis pipeline for mother machine data, which takes advantage of the high-level interactivity of napari. Here, we give an overview of napari-MM3 and test it against several well-designed and widely used image analysis pipelines, including BACMMAN and DeLTA. Researchers often analyze mother machine data with custom scripts using varied image analysis methods, but a quantitative comparison of the output of different pipelines has been lacking. To this end, we show that key single-cell physiological parameter correlations and distributions are robust to the choice of analysis method. However, we also find that small changes in thresholding parameters can systematically alter parameters extracted from single-cell imaging experiments. Moreover, we explicitly show that in deep learning-based segmentation, 'what you put is what you get' (WYPIWYG) - that is, pixel-level variation in training data for cell segmentation can propagate to the model output and bias spatial and temporal measurements. Finally, while the primary purpose of this work is to introduce the image analysis software that we have developed over the last decade in our lab, we also provide information for those who want to implement mother machine-based high-throughput imaging and analysis methods in their research.

Characterization of blaNDM-19-producing IncX3 plasmid isolated from carbapenem-resistant Escherichia coli and Klebsiellapneumoniae.

The increase in the prevalence of carbapenem-producing Enterobacterales (CPE) is a major threat, with the New Delhi metallo-ß-lactamase (NDM) enzyme-producing CPEs being one of the major causative agents of healthcare settings infections. In this study, we characterized an IncX3 plasmid harboring blaNDM-19 in Lebanon, recovered from three Escherichia coli belonging to ST167 and one Klebsiella pneumoniae belonging to ST16 isolated from a clinical setting. Plasmid analysis using PBRT, Plasmid Finder, and PlasmidSPAdes showed that all four isolates carried a conjugative 47-kb plasmid having blaNDM-19, and was designated as pLAU-NDM19. We constructed a sequence-based maximum likelihood phylogenetic tree and compared pLAU-NDM19 to other representative IncX3 plasmids carrying NDM-variants and showed that it was closely linked to NDM-19 positive IncX3 plasmid from K. pneumoniae reported in China. Our findings also revealed the route mediating resistance transmission, the IncX3 dissemination among Enterobacterales, and the NDM-19 genetic environment. We showed that mobile elements contributed to the variability of IncX3 genomic environment and highlighted that clonal dissemination in healthcare settings facilitated the spread of resistance determinants. Antimicrobial stewardship programs implemented in hospitals should be coupled with genomic surveillance to better understand the mechanisms mediating the mobilization of resistance determinants among nosocomial pathogens and their subsequent clonal dissemination.

Convergent genetic adaptation of Escherichia coli in minimal media leads to pleiotropic divergence.

Adaptation in an environment can either be beneficial, neutral or disadvantageous in another. To test the genetic basis of pleiotropic behaviour, we evolved six lines of E. coli independently in environments where glucose and galactose were the sole carbon sources, for 300 generations. All six lines in each environment exhibit convergent adaptation in the environment in which they were evolved. However, pleiotropic behaviour was observed in several environmental contexts, including other carbon environments. Genome sequencing reveals that mutations in global regulators rpoB and rpoC cause this pleiotropy. We report three new alleles of the rpoB gene, and one new allele of the rpoC gene. The novel rpoB alleles confer resistance to Rifampicin, and alter motility. Our results show how single nucleotide changes in the process of adaptation in minimal media can lead to wide-scale pleiotropy, resulting in changes in traits that are not under direct selection.

Efficacy evaluation of hydrogen peroxide disinfectant based zinc oxide nanoparticles against diarrhea causing Escherichia coli in ruminant animals and broiler chickens.

Different strains of Escherichia coli that exhibit genetic characteristics linked to diarrhea pose a major threat to both human and animal health. The purpose of this study was to determine the prevalence of pathogenic Escherichia coli (E. coli), the genetic linkages and routes of transmission between E. coli isolates from different animal species. The efficiency of disinfectants such as hydrogen peroxide (H2O2), Virkon®S, TH4+, nano zinc oxide (ZnO NPs), and H2O2-based zinc oxide nanoparticles (H2O2/ZnO NPs) against isolated strains of E. coli was evaluated. Using 100 fecal samples from different diarrheal species (cow n = 30, sheep n = 40, and broiler chicken n = 30) for E. coli isolation and identification using the entero-bacterial repetitive intergenic consensus (ERIC-PCR) fingerprinting technique. The E. coli properties isolated from several diarrheal species were examined for their pathogenicity in vitro. Scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), Fourier-transform infrared spectrum (FT-IR), X-ray diffraction (XRD), zeta potential, and particle size distribution were used for the synthesis and characterization of ZnO NPs and H2O2/ZnO NPs. The broth macro-dilution method was used to assess the effectiveness of disinfectants and disinfectant-based nanoparticles against E. coli strains. Regarding the results, the hemolytic activity and Congo red binding assays of pathogenic E. coli isolates were 55.3 and 44.7%, respectively. Eleven virulent E. coli isolates were typed into five ERIC-types (A1, A2, B1, B2, and B3) using the ERIC-PCR method. These types clustered into two main clusters (A and B) with 75% similarity. In conclusion, there was 90% similarity between the sheep samples' ERIC types A1 and A2. On the other hand, 89% of the ERIC types B1, B2, and B3 of cows and poultry samples were comparable. The H2O2/ZnO NPs composite exhibits potential antibacterial action against E. coli isolates at 0.04 mg/ml after 120 min of exposure.

Bacterial Flora and Treatment Strategies in Women With Escherichia coli Urinary Tract Infections.

Introduction This study explores the intricate relationship between bacterial flora and the occurrence of Escherichia coli (E. coli) infections in gynecological patients. It aims to provide insights into the various treatment strategies used to effectively manage bacterial pathogens, especially E. coli infections. By conducting a comprehensive analysis of the bacterial flora in gynecological patients, the study highlights the notable presence of E. coli, prompting further investigation into the factors that contribute to its colonization. The objective of the study is to comprehensively investigate and detect urinary tract infections (UTIs) specifically caused by E. coli among gynecological patients. The study aims to delve into bacterial flora prevalence, antibiotic resistance patterns, and potential virulence factors. Through this analysis, the study intends to identify effective strategies for rapid detection and diagnosis of UTIs caused by E. coli by utilizing advanced microbiological and molecular techniques. Furthermore, the study aims to formulate and propose a strategic treatment approach with a particular emphasis on selecting appropriate antibiotics to reduce the risk of severe infections and associated complications. Materials and methods The methodology employed in this study included the isolation and characterization of bacterial strains from clinical samples obtained from gynecological patients. A total of 52 urine specimens were collected from patients with complaints of infection in the urinary tract and infertility. These samples underwent both preliminary and confirmatory microbiological analysis, such as gram staining, biochemical confirmation test, and antibiotic susceptibility, and further proceeded with the multiplex polymerase chain reaction (PCR) technique. The results of PCR and antibiotic susceptibility revealed the specific gene involvement and resistant characteristics of E. coli. Results The findings revealed a total of 32 specimens positive for E. coli, of which 10 patients had infertility complaints and 22 patients had UTIs. The preliminary test, gram staining, showed the gram-negative bacilli E. coli, and the nutrient agar plate revealed smooth circular translucent colonies; MacConkey agar showed pink-colored lactose-fermented colonies; and the blood and chocolate agar plates showed grayish white moist gamma-hemolytic colonies. The biochemical confirmation of E. coli resulted in positive for indole and methyl red tests and negative for Voges-Proskauer and citrate utilization tests. The multiplex PCR analysis confirmed the E. coli strains with the presence of two target genes, stx2d and stx2e. Conclusion To summarize, this study offers valuable insights into the bacterial flora of gynecological patients impacted by E. coli infections, which provides a foundation for the development of precise and efficient treatment strategies. The results emphasize the importance of personalized treatment approaches that consider both the microbiological characteristics of the infection and the evolving landscape of antibiotic resistance. The implication of this research extends to enhancing clinical outcomes and alleviating the burden of E. coli infections in gynecological settings.

A Successful Approach to Diagnosing Shiga-like Toxin-Producing Escherichia coli-Induced Colitis.

Shiga-like toxin-producing Escherichia coli (STEC) is a well-known cause of foodborne acute diarrheic diseases, especially in children and the elderly. The potentially fatal complications associated with toxin production range from bloody diarrhea and ischemic colitis to kidney failure, hemolytic-uremic syndrome (HUS), and colon perforation. Here, we describe a case and literature review of STEC-induced colitis, highlighting the clinical features and the necessary tools for the best diagnostic approach and management. Facing challenging differential diagnosis, ranging from ischemic colitis and inflammatory bowel disease to infectious processes due to a pathogenic or opportunistic agent, we conducted a step-by-step exploration. Following bacteriological investigation, imagistic screening, and colonoscopy, we ruled out some of the initial suppositions and reached a final diagnosis, while also considering the pathological results. Although antibiotics are not indicated in this pathology, our patient did receive antibiotics, given the risk of translocation and colon perforation, without any associated complications such as HUS or peritonitis. Detailed and rigorous investigations conducted by a multi-specialty team are required for prompt medical support. Coping with the symptoms and refraining from further complications are the mainstem aims of treatment.

A Lignin Silver Nanoparticles/Polyvinyl Alcohol/Sodium Alginate Hybrid Hydrogel with Potent Mechanical Properties and Antibacterial Activity.

Antibacterial hydrogels have attracted significant attention due to their diverse applications, efficient antimicrobial properties, and adaptability to various environments and requirements. However, their relatively fragile structure, coupled with the potential for environmental toxicity when exposed to their surroundings for extended periods, may significantly limit their practical application potential. In this work, a composite hydrogel was synthesized with outstanding mechanical features and antibacterial capability. The hydrogel was developed through the combination of the eco-friendly and enduring antibacterial agent, lignin silver nanoparticles (Lig-Ag NPs), with polyvinyl alcohol (PVA) and sodium alginate (SA), in varying proportions. The successful synthesis of the hydrogel and the dispersed distribution of Lig-Ag NPs within the hydrogel were confirmed by various analytical techniques, including field emission scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), mercury intrusion porosimetry (MIP), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The formation of multiple hydrogen bonds between Lig-Ag NPs and the composites contributed to a more stable and dense network structure of the hydrogel, consequently enhancing its mechanical properties. Rheological tests revealed that the hydrogel exhibited an elastic response and demonstrated outstanding self-recovery properties. Significantly, the antibacterial hydrogel demonstrated effectiveness against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), achieving a <5% survival of bacteria within 12 h. This study presented a green and straightforward synthetic strategy for the application of antibacterial composite hydrogels in various fields.

Quantitative Analysis of Genomic DNA Degradation of E. coli Using Automated Gel Electrophoresis under Various Levels of Microwave Exposure.

The problem that this study addresses is to understand how microwave radiation is able to degrade genomic DNA of E. coli. In addition, a comparative study was made to evaluate the suitability of a high-throughput automated electrophoresis platform for quantifying the DNA degradation under microwave radiation. Overall, this study investigated the genomic DNA degradation of E. coli under microwave radiation using automated gel electrophoresis. To examine the viable organisms and degradation of genomic DNA under microwave exposure, we used three methods: (1) post-microwave exposure, where E. coli was enumerated using modified mTEC agar method using membrane filtration technique; (2) extracted genomic DNA of microwaved sample was quantified using the Qubit method; and (3) automated gel electrophoresis, the TapeStation 4200, was used to examine the bands of extracted DNA of microwaved samples. In addition, to examine the impacts of microwaves, E. coli colonies were isolated from a fecal sample (dairy cow manure), these colonies were grown overnight to prepare fresh E. coli culture, and this culture was exposed to microwave radiation for three durations: (1) 2 min; (2) 5 min; and (3) 8 min. In general, Qubit values (ng/µL) were proportional to the results of automated gel electrophoresis, TapeStation 4200, DNA integrity numbers (DINs). Samples from exposure studies (2 min, 5 min, and 8 min) showed no viable E. coli. Initial E. coli levels (at 0 min microwave exposure) were 5 × 108 CFU/mL, and the E. coli level was reduced to a non-detectable level within 2 min of microwave exposure. The relationships between Qubit and TapeStation measurements was linear, except for when the DNA level was lower than 2 ng/µL. In 8 min of microwave exposure, E. coli DNA integrity was reduced by 61.7%, and DNA concentration was reduced by 81.6%. The overall conclusion of this study is that microwave radiation had a significant impact on the genomic DNA of E. coli, and prolonged exposure of E. coli to microwaves can thus lead to a loss of genomic DNA integrity and DNA concentrations.

Identification of Escherichia coli multidrug resistance transporters involved in anthocyanin biosynthesis.

The anthocyanin compound cyanidin 3-O-glucoside (C3G) is a natural pigment widely used in food and nutraceutical industries. Its microbial synthesis by E. coli is a promising alternative to the traditional extraction methods. However, part of the synthesized C3G accumulates in the cytoplasm, thus potentially causing growth inhibition and product degradation. Therefore, it is necessary to enhance C3G secretion via exploration of native transporters facilitating C3G export. In this study, we report the screening and verification of native multidrug resistance transporters from 40 candidates in E. coli that can improve the extracellular C3G production when using catechin as the substrate. Overexpression of single transporter genes including fsr, yebQ, ynfM, mdlAB, and emrKY were found to increase C3G production by 0.5- to 4.8-fold. Genetic studies indicated that mdlAB and emrKY are vital transporters in the secretion of C3G. Our study reveals a set of new multidrug resistance transporters for the improvement of microbial biosynthesis of C3G and other anthocyanins.

Advancements in understanding bacterial enteritis pathogenesis through organoids.

Bacterial enteritis has a substantial role in contributing to a large portion of the global disease burden and serves as a major cause of newborn mortality. Despite advancements gained from current animal and cell models in improving our understanding of pathogens, their widespread application is hindered by apparent drawbacks. Therefore, more precise models are imperatively required to develop more accurate studies on host-pathogen interactions and drug discovery. Since the emergence of intestinal organoids, massive studies utilizing organoids have been conducted to study the pathogenesis of bacterial enteritis, revealing new mechanisms and validating established ones. In this review, we focus on the advancements of several bacterial pathogenesis mechanisms observed in intestinal organoid/enteroid models, exploring the host response and bacterial effectors during the infection process. Finally, we address the features that warrant additional investigation or could be enhanced in existing organoid models in order to guide future research endeavors.

A new look at the fluorescent protein-based approach for identifying optimal coding sequence for recombinant protein expression in E. coli.

Due to the degeneracy of the genetic code, most amino acids are encoded by several codons. The choice among synonymous codons at the N-terminus of genes has a profound effect on protein expression in Escherichia coli. This is often explained by the different contributions of synonymous codons to mRNA secondary structure formation. Strong secondary structures at the 5'-end of mRNA interfere with ribosome binding and affect the process of translation initiation. In silico optimization of the gene 5'-end can significantly increase the level of protein expression; however, this method is not always effective due to the uncertainty of the exact mechanism by which synonymous substitutions affect expression; thus, it may produce nonoptimal variants as well as miss some of the best producers. In this paper, an alternative approach is proposed based on screening a partially randomized library of expression constructs comprising hundreds of selected synonymous variants. The effect of such substitutions was evaluated using the gene of interest fused to the reporter gene of the fluorescent protein with subsequent screening for the most promising candidates according to the reporter's signal intensity. The power of the approach is demonstrated by a significant increase in the prokaryotic expression of three proteins: canine cystatin C, human BCL2-associated athanogene 3 and human cardiac troponin I. This simple approach was suggested which may provide an efficient, easy, and inexpensive optimization method for poorly expressed proteins in bacteria.

Cryo-EM structure of cytochrome bo3 quinol oxidase assembled in peptidiscs reveals an "open" conformation for potential ubiquinone-8 release.

Cytochrome bo3 quinol oxidase belongs to the heme­copper-oxidoreductase (HCO) superfamily, which is part of the respiratory chain and essential for cell survival. While the reaction mechanism of cyt bo3 has been studied extensively over the last decades, specific details about its substrate binding and product release have remained unelucidated due to the lack of structural information. Here, we report a 2.8 Å cryo-electron microscopy structure of cyt bo3 from Escherichia coli assembled in peptidiscs. Our structural model shows a conformation for amino acids 1-41 of subunit I different from all previously published structures while the remaining parts of this enzyme are similar. Our new conformation shows a "U-shape" assembly in contrast to the transmembrane helix, named "TM0", in other reported structural models. However, TM0 blocks ubiquinone-8 (reaction product) release, suggesting that other cyt bo3 conformations should exist. Our structural model presents experimental evidence for an "open" conformation to facilitate substrate/product exchange. This work helps further understand the reaction cycle of this oxidase, which could be a benefit for potential drug/antibiotic design for health science.

Characterization of diarrheagenic Escherichia coli from different cattle production systems in Brazil.

Diarrheagenic E. coli (DEC) can cause severe diarrhea and is a public health concern worldwide. Cattle are an important reservoir for this group of pathogens, and once introduced into the abattoir environment, these microorganisms can contaminate consumer products. This study aimed to characterize the distribution of DEC [Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC)] from extensive and intensive cattle production systems in Brazil. Samples (n = 919) were collected from animal feces (n = 200), carcasses (n = 600), meat cuts (n = 90), employee feces (n = 9), and slaughterhouse water (n = 20). Virulence genes were detected by PCR in 10% of animal samples (94/919), with STEC (n = 81) as the higher prevalence, followed by EIEC (n = 8), and lastly EPEC (n = 5). Animals raised in an extensive system had a higher prevalence of STEC (average 48%, sd = 2.04) when compared to animals raised in an intensive system (23%, sd = 1.95) (Chi-square test, P < 0.001). From these animals, most STEC isolates only harbored stx2 (58%), and 7% were STEC LEE-positive isolates that were further identified as O157:H7. This study provides further evidence that cattle are potential sources of DEC, especially STEC, and that potentially pathogenic E. coli isolates are widely distributed in feces and carcasses during the slaughter process.

Survival of O157 and non-O157 shiga toxin-producing Escherichia coli in Korean style kimchi.

Korean style kimchi contaminated with Shiga toxin-producing Escherichia coli (STEC) O157:H7 was the cause of an outbreak in Canada from December 2021 to January 2022. To determine if this STEC O157:H7 has greater potential for survival in kimchi than other STEC, the outbreak strain and six other STEC strains (O26:H11, O91:H21, O103:H2, O121:H19, and two O157:H7) were inoculated individually at 6 to 6.5 log CFU/g into commercially sourced kimchi and incubation at 4 °C. At intervals of seven days inoculated and control kimchi was plated onto MacConkey agar to enumerate lactose utilising bacteria. The colony counts were interpreted as enumerating the inoculated STEC, since no colonies were observed on MacConkey agar plated with uninoculated kimchi. Over eight weeks of incubation the pH was stable at 4.10 to 4.05 and the STEC strains declined by 0.7-1.0 log, with a median reduction of 0.9 log. The linear rate of reduction of kimchi outbreak STEC O157:H7 was -0.4 log per 30 days (Slope Uncertainty 0.05), which was not significantly different from the other O157 and nonO157 STEC strains (P = 0.091). These results indicate that the outbreak was not due to the presence of strain better adapted to survival in kimchi than other STEC, and that STEC can persist in refrigerated Korean style kimchi with a minimal decline over the shelf-life of the product.